Development of radiometal-based labelling techniques and tracers for non-invasive molecular imaging

Metallic radionuclides, radiometals, have an important role in nuclear medicine. Their straightforward coordination radiochemistry allows for a large variety of applications. The similarities and differences between the radiometals can be utilised to expand the window of diagnostic imaging or transfer diagnostic methods from one imaging modality to another. Radiometals from the same or from different elements (both therapeutic and diagnostic) may be coordinated to similar probes, as a theranostic pair. Radionuclide-based molecular imaging is a non-invasive in vivo imaging technique that quantifies the concentrations of radioactive probes in biological processes occurring at cellular and subcellular levels in living organisms. The two major diagnostic in vivo imaging techniques used are Single-Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET). In this thesis, radiometal production using a cyclotron solid target system and some fundamental aspects of radiometal labelling are explored, using two of the most common positron-emitting radiometals, gallium-68 (68Ga) and zirconium-89 (89Zr). In paper I an albumin targeting Affibody molecule, ABY-028, was successfully developed, 68Ga-labelled and in vivo evaluated using a small animal PET camera. We showed that the biodistribution was consistent with the binding of [68Ga]Ga-ABY-028 to plasma albumin. Uptake patterns differed between tumours at different stages and of different phenotypes. Tracer uptake responses to permeability-altering therapeutics and during cerebral infarction could be observed. This novel radiotracer is a promising tool for in vivo molecular imaging of variations and alterations of vascular permeability and has the potential to function as a baseline control of the non-specific uptake of other albumin-binding domain (ABD)-based diagnostic or therapeutic agents. In paper II cells were 89Zr-labelled, using two different metal complexes, with two distinctive labelling mechanisms, [89Zr]Zr-(oxine)4 and [89Zr]Zr-DFO-NCS. Synthesis protocols were successfully optimised to yield high radiochemical conversions of both 89Zr-complexes. Both radiotracers presented in this head-to-head study showed feasibility for universal radiolabellings of different cell types. The results suggested that [89Zr]Zr-(oxine)4 is most likely superior. In papers III and IV methods to meet the generally increasing demand for 68Ga have been developed. In paper III a cyclotron-based solid target system was used for production and purification of the radionuclide. In paper IV a refinement method of the radionuclide’s quality (regarding content of competing metal ions) was developed for clinical applicability for use in radiolabelling of DOTA-based radiopharmaceuticals, [68Ga]Ga-DOTATOC and [68Ga]Ga-FAPI-46. Compared to generator-derived 68Ga, we successfully produced 10 times more product of both the radiopharmaceuticals using our solid target cyclotron-produced 68Ga. The strategies and approaches investigated and developed in this thesis have potential for translation to more exotic radiometals in the future, to potentially expanding the palette of chemical properties that can be used in radiolabelling, as well as the decay characteristics and time-windows for imaging. The methods and techniques for radiometal labelling explored in this thesis might also be translated to other specific tissue targeting molecules or cells

Clinically Applicable Cyclotron-Produced Gallium-68 Gives High-Yield Radiolabeling of DOTA-Based Tracers

By using solid targets in medical cyclotrons, it is possible to produce large amounts of 68GaCl3. Purification of Ga3+ from metal ion impurities is a critical step, as these metals compete with Ga3+ in the complexation with different chelators, which negatively affects the radiolabeling yields. In this work, we significantly lowered the level of iron (Fe) impurities by adding ascorbate in the purification, and the resulting 68GaCl3could be utilized for high-yield radiolabeling of clinically relevant DOTA-based tracers. 68GaCl3 was cyclotron-produced and purified with ascorbate added in the wash solutions through the UTEVA resins. The 68Ga eluate was analyzed for radionuclidic purity (RNP) by gamma spectroscopy, metal content by ICP-MS, and by titrations with the chelators DOTA, NOTA, and HBED. The 68GaCl3eluate was utilized for GMP-radiolabeling of the DOTA-based tracers DOTATOC and FAPI-46 using an automated synthesis module. DOTA chelator titrations gave an apparent molar activity (AMA) of 491 ± 204 GBq/µmol. GMP-compliant syntheses yielded up to 7 GBq/batch [68Ga]Ga-DOTATOC and [68Ga]Ga-FAPI-46 (radiochemical yield, RCY ~ 60%, corresponding to ten times higher compared to generator-based productions). Full quality control (QC) of 68Ga-labelled tracers showed radiochemically pure and stable products at least four hours from end-of-synthesis

Hallmarks in prostate cancer imaging with Ga68-PSMA-11-PET/CT with reference to detection limits and quantitative properties

