Novel 68Ga/89Zr-chelators for targeted biomolecules in PET

Over the last years Gallium-68 (68Ga) has received tremendous attention for labelling of radiopharmaceuticals for Positron Emission Tomography (PET). As a generator nuclide with a half life of 68min and high positron emission 68Ga has become an interesting alternative to Fluor-18. 68Ga labeling of biomolecules currently in based on the use of bifunctional chelators based on aminocarboxylates (mainly DOTA and NOTA). We recently could show that cyclic peptide siderophores have very good complexing properties for 68Ga resulting in high specific activities and excellent metabolic stability. Starting from a cyclic derivative we developed a synthetic pathway to conjugate amino acids and a model RGD peptide to this peptide siderophore. A trimeric RGD conjugate could be labelled with 68Ga under mild conditions achieving extraordinary high specific activities. It showed excellent stability and efficient binding to alpha-v-Beta3 Integrin expressing tumour cells, much higher than established monomeric RGD-derivatives. Besides 68Ga also 89Zr has attracted great interest for applications in PET. Its half life of 78.4hr allows imaging of biological targets even days after application. In preliminary tests we could show that various peptide siderophores can be labeled with 89Zr achieving high yields and stability providing an alternative to the currently used Desferrioxamine (DFO) for 89Zr radiolabelling. Based on these preliminary results we postulated the following: A) Cyclic peptide siderophores are very promising bifuctional chelators to prepare bioconjugates for 68Ga labelling. B) Coupling of peptides TAFC derivatives results in a metabolic stabilization of the chelate. C) Such cyclic peptide siderophore conjugates can also be radiolabelled with 89Zr providing advantages over linear chelators. This project aims to use selected peptide siderophores for the design, synthesis and evaluation of targeted biomolecules for 68Ga and 89Zr labelling. This will be achieved in 6 working packages (WP). The first two WP include RGD conjugates with different linkers, WP3 a minigastrin- and WP4 a conjugate with an EGFR targeted affibody. These WP include synthesis, radiolabelling and characterization regarding stability, receptor binding and targeting in respective tumour models. In an additional WP selected conjugates will be labelled with 89Zr and compared with their 68Ga-counterparts. WP6 deals with evaluation of synthetic strategies to access bioconjugates with other peptide siderophores for 68Ga and 89 Zr labelling. Cooperations with the University of Nijmegen (RGD and Minigastrin) and Uppsala (Affibodies) have been established, in particular also for characterization of the radiolabelled bioconjugates and their imaging properties by means of Micro-PET. This project explores novel opportunities to prepare highly specific radiolabelled biomolecules for molecular imaging with PET in particular for oncological applications thereby opening new opportunities for nuclear medicine diagnostics.

Over the last years Gallium-68 (68Ga) and Zirkonium-89 (89Zr) have received tremendous attention for labelling of radiopharmaceuticals for Positron Emission Tomography (PET), a modern molecular imaging method. As a generator nuclide with a half life of 68min 68Ga has become an interesting alternative to Fluor-18, 89Zr especially for labelling proteins such as antibodies. In this project, the application of cyclic peptide siderophores, especially fusarinine C (FSC), as a scaffold for the development of targeted biomolecules for radiolabelling with 68Ga and 89Zr was investigated. In a first step, RGD peptides that bind to integrin target structures expressed on blood vessels in tumor tissue, were coupled to FSC. These FSC (RGD)3 bioconjugates (3 peptides per FSC molecule) showed excellent binding of 68Ga and 89Zr and markedly enhanced binding to integrins as compared to single labeled RGD peptides. In a second step, minigastrin peptides that recognize specific tumors, such as medullary thyroid carcinoma, were conjugated to FSC. It was shown that by attaching several peptides, the accumulation in the tumor could be increased and an improved diagnostic of tumors is possible, also by the fact that these molecules are longer stable in the body and less rapidly lose their binding properties. The use of the new FSC-based chelators has also been demonstrated by attaching a so-called Affibody, a small protein that selectively recognizes a growth factor of tumor cells, to FSC. In particular, FSC revealed to be a promising alterative to previously used 89Zr labels. Initial chemical modifications of the FSC basic structure were also performed. A new research area today is the combination of radioactive labels with fluorescent markers that allow optical imaging with PET using a diagnostic marker. We were able to modify the FSC molecule that on the one hand it detects tumor cells by radioactive decay and detection by PET, on the other hand it also emits fluorescence and thus can also be visualized with optical methods, for example, intraoperatively. The project has been successfully realized through several international collaborations (Uppsala, Nijmegen, London and Olomouc) resulting in more than 10 scientific publications, 2 dissertations and 3 diploma theses, as well as numerous conference presentations. It provided new opportunities to develop targeted molecules for molecular imaging, especially in oncology, with improved properties for PET towards improved diagnostic methods in personalized medicine.