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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.

ABSTRACT:

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 [⁸⁹Zr]Zr-oxine (8-hydroxyquinoline) and [⁸⁹Zr]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 [⁸⁹Zr]Zr-oxine and [⁸⁹Zr]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 [⁸⁹Zr]Zr-oxine or [⁸⁹Zr]Zr-DFO-NCS. The cellular retention of ⁸⁹Zr 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 [⁸⁹Zr]Zr-oxine and [⁸⁹Zr]Zr-DFO-NCS resulted in straightforward protocols not requiring additional purification. [⁸⁹Zr]Zr-oxine and [⁸⁹Zr]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. ⁸⁹Zr 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 [⁸⁹Zr]Zr-oxine and [⁸⁹Zr]Zr-DFO-NCS are equally effective in cell labelling. However, [⁸⁹Zr]Zr-oxine was superior to [⁸⁹Zr]Zr-DFO-NCS with regard to long-term stability, cellular retention, minimal variation between cell types and cell labelling efficiency.

SUBMITTER: Friberger I

PROVIDER: S-EPMC8578071 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundIn vivo monitoring of cell biodistribution using positron emission tomography (PET) provides a quantitative non-invasive method to further optimize cell therapies and related new developments in the field. Our group has earlier optimized and evaluated the in vitro properties of two radiotracers,[89Zr]Zr-(oxinate)4 and [89Zr]Zr-DFO-NCS, for the radiolabelling of different cell types. Here, we performed a microPET study to assess the in vivo biodistribution of cells in rats using these two radiotracers. Human decidual stromal cells (hDSC) and rat macrophages (rMac) were radiolabelled with [89Zr]Zr-(oxinate)4 or [89Zr]Zr-DFO-NCS. Rats were intravenously injected with radiolabelled cells, and the in vivo biodistribution was monitored with microPET/CT imaging for up to day 7. Organ uptake was evaluated and presented as a percentage of injected activity per gram tissue (%IA/g) and total absorbed organ doses (mSv/MBq).ResultsThe biodistribution in vivo showed an immediate uptake in the lungs. Thereafter, [89Zr]Zr-(oxinate)4 labelled cells migrated to the liver, while the signal from [89Zr]Zr-DFO-NCS labelled cells lingered in the lungs. The differences in the in vivo behaviour for the same cell type appeared related to the radiotracer labelling. After 24 h, [89Zr]Zr-(oxinate)4 labelled cells had over 70% higher liver uptake for both hDSC and rMac compared to [89Zr]Zr-DFO-NCS labelled cells, whereas [89Zr]Zr-DFO-NCS labelled cells showed over 60% higher uptake in the lungs compared to [89Zr]Zr-(oxinate)4 labelled cells. This difference in both lung and liver uptake continued until day 7. Dosimetry calculations showed a higher effective dose (mSv/MBq) for [89Zr]Zr-DFO-NCS compared to [89Zr]Zr-(oxinate)4, for both cell types. Although the bone uptake was higher for [89Zr]Zr-(oxinate)4 labelled cells, the prolonged uptake in the lungs contributed to a significant crossfire to bone marrow resulting in a higher bone dose.ConclusionThe [89Zr]Zr-DFO-NCS labelled cells suggest a prolonged accumulation in the lungs, while [89Zr]Zr-(oxinate)4 suggests quicker clearance of the lungs followed by accumulation in the liver. Accumulation of radiolabelled cells in the liver corresponds to other cell-tracking methods. Further studies are required to determine the actual location of the [89Zr]Zr-DFO-NCS labelled cell.