Abstract Background Gallium-68-labeled prostate-specific antigen positron emission tomography/computed tomography imaging (Ga68-PSMA-11-PET/CT) has emerged as a potential gold standard for prostate cancer (PCa) diagnosis. However, the imaging limitations of this technique at the early state of PCa recurrence/metastatic spread are still not well characterized. The aim of this study was to determine the quantitative properties and the fundamental imaging limits of Ga68-PSMA-11-PET/CT in localizing small PCa cell deposits. Methods The human PCa LNCaP cells (PSMA expressing) were grown and collected as single cell suspension or as 3D-spheroids at different cell numbers and incubated with Ga68-PSMA-11. Thereafter, human HCT116 cells (PSMA negative) were added to a total cell number of 2 × 105 cells per tube. The tubes were then pelleted and the supernatant aspirated. A whole-body PET/CT scanner with a clinical routine protocol was used for imaging the pellets inside of a cylindrical water phantom with increasing amounts of background activity. The actual activity bound to the cells was also measured in an automatic gamma counter. Imaging detection limits and activity recovery coefficients as a function of LNCaP cell number were obtained. The effect of Ga68-PSMA-11 mass concentration on cell binding was also investigated in samples of LnCaP cells incubated with increasing concentrations of radioligand. Results A total of 1 × 104 LNCaP cells mixed in a pellet of 2 × 105 cells were required to reach a 50% detection probability with Ga68-PSMA-11-PET/CT without background. With a background level of 1 kBq/ml, between 4 × 105 and 1 × 106 cells are required. The radioligand equilibrium dissociation constant was 27.05 nM, indicating high binding affinity. Hence, the specific activity of the radioligand has a profound effect on image quantification. Conclusions Ga68-PSMA-11-PET detects a small number of LNCaP cells even when they are mixed in a population of non-PSMA expressing cells and in the presence of background. The obtained image detection limits and characteristic quantification properties of Ga68-PSMA-11-PET/CT are essential hallmarks for the individualization of patient management. The use of the standardized uptake value for Ga68-PSMA-11-PET/CT image quantification should be precluded

The cellular basis of increased PET hypoxia tracer uptake in focal cerebral ischemia with comparison between [¹⁸F]FMISO and [⁶⁴Cu]CuATSM

PET hypoxia imaging can assess tissue viability in acute ischemic stroke (AIS). [(18)F]FMISO is an established tracer but requires substantial accumulation time, limiting its use in hyperacute AIS. [(64)Cu]CuATSM requires less accumulation time and has shown promise as a hypoxia tracer. We compared these tracers in a M2-occlusion model (M2CAO) with preserved collateral blood flow. Rats underwent M2CAO and [(18)F]FMISO (n = 12) or [(64)Cu]CuATSM (n = 6) examinations. [(64)Cu]CuATSM animals were also examined with MRI. Pimonidazole was used as a surrogate for [(18)F]FMISO in an immunofluorescence analysis employed to profile levels of hypoxia in neurons (NeuN) and astrocytes (GFAP). There was increased [(18)F]FMISO uptake in the M2CAO cortex. No increase in [(64)Cu]CuATSM activity was found. The pimonidazole intensity of neurons and astrocytes was increased in hypoxic regions. The pimonidazole intensity ratio was higher in neurons than in astrocytes. In the majority of animals, immunofluorescence revealed a loss of astrocytes within the core of regions with increased pimonidazole uptake. We conclude that [(18)F]FMISO is superior to [(64)Cu]CuATSM in detecting hypoxia in AIS, consistent with an earlier study. [(18)F]FMISO may provide efficient diagnostic imaging beyond the hyperacute phase. Results do not provide encouragement for the use of [(64)Cu]CuATSM in experimental AIS

Optimized, automated and cGMP-compliant synthesis of the HER2 targeting [68Ga]Ga-ABY-025 tracer

