Dataset Information

Preclinical Development of CD38-Targeted [⁸⁹Zr]Zr-DFO-Daratumumab for Imaging Multiple Myeloma.

ABSTRACT: 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 CD38^low cells. Daratumumab bioconjugates are being evaluated for image-guided delivery of therapeutic radionuclides.

SUBMITTER: Ghai A

PROVIDER: S-EPMC5807532 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Preclinical Development of CD38-Targeted [<sup>89</sup>Zr]Zr-DFO-Daratumumab for Imaging Multiple Myeloma.

Ghai Anchal A Maji Dolonchampa D Cho Nicholas N Chanswangphuwana Chantiya C Rettig Michael M Shen Duanwen D DiPersio John J Akers Walter W Dehdashti Farrokh F Achilefu Samuel S Vij Ravi R Shokeen Monica M

Journal of nuclear medicine : official publication, Society of Nuclear Medicine 20171012 2

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 pa ...[more]

PMID: 29025987

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Project description:PurposeCluster of differentiation 38 (CD38) is a promising therapeutic target in multiple myeloma (MM) patients and has resulted in the development of several CD38 immunotherapies. Current methods to evaluate CD38 expression in the preclinical setting include ex vivo flow cytometry and immunohistochemistry, which can be cumbersome and do not give whole-body information. In vivo imaging technologies such as positron emission tomography rely on decay of radioisotopes, limiting the number of molecular interactions observed at any given time point. Here, we demonstrate the use of near-infrared (NIR) fluorescence imaging for spatiotemporal monitoring of CD38 expression in preclinical MM using the anti-CD38 daratumumab (DARA) conjugated to the NIR fluorophore IRDye800CW (DARA-IRDye800).ProceduresStability studies with human serum and binding assays with human myeloma cells were performed with DARA-IRDye800. Immunocompromised mice with intra- and extramedullary tumors (n = 5/group) were administered with DARA-IRDye800 for in vivo imaging up to 7 days after injection. Ex vivo biodistribution and flow cytometry studies were performed to validate in vivo imaging results. A separate therapy study was performed in mice with intramedullary tumors that were treated and not treated with DARA at a therapeutic dose (n = 7/group). DARA-IRDye800 was administered for subsequent in vivo and ex vivo imaging in both cohorts of mice.ResultsDARA-IRDye800 maintained stability and had high affinity for CD38 (KD = 3.5 ± 0.05 nM). DARA-IRDye800 demonstrated a 5- and 18-fold increase in contrast in tumor-bearing regions of mice with extra- and intramedullary MM. Finally, mice treated with therapeutic doses of DARA and imaged with DARA-IRDye800 showed an 11-fold decrease in fluorescence intensities in vivo compared with untreated controls.ConclusionsOur studies establish DARA-IRDye800 as a promising contrast agent for preclinical evaluation of CD38 expression and for further investigating myeloma engraftment and kinetics in relation to anti-CD38 therapies.

