18F-Labelling of biomolecules for PET

Positron emission tomography (PET) is a sensitive, non-invasive imaging technology that produces three-dimensional images showing the in vivo concentration of radiotracers, which are usually labelled with short-lived positron emitting radioisotopes. Among β+-emitting radioisotopes, fluorine-18 is currently the radionuclide of choice for PET because of its favourable nuclear decay characteristics and ease of production. Clinicians in the molecular imaging community are not only interested in in using small organic molecules but also high molecular weight biomolecules and peptides are increasingly being considered for use as PET-radiopharmaceuticals. However, the incorporation of fluorine-18 into heat-sensitive and complex biomolecules creates substantial challenges for radiochemists. The Al18F-labelling method is a relatively new approach that allows radiofluorination of biomolecules such as peptides and proteins in a one-step procedure and in aqueous solution. McBride et al. published the first study using a chelator for {Al18F}2+ labelling in 2009. Although many studies demonstrated the versatility of the Al18F approach to radiolabel peptides, the approach has limited applicability for heat sensitive biomolecules due to high temperatures required for the complexation reaction (≥ 100 °C) when used with macrocyclic complexing agents such as NOTA and NODA. Therefore the objective of this thesis was to develop new polydentate ligands that allow chelation of {Al18F}2+ at moderate temperatures (<40 °C), whilst maintaining in vivo stability. In this thesis, we identified new chelators that allow efficient complexation of {Al18F}2+ using mild labelling conditions, whilst maintaining in vivo stability. The first set of chelators that we developed are acyclic ligands with an N2O3 coordinative set or N3O2 bis-phenol structure. It was observed that only ligands containing the fragment ethylenediamine-N,N’-diacetic acid (H3L1-L4) were able to efficiently chelate aluminium fluoride. Ligands without this arrangement of functional groups (H3L5-8) did not complex aluminium fluoride with good RCY under the conditions tested. Successful labelling of two compounds, (H3L1) and (H3L3) was established. H3L1 and H3L3 both have excellent labelling properties but, unfortunately, [18F]AlF-L1 was not stable in vitro. [18F]AlF-L3 complex demonstrated a stability in PBS and in rat plasma comparable to that of the previously reported [18F]AlF-N-benzyl-NODA complex, up to 60 minutes, but suffered from decomplexation after extended incubation times. Although in vitro incubation in rat plasma showed slow decomposition, biodistribution of [18F]AlF-L3 showed absence of bone uptake indicating that in vivo defluorination or demetalation if any, is limited. Optimal labelling results were obtained at pH 4.5-5 and it has been shown that the specific activity of fluorine-18 proves to be an important parameter for the Al18F chelation yield. The acyclic chelator H3L3 demonstrated to be a good lead candidate for labelling of heat-sensitive biomolecules with fluorine-18. However, there was still room for improvement regarding the stability of the [18F]AlF-L3 complex, so that the synthesis of improved derivatives was warranted. We explored whether incorporation of a trans-cyclohexyl moiety into H3L1, to form restrained complexing agent 1 (RESCA1), would increase steric rigidity of the AlF-complex, improving the orientation of the chelating groups and as a result improving the stability of the resulting AlF-complex. RESCA1 is an acyclic pentadentate ligand with an N2O3 coordinative set of donor atoms and was synthesised starting from commercially available chemicals. RESCA1 as such showed excellent labelling properties and its Al18F-complex demonstrated high in vitro and in vivo stability. Small changes in the backbone of RESCA1, to form RESCA2-5, resulted in minor to major loss of stability of the corresponding Al18F-complexes and thus, RESCA1 was found to be the most promising lead candidate for radiolabelling heat-sensitive biomolecules via the Al18F-method. To demonstrate the generic applicability of the proposed method we successfully labelled the heat-sensitive biomolecule Human Serum Albumin (HSA) at ambient temperature with {Al18F}2+ with high radiochemical purity in less than 30 minutes. [18F]AlF-RESCA1-HSA showed excellent in vitro and in vivo stability. Moreover, [18F]AlF-RESCA1-HSA showed favourable properties for PET blood pool imaging applications. Prostate-specific membrane antigen (PSMA) is overexpressed in a majority of primary and metastatic prostate cancer patients and is a very promising target for specific prostate cancer imaging and therapy. Currently, [68Ga]Ga-PSMA-HBEDD-CC is the most widely used PSMA-ligand. This urea-based PSMA inhibitor demonstrates excellent pharmacokinetics as well as stability in vivo, leading to its clinical applications worldwide for imaging of prostate cancer. However, labelling with fluorine-18 would offer advantages with respect to availability, batch size, and image resolution compared to labelling with gallium-68. In this thesis we have developed three new Al18F-labelled urea-based PSMA inhibitors, [18F]AlF-PSMA-L3, [18F]AlF-PSMA-NODA-MPAA and [18F]AlF-PSMA-RESCA1. Cell uptake and internalisation experiments with LNCaP (PSMA+) and PC-3 (PSMA-) cells revealed similar specific internalisation and cell accumulation for [18F]AlF-PSMA-RESCA1 and [68Ga]Ga-PSMA-HBED-CC. In contrast, [18F]AlF-PSMA-NODA-MPAA and [18F]AlF-PSMA-L3 showed considerable lower uptake in LNCaP cells. Moreover, [18F]AlF-PSMA-L3 suffers from instability over time which compromises the utility of this tracer. All tracers were cleared fast from plasma and PSMA-negative tissue mainly by the kidneys but also hepatobiliary excretion was observed in the case of [18F]AlF-RESCA1-PSMA. High specific accumulation of all tracers was observed on PSMA+ LNCaP tumour slices and autoradiography on human prostate tumour tissue showed specific affinity for the human PSMA receptor. Comparable specific tumor uptake was observed for [18F]AlF-PSMA-RESCA1 and [68Ga]Ga-HBEDD-CC-PSMA in a head-to-head comparison in PSMA+ tumor bearing mice. To conclude, [18F]AlF-PSMA-RESCA1 might be a promising tool for early detection of prostate cancer recurrence. Nanobodies are promising tools in molecular imaging and have many advantages in comparison with full-size antibodies. We demonstrated that the Al18F-method using RESCA1 provides an efficient approach to radiolabel nanobodies with fluorine-18 at ambient temperature. We successfully derivatised nanobodies targeting the CRIg receptor with RESCA1 and labelled the conjugate with {Al18F}2+ in good radiochemical yields at ambient temperature. [18F]AlF-RESCA1-NbV4m119 was purified with preparative HPLC resulting in the Al18F-tracer with a radiochemical purity higher than 97% and the compound showed excellent in vitro stability. Furthermore, we successfully optimised a generic radio-HPLC-HRMS system for the analysis and quality control of radiolabelled nanobodies. Biodistribution studies in mice showed that [18F]AlF-RESCA1-NbV4m119 was mainly excreted via the renal pathway but also high liver uptake was observed in normal WT mice, which is expected because CRIg receptors are highly expressed on Kupffer cells. Biodistribution and microPET studies in CRIg-/- mice demonstrated absence of significant uptake in liver, indicating excellent specificity of the Al18F-labelled tracer. Almost no intact [18F]AlF-RESCA1-NbV4m119 was found in urine at 3 h p.i. and relatively high bone uptake was observed, indicating in vivo instability of the tracer. However, in vivo PET imaging in mice did not show significant bone uptake at 1 h p.i. and high liver-to background images were obtained, indicating favourable in vivo imaging properties of [18F]AlF-RESCA1-NbV4m119 with high specificity for CRIg. This generic radiolabelling, purification and characterisation method provides a general approach for radiolabelling of nanobodies with fluorine-18. In conclusion, the acyclic chelator RESCA1 demonstrated to be a good lead candidate for labelling of heat-sensitive biomolecules with fluorine-18. This new class of AlF-chelators may have a great impact on PET radiochemical space as it will stimulate the rapid development of new fluorine-18 labelled peptides and other heat-sensitive biomolecules. However, more basic research is needed before this method can result in a kit-based, radiolabelling strategy.

Jaar van publicatie:2016

Toegankelijkheid:Open