Project description:PurposeAll clinical 89Zr-immuno-PET studies are currently performed with the chelator desferrioxamine (DFO). This chelator provides hexadentate coordination to zirconium, leaving two coordination sites available for coordination with, e.g., water molecules, which are relatively labile ligands. The unsaturated coordination of DFO to zirconium has been suggested to result in impaired stability of the complex in vivo and consequently in unwanted bone uptake of 89Zr. Aiming at clinical improvements, we report here on a bifunctional isothiocyanate variant of the octadentate chelator DFO* and the in vitro and in vivo comparison of its 89Zr-DFO*-mAb complex with 89Zr-DFO-mAb.MethodsThe bifunctional chelator DFO*-pPhe-NCS was prepared from previously reported DFO* and p-phenylenediisothiocyanate. Subsequently, trastuzumab was conjugated with either DFO*-pPhe-NCS or commercial DFO-pPhe-NCS and radiolabeled with Zr-89 according to published procedures. In vitro stability experiments were carried out in saline, a histidine/sucrose buffer, and blood serum. The in vivo performance of the chelators was compared in N87 tumor-bearing mice by biodistribution studies and PET imaging.ResultsIn 0.9 % NaCl 89Zr-DFO*-trastuzumab was more stable than 89Zr-DFO-trastuzumab; after 72 h incubation at 2-8 °C 95 % and 58 % intact tracer were left, respectively, while in a histidine-sucrose buffer no difference was observed, both products were???92 % intact. In vivo uptake at 144 h post injection (p.i.) in tumors, blood, and most normal organs was similar for both conjugates, except for skin, liver, spleen, ileum, and bone. Tumor uptake was 32.59?±?11.95 and 29.06?±?8.66 % ID/g for 89Zr-DFO*-trastuzumab and 89Zr-DFO-trastuzumab, respectively. The bone uptake was significantly lower for 89Zr-DFO*-trastuzumab compared to 89Zr-DFO-trastuzumab. At 144 h p.i. for 89Zr-DFO*-trastuzumab and 89Zr-DFO-trastuzumab, the uptake in sternum was 0.92?±?0.16 and 3.33?±?0.32 % ID/g, in femur 0.78?±?0.11 and 3.85,?±?0.80 and in knee 1.38?±?0.23 and 8.20?±?2.94 % ID/g, respectively. The uptake in bone decreased from 24 h to 144 h p.i. about two fold for the DFO* conjugate, while it increased about two fold for the DFO conjugate.ConclusionsZr-DFO*-trastuzumab showed superior in vitro stability and in vivo performance when compared to 89Zr-DFO-trastuzumab. This makes the new octadentate DFO* chelator a candidate successor of DFO for future clinical 89Zr-immuno-PET.

Project description:Multiple myeloma (MM) is a plasma B-cell hematologic cancer that causes significant skeletal morbidity. Despite improvements in survival, heterogeneity in response remains a major challenge in MM. Cluster of differentiation 38 (CD38) is a type II transmembrane glycoprotein overexpressed in myeloma cells and is implicated in MM cell signaling. Daratumumab is a U.S. Food and Drug Administration-approved high-affinity monoclonal antibody targeting CD38 that is clinically benefiting refractory MM patients. Here, we evaluated [89Zr]Zr-desferrioxamine (DFO)-daratumumab PET/CT imaging in MM tumor models. Methods: Daratumumab was conjugated to DFO-p-benzyl-isothiocyanate (DFO-Bz-NCS) for radiolabeling with 89Zr. Chelator conjugation was confirmed by electrospray ionization-mass spectrometry, and radiolabeling was monitored by instant thin-layer chromatography. Daratumumab was conjugated to Cyanine5 (Cy5) dye for cell microscopy. In vitro and in vivo evaluation of [89Zr]Zr-DFO-daratumumab was performed using CD38+ human myeloma MM1.S-luciferase (MM1.S) cells. Cellular studies determined the affinity, immunoreactivity, and specificity of [89Zr]Zr-DFO-daratumumab. A 5TGM1-luciferase (5TGM1)/KaLwRij MM mouse model served as control for imaging background noise. [89Zr]Zr-DFO-daratumumab PET/CT small-animal imaging was performed in severe combined immunodeficient mice bearing solid and disseminated MM tumors. Tissue biodistribution (7 d after tracer administration, 1.11 MBq/animal, n = 4-6/group) was performed in wild-type and MM1.S tumor-bearing mice. Results: A specific activity of 55.5 MBq/nmol (0.37 MBq/?g) was reproducibly obtained with [89Zr]Zr-daratumumab-DFO. Flow cytometry confirmed CD38 expression (>99%) on the surface of MM1.S cells. Confocal microscopy with daratumumab-Cy5 demonstrated specific cell binding. Dissociation constant, 3.3 nM (±0.58), and receptor density, 10.1 fmol/mg (±0.64), was obtained with a saturation binding assay. [89Zr]Zr-DFO-daratumumab/PET demonstrated specificity and sensitivity for detecting CD38+ myeloma tumors of variable sizes (8.5-128 mm3) with standardized uptake values ranging from 2.1 to 9.3. Discrete medullar lesions, confirmed by bioluminescence images, were efficiently imaged with [89Zr]Zr-DFO-daratumumab/PET. Biodistribution at 7 d after administration of [89Zr]Zr-DFO-daratumumab showed prominent tumor uptake (27.7 ± 7.6 percentage injected dose per gram). In vivo blocking was achieved with a 200-fold excess of unlabeled daratumumab. Conclusion: [89Zr]Zr-DFO- and Cy5-daratumumab demonstrated superb binding to CD38+ human MM cells and significantly low binding to CD38low cells. Daratumumab bioconjugates are being evaluated for image-guided delivery of therapeutic radionuclides.