Abstract Background The Affibody molecule, ABY-025, has demonstrated utility to detect human epidermal growth factor receptor 2 (HER2) in vivo, either radiolabelled with indium-111 (111In) or gallium-68 (68Ga). Using the latter, 68Ga, is preferred due to its use in positron emission tomography with superior resolution and quantifying capabilities in the clinical setting compared to 111In. For an ongoing phase II study (NCT05619016) evaluating ABY-025 for detecting HER2-low lesions and selection of patients for HER2-targeted treatment, the aim was to optimize an automated and cGMP-compliant radiosynthesis of [68Ga]Ga-ABY-025. [68Ga]Ga-ABY-025 was produced on a synthesis module, Modular-Lab PharmTracer (Eckert & Ziegler), commonly used for 68Ga-labelings. The radiotracer has previously been radiolabeled on this module, but to streamline the production, the method was optimized. Steps requiring manual interactions to the radiolabeling procedure were minimized including a convenient and automated pre-concentration of the 68Ga-eluate and a simplified automated final formulation procedure. Every part of the radiopharmaceutical production was carefully developed to gain robustness and to avoid any operator bound variations to the manufacturing. The optimized production method was successfully applied for 68Ga-labeling of another radiotracer, verifying its versatility as a universal and robust method for radiosynthesis of Affibody-based peptides. Results A simplified and optimized automated cGMP-compliant radiosynthesis method of [68Ga]Ga-ABY-025 was developed. With a decay corrected radiochemical yield of 44 ± 2%, a radiochemical purity (RCP) of 98 ± 1%, and with an RCP stability of 98 ± 1% at 2 h after production, the method was found highly reproducible. The production method also showed comparable results when implemented for radiolabeling another similar peptide. Conclusion The improvements made for the radiosynthesis of [68Ga]Ga-ABY-025, including introducing a pre-concentration of the 68Ga-eluate, aimed to utilize the full potential of the 68Ge/68Ga generator radioactivity output, thereby reducing radioactivity wastage. Furthermore, reducing the number of manually performed preparative steps prior to the radiosynthesis, not only minimized the risk of potential human/operator errors but also enhanced the process’ robustness. The successful application of this optimized radiosynthesis method to another similar peptide underscores its versatility, suggesting that our method can be adopted for 68Ga-labeling radiotracers based on Affibody molecules in general. Trial registration: NCT, NCT05619016, Registered 7 November 2022, https://clinicaltrials.gov/study/NCT05619016?term=HER2&cond=ABY025&rank=

Optimisation of the Synthesis and Cell Labelling Conditions for [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS: a Direct In Vitro Comparison in Cell Types with Distinct Therapeutic Applications

Erratum inCorrection: Optimisation of the Synthesis and Cell Labelling Conditions for [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS: a Direct In Vitro Comparison in Cell Types with Distinct Therapeutic Applications.Friberger I, Jussing E, Han J, Goos JACM, Siikanen J, Kaipe H, Lambert M, Harris RA, Samén E, Carlsten M, Holmin S, Tran TA.Mol Imaging Biol. 2022 Jun;24(3):510. doi: 10.1007/s11307-022-01709-1.PMID: 35174429 Free PMC article. No abstract available.International audienceAbstract Background There is a need to better characterise cell-based therapies in preclinical models to help facilitate their translation to humans. Long-term high-resolution tracking of the cells in vivo is often impossible due to unreliable methods. Radiolabelling of cells has the advantage of being able to reveal cellular kinetics in vivo over time. This study aimed to optimise the synthesis of the radiotracers [ 89 Zr]Zr-oxine (8-hydroxyquinoline) and [ 89 Zr]Zr-DFO-NCS (p-SCN-Bn-Deferoxamine) and to perform a direct comparison of the cell labelling efficiency using these radiotracers. Procedures Several parameters, such as buffers, pH, labelling time and temperature, were investigated to optimise the synthesis of [ 89 Zr]Zr-oxine and [ 89 Zr]Zr-DFO-NCS in order to reach a radiochemical conversion (RCC) of >95 % without purification. Radio-instant thin-layer chromatography (iTLC) and radio high-performance liquid chromatography (radio-HPLC) were used to determine the RCC. Cells were labelled with [ 89 Zr]Zr-oxine or [ 89 Zr]Zr-DFO-NCS. The cellular retention of 89 Zr and the labelling impact was determined by analysing the cellular functions, such as viability, proliferation, phagocytotic ability and phenotypic immunostaining. Results The optimised synthesis of [ 89 Zr]Zr-oxine and [ 89 Zr]Zr-DFO-NCS resulted in straightforward protocols not requiring additional purification. [ 89 Zr]Zr-oxine and [ 89 Zr]Zr-DFO-NCS were synthesised with an average RCC of 98.4 % (n = 16) and 98.0 % (n = 13), respectively. Cell labelling efficiencies were 63.9 % (n = 35) and 70.2 % (n = 30), respectively. 89 Zr labelling neither significantly affected the cell viability (cell viability loss was in the range of 1–8 % compared to its corresponding non-labelled cells, P value > 0.05) nor the cells’ proliferation rate. The phenotype of human decidual stromal cells (hDSC) and phagocytic function of rat bone-marrow-derived macrophages (rMac) was somewhat affected by radiolabelling. Conclusions Our study demonstrates that [ 89 Zr]Zr-oxine and [ 89 Zr]Zr-DFO-NCS are equally effective in cell labelling. However, [ 89 Zr]Zr-oxine was superior to [ 89 Zr]Zr-DFO-NCS with regard to long-term stability, cellular retention, minimal variation between cell types and cell labelling efficiency