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:Methods that provide rapid access to radiolabeled antibodies are vital in the development of diagnostic and radiotherapeutic agents for PET or radioimmunotherapy. The human hepatocyte growth factor receptor (c-MET) signaling pathway is dysregulated in several malignancies, including gastric cancer, and is an important biomarker in drug discovery. Here, we used a photoradiochemical approach to produce 89Zr-radiolabeled onartuzumab (a monovalent, antihuman c-MET antibody), starting directly from the fully formulated drug (MetMAb). Methods: Simultaneous 89Zr-radiolabeling and protein conjugation was performed in one-pot reactions containing 89Zr-oxalate, the photoactive chelate desferrioxamine B (DFO)-aryl azide (DFO-ArN3), and MetMAb to give 89Zr-DFO-azepin-onartuzumab. As a control, 89Zr-DFO-benzyl Bn-isothiocyanate Bn-NCS-onartuzumab was prepared via a conventional two-step process using prepurified onartuzumab and DFO-Bn-NCS. Radiotracers were purified by using size-exclusion methods and evaluated by radiochromatography. Radiochemical stability was studied in human serum, and immunoreactivity was determined by cellular binding assays using MKN-45 gastric carcinoma cells. PET imaging at multiple time points (0-72 h) was performed on female athymic nude mice bearing subcutaneous MKN-45 xenografts. Biodistribution experiments were performed after the final image was obtained. The tumor specificity of 89Zr-DFO-azepin-onartuzumab was assessed in vivo by competitive inhibition (blocking) studies. Results: Initial photoradiosynthesis experiments produced 89Zr-DFO-azepin-onartuzumab in less than 15 min, with an isolated decay-corrected radiochemical yield (RCY) of 24.8%, a radiochemical purity of approximately 90%, and a molar activity of approximately 1.5 MBq nmol-1 Reaction optimization improved the radiochemical conversion of 89Zr-DFO-azepin-onartuzumab to 56.9% ± 4.1% (n = 3), with isolated RCYs of 41.2% ± 10.6% (n = 3) and radiochemical purity of more than 90%. Conventional methods produced 89Zr-DFO-Bn-NCS-onartuzumab with an isolated RCY of more than 97%, radiochemical purity of more than 97% and molar activity of approximately 14.0 MBq nmol-1 Both radiotracers were immunoreactive and stable in human serum. PET imaging and biodistribution studies showed high tumor uptake for both radiotracers. By 72 h, tumor and liver uptake (percentage injected dose [%ID]) reached 15.37 ± 5.21 %ID g-1 and 6.56 ± 4.03 %ID g-1, respectively, for 89Zr-DFO-azepin-onartuzumab (n = 4) and 21.38 ± 11.57 %ID g-1 and 18.84 ± 6.03 %ID g-1, respectively, for 89Zr-DFO-Bn-NCS-onartuzumab (n = 4). Blocking experiments gave a statistically significant reduction in tumor uptake (6.34 ± 0.47 %ID g-1) of 89Zr-DFO-azepin-onartuzumab (n = 4). Conclusion: The experiments demonstrated that photoradiosynthesis is a viable alternative approach for producing 89Zr-radiolabeled antibodies directly in protein formulation buffer, reducing protein aggregation and liver uptake.

Project description:Avian pathogenic Escherichia coli (APEC) is a serious systemic infectious disease in poultry infections, causing severe economic losses to the poultry industry. Previous studies have shown that secretion of virulence proteins was required for the pathogenicity of APEC through the secretion system. Outer membrane vesicles (OMVs) are a generalized secretion system of Gram-negative bacteria that play a key role in the long-distance delivery of virulence factors, but whether they are associated with the pathogenic mechanism of APEC has not been determined. In this study, OMVs were purified and characterized from AE17 (O2 serotype) by ultracentrifugation and density gradient centrifugation and their protein cargo was identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, 89Zr was labeled after chelating AE17 OMVs by DFO and positron emission tomography PET imaging was used to track 89Zr-DFO-OMVs in chickens and to pathologically analyze the distribution sites. This study showed that AE17 OMVs were membrane vesicles ranging in size from 20 to 200 nm and proteomic analysis revealed the presence of virulence proteins, including adhesion proteins OmpA, OmpC, OmpF, OmpX, FimH, FimC and FigE, and serum resistance proteins OmpT and MliC and immune response regulator proteins (FliC). In addition, in vivo PET imaging to track the biodistribution of AE17 OMVs showed that AE17 OMVs were taken up by the lung region and the gastrointestinal and renal regions but were not detected in other areas. Pathological analysis of the tissue sites where AE17 OMVs were ingested showed inflammatory responses and damage. These findings suggested that AE17 OMVs not only contained a group of virulence proteins associated with AE17 infection but can also deliver these virulence proteins over long distances and caused tissue inflammatory damage. Our study revealed a previously unidentified causative microbial signal in the pathogenesis of APEC that could aid in the development of vaccines and antibiotics effective against APEC.

Dataset Information

Preclinical Development of CD38-Targeted [⁸⁹Zr]Zr-DFO-Daratumumab for Imaging Multiple Myeloma.

Publications

Preclinical Development of CD38-Targeted [<sup>89</sup>Zr]Zr-DFO-Daratumumab for Imaging Multiple Myeloma.

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Dataset Information

Preclinical Development of CD38-Targeted [89Zr]Zr-DFO-Daratumumab for Imaging Multiple Myeloma.

Publications

Preclinical Development of CD38-Targeted [<sup>89</sup>Zr]Zr-DFO-Daratumumab for Imaging Multiple Myeloma.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

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Preclinical Development of CD38-Targeted [⁸⁹Zr]Zr-DFO-Daratumumab for Imaging Multiple Myeloma.