Project description:PurposeTo develop a clinically translatable method of cell labeling with zirconium 89 ((89)Zr) and oxine to track cells with positron emission tomography (PET) in mouse models of cell-based therapy.Materials and methodsThis study was approved by the institutional animal care committee. (89)Zr-oxine complex was synthesized in an aqueous solution. Cell labeling conditions were optimized by using EL4 mouse lymphoma cells, and labeling efficiency was examined by using dendritic cells (DCs) (n = 4), naïve (n = 3) and activated (n = 3) cytotoxic T cells (CTLs), and natural killer (NK) (n = 4), bone marrow (n = 4), and EL4 (n = 4) cells. The effect of (89)Zr labeling on cell survival, proliferation, and function were evaluated by using DCs (n = 3) and CTLs (n = 3). Labeled DCs (444-555 kBq/[5 × 10(6)] cells, n = 5) and CTLs (185 kBq/[5 × 10(6)] cells, n = 3) transferred to mice were tracked with microPET/CT. In a melanoma immunotherapy model, tumor targeting and cytotoxic function of labeled CTLs were evaluated with imaging (248.5 kBq/[7.7 × 10(6)] cells, n = 4) and by measuring the tumor size (n = 6). Two-way analysis of variance was used to compare labeling conditions, the Wilcoxon test was used to assess cell survival and proliferation, and Holm-Sidak multiple tests were used to assess tumor growth and perform biodistribution analyses.Results(89)Zr-oxine complex was synthesized at a mean yield of 97.3% ± 2.8 (standard deviation). It readily labeled cells at room temperature or 4°C in phosphate-buffered saline (labeling efficiency range, 13.0%-43.9%) and was stably retained (83.5% ± 1.8 retention on day 5 in DCs). Labeling did not affect the viability of DCs and CTLs when compared with nonlabeled control mice (P > .05), nor did it affect functionality. (89)Zr-oxine complex enabled extended cell tracking for 7 days. Labeled tumor-specific CTLs accumulated in the tumor (4.6% on day 7) and induced tumor regression (P < .05 on day 7).ConclusionWe have developed a (89)Zr-oxine complex cell tracking technique for use with PET that is applicable to a broad range of cell types and could be a valuable tool with which to evaluate various cell-based therapies.

