Advanced chelators for innovative diagnosis technologies – ACHETE

The aim of the project is to develop an effective method for tetra-N-functionalisation of cyclam, allowing the introduction of four bidentate coordinating groups, such as hydroxamate or HOPO, to design octadentate ligands for Zr4+ coordination. The originality of this approach lies in the use of cyclam as a platform for the pre-organisation of the four bidentate coordination units to ensure rapid and efficient complexation while ensuring the aqueous solubility of the complexes obtained. The 89Zr radiolabelling studies aim to develop an effective radiolabelling protocol and to evaluate the stability of the radiochelates obtained. Furthermore, cyclam has the advantage of being easily derived into a bifunctional version, i.e. possessing an additional reactive group, using well-established C-functionalisation methods. The objective is to synthesise a bifunctional version of the most effective chelators in order to graft them onto antibodies or polymers to obtain radiopharmaceuticals for immuno-PET or metal polymers for mass cytometry, respectively.

Two families of octadentate ligands, based on cyclam bearing four hydroxamate (L1 and L2) or HOPO units (L3 and L4), were designed. The syntheses were optimised for reproducibility. The chelators were subjected to coordination and radiolabelling studies using 89Zr. Initial stability assessments guided the design of future chelators for in vivo application. Work on the synthesis of bifunctional derivatives has also been initiated, enabling the development of radiopharmaceuticals for immuno-PET or metal polymers for mass cytometry. However, preliminary studies in mass cytometry have revealed limitations in terms of sensitivity when detecting stable zirconium isotopes, and priority will be given to the use of bifunctional chelates for application in PET imaging.

According to the initial objectives, the project led to the design of two families of octadentate ligands for Zr4+ complexation. The solubility issues usually encountered with Zr4+ chelates were overcome with AHA derivatives, albeit at the expense of lower stability of the resulting radiocomplexes. The opposite behaviour was observed for HOPO derivatives. The chelates are limited by their water solubility but show stability similar to that of the DFO reference radiocomplex. These results pave the way for future structural modifications, such as the introduction of more rigid AHA motifs (PIPOH) or the AHA/HOPO combination, with the aim of achieving a balance between solubility in biological media and the stability of (radio)complexes.
The project has led to advances in the field of zirconium coordination chemistry, which remains largely unexplored due to the particular properties of this highly charged ion. Particular emphasis was placed on attempts to characterise the complexes, although further efforts are still needed. However, the design of these new ligands, and in particular HOPO ligands, also opens up the possibility of exploring metal cations with similar properties, such as Ti4+, Hf4+ and Sc3+. The latter two are particularly interesting for applications in nuclear medicine. HOPO ligands could thus be tested for scandium coordination. This rare earth metal is considered a hard Lewis acid due to its small ionic radius and +3 charge. It has several radionuclides for which interest is growing. In particular, 43Sc and 44Sc, which are positron emitters, are suitable for PET imaging, while 47Sc, a ß-emitter, has great potential in radiotherapy. All of these isotopes thus form promising theranostic pairs (44Sc/47Sc or 43Sc/47Sc).
The transposition of cyclam tetrafunctionalisation methods to bifunctional platforms has been validated, opening up interesting prospects within the framework of this project, but also in other fields such as catalysis and functional materials.
With regard to CM, although no conclusive results were obtained with Zr4? due to its low isotopic sensitivity, the project led to the initiation of a partnership with the Hyperion platform and the LBAI laboratory at UBO, focusing on a project to develop new labelling agents based on other metals (In3+, La3+ and Bi3+). If successful, this work could lead to a patent application or generate industrial interest.

The work has been presented at national and international conferences in the form of oral presentations (annual scientific day of CGO ‘Vectorisation, Imaging, Radiotherapies’, 16th international CGO workshop ‘Tumour Targeting, Imaging, Radiotherapies’, GECOM-CONCOORD) or posters (SCF 2023, UBO Research Day, SCF-BPL 2024). Two publications are currently being drafted and will be submitted before the end of 2025.

In order to ensure the follow-up of a cancer patient, from the diagnosis and throughout the (post)treatment process, anatomical imagery is no longer sufficient and needs to be associated to single-cell analysis techniques to better understand the characteristics of cancer cells.
Among the imaging modalities, PET (Positron Emission Tomography) is particularly sensitive and coupled with the specificity of an antibody, it allows a targeted diagnosis (immunoPET). Metallic radioisotopes are currently the subject of intense researches because their wide variety enables to broaden the field of detectable tumors.
In terms of single-cell analyses, mass cytometry is particularly powerful. It directly emanates from flow cytometry, but the detection of tagged cells is done by mass spectrometry which has a resolution within one atomic mass unit. The unique properties of mass cytometry allow the identification of rare cell populations through the detection of antibodies labeled with stable metal isotopes.
For both in vivo and in vitro diagnostic methods, the use of Zr(IV) is particularly interesting. But like any metal, it cannot be used under its free form but as a stable metal chelate that is compatible with biological media (ie. water-soluble).
Zr(IV) has a beta+ emitting isotope, Zr-89, which thanks to its average energy (397 kV) enables to obtain PET images with a very good signal/noise ratio. Its relatively long half-life (78.4 h) allows its association to antibodies having slow biodistribution kinetics and therefore to detect cancers long and/or difficult to access, or to evaluate the response of patients to radiotherapy. Currently, the labelling of antibodies with Zr-89 uses the linear ligand Deferoxamine B (DFO) as chelator. However, several studies have shown the in vivo release of free Zr-89 which accumulates in bones.
The labelling agents employed for mass cytometry are more precisely based on antibodies labelled with polymers functionalized by multiple copies of a same metal chelate. The wide choice of existing metallic isotopes theoretically allows access to 135 detection channels, and therefore to identify as many cellular components, without signal overlap, for a complete study of the functionalities of biological systems. However, in reality, only the isotopes of lanthanide ions are fully exploited (38 parameters). To respond to cellular complexity, access to more metals, such as Zr(IV) which has 4 exploitable stable isotopes, is essential. Nevertheless, this requires the development of new chelating polymers and more specifically of chelating units specific of the metal to considered in order to have stable complexes which do not dissociate in the cellular medium. In the literature, only one polymer has been proposed for the use of Zr(IV), it is based on DFO but is limited by water-solubility issues.
Because of the limitations of DFO in biological media, it is essential to propose new Zr(IV) chelators. On the other hand, it seems judicious to be able to dispose of a unique family of ligands which could be used both for immunoPET imaging and for mass cytometry. Cyclic polyamines are known for their excellent coordination properties. Moreover, adequately functionalized, they can be specific of a given metal. The objective of the project is to develop new polyazamacrocyclic Zr(IV) chelators stable and soluble in biological media as well as their bifunctional analogues which will then be directly coupled to antibodies and tested as radiopharmaceuticals for immunoPET imaging with Zr-89 but also used for the design of chelating polymers for mass cytometry.