Project description:The encapsulation of Glucocorticoids (GCs) into long-circulating liposomes (LCLs) is a proven strategy to reduce the side effects of glucocorticoids and improve the treatment of inflammatory diseases, such as rheumatoid arthritis (RA). With the aim of supporting the development of GC-loaded LCLs, and potentially predict patient response to therapy clinically, we evaluated a direct PET imaging radiolabelling approach for preformed GC-LCLs in an animal model of human inflammatory arthritis. Methods: A preformed PEGylated liposomal methylprednisolone hemisuccinate (NSSL-MPS) nanomedicine was radiolabelled using [89Zr]Zr(oxinate)4 (89Zr-oxine), characterised and tracked in vivo using PET imaging in a K/BxN serum-transfer arthritis (STA) mouse model of inflammatory arthritis and non-inflamed controls. Histology and joint size measurements were used to confirm inflammation. The biodistribution of 89Zr-NSSL-MPS was compared to that of free 89Zr in the same model. A therapeutic study using NSSL-MPS using the same time points as the PET/CT imaging was carried out. Results: The radiolabelling efficiency of NSSL-MPS with [89Zr]Zr(oxinate)4 was 69 ± 8 %. PET/CT imaging of 89Zr-NSSL-MPS showed high uptake (3.6 ± 1.5 % ID; 17.4 ± 9.3 % ID/mL) at inflamed joints, with low activity present in non-inflamed joints (0.5 ± 0.1 % ID; 2.7 ± 1.1 % ID/mL). Importantly, a clear correlation between joint swelling and high 89Zr-NSSL-MPS uptake was observed, which was not observed with free 89Zr. STA mice receiving a therapeutic dose of NSSL-MPS showed a reduction in inflammation at the time points used for the PET/CT imaging compared with the control group. Conclusions: PET imaging was used for the first time to track a liposomal glucocorticoid, showing high uptake at visible and occult inflamed sites and a good correlation with the degree of inflammation. A subsequent therapeutic response matching imaging time points in the same model demonstrated the potential of this radiolabeling method as a theranostic tool for the prediction of therapeutic response - with NSSL-MPS and similar nanomedicines - in the treatment of inflammatory diseases.

Project description:PurposeCurrently, the most commonly used chelator for labelling antibodies with 89Zr for immunoPET is desferrioxamine B (DFO). However, preclinical studies have shown that the limited in vivo stability of the 89Zr-DFO complex results in release of 89Zr, which accumulates in mineral bone. Here we report a novel chelator DFOcyclo*, a preorganized extended DFO derivative that enables octacoordination of the 89Zr radiometal. The aim was to compare the in vitro and in vivo stability of [89Zr]Zr-DFOcyclo*, [89Zr]Zr-DFO* and [89Zr]Zr-DFO.MethodsThe stability of 89Zr-labelled chelators alone and after conjugation to trastuzumab was evaluated in human plasma and PBS, and in the presence of excess EDTA or DFO. The immunoreactive fraction, IC50, and internalization rate of the conjugates were evaluated using HER2-expressing SKOV-3 cells. The in vivo distribution was investigated in mice with subcutaneous HER2+ SKOV-3 or HER2- MDA-MB-231 xenografts by PET/CT imaging and quantitative ex vivo tissue analyses 7 days after injection.Results89Zr-labelled DFO, DFO* and DFOcyclo* were stable in human plasma for up to 7 days. In competition with EDTA, DFO* and DFOcyclo* showed higher stability than DFO. In competition with excess DFO, DFOcyclo*-trastuzumab was significantly more stable than the corresponding DFO and DFO* conjugates (p < 0.001). Cell binding and internalization were similar for the three conjugates. In in vivo studies, HER2+ SKOV-3 tumour-bearing mice showed significantly lower bone uptake (p < 0.001) 168 h after injection with [89Zr]Zr-DFOcyclo*-trastuzumab (femur 1.5 ± 0.3%ID/g, knee 2.1 ± 0.4%ID/g) or [89Zr]Zr-DFO*-trastuzumab (femur 2.0 ± 0.3%ID/g, knee 2.68 ± 0.4%ID/g) than after injection with [89Zr]Zr-DFO-trastuzumab (femur 4.5 ± 0.6%ID/g, knee 7.8 ± 0.6%ID/g). Blood levels, tumour uptake and uptake in other organs were not significantly different at 168 h after injection. HER2- MDA-MB-231 tumour-bearing mice showed significantly lower tumour uptake (p < 0.001) after injection with [89Zr]Zr-DFOcyclo*-trastuzumab (16.2 ± 10.1%ID/g) and [89Zr]Zr-DFO-trastuzumab (19.6 ± 3.2%ID/g) than HER2+ SKOV-3 tumour-bearing mice (72.1 ± 14.6%ID/g and 93.1 ± 20.9%ID/g, respectively), while bone uptake was similar.Conclusion89Zr-labelled DFOcyclo* and DFOcyclo*-trastuzumab showed higher in vitro and in vivo stability than the current commonly used 89Zr-DFO-trastuzumab. DFOcyclo* is a promising candidate to become the new clinically used standard chelator for 89Zr immunoPET.

Dataset Information

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.

Background

Procedures

Results

Conclusions

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Dataset Information

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.

Background

Procedures

Results

Conclusions

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

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.