89Zr-Labeled Domain II-Specific scFv-Fc ImmunoPET Probe for Imaging Epidermal Growth Factor Receptor In Vivo

Alizadeh, Elahe; Ahmed, Khawaja Ashfaque; Solomon, V. Raja; Gaja, Vijay; Bernhard, Wendy; Makhlouf, Amal; González, Carolina; Barreto, Kris; Casacó, Á.; Geyer, C. Ronald; Fonge, Humphrey

doi:10.3390/cancers13030560

Cited by 7 publications

(8 citation statements)

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“…Incucyte live cell imaging S3 (Essen BioScience, Ann Arbor, MI, USA) was used to study the internalization of the DFO-M16Ab in MUC16-expressing OVCAR3 and SW1990 cells and MUC-16-negative SKOV3 cells as previously reported [ref]. Following live-cell imaging, the mean red object area (µm 2 /well) was calculated using the IncuCyte software, which is used to quantify the internalization uptake of the antibody [29].…”

Section: Antibody Internalizationmentioning

confidence: 99%

Engineering of a Fully Human Anti-MUC-16 Antibody and Evaluation as a PET Imaging Agent

et al. 2022

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Antibodies that recognize cancer biomarkers, such as MUC16, can be used as vehicles to deliver contrast agents (imaging) or cytotoxic payloads (therapy) to the site of tumors. MUC16 is overexpressed in 80% of epithelial ovarian cancer (EOC) and 65% of pancreatic ductal adenocarcinomas (PDAC), where effective ‘theranostic’ probes are much needed. This work aims to develop fully human antibodies against MUC16 and evaluate them as potential immuno-PET imaging probes for detecting ovarian and pancreatic cancers. We developed a fully human monoclonal antibody, M16Ab, against MUC16 using phage display. M16Ab was conjugated with p-SCN-Bn-DFO and radiolabeled with 89Zr. 89Zr-DFO-M16Ab was then evaluated for binding specificity and affinity using flow cytometry. In vivo evaluation of 89Zr-DFO-M16Ab was performed by microPET/CT imaging at different time points at 24–120 h post injection (p.i.) and ex vivo biodistribution studies in mice bearing MUC16-expressing OVCAR3, SKOV3 (ovarian) and SW1990 (pancreatic) xenografts. 89Zr-DFO-M16Ab bound specifically to MUC16-expressing cancer cells with an EC50 of 10nM. 89Zr-DFO-M16Ab was stable in serum and showed specific uptake and retention in tumor xenografts even after 120 h p.i. (microPET/CT) with tumor-to-blood ratios > 43 for the SW1990 xenograft. Specific tumor uptake was observed for SW1990/OVCAR3 xenografts but not in MUC16-negative SKOV3 xenografts. Pharmacokinetic study shows a relatively short distribution (t1/2α) and elimination half-life (t1/2ß) of 4.4 h and 99 h, respectively. In summary, 89Zr-DFO-M16Ab is an effective non-invasive imaging probe for ovarian and pancreatic cancers and shows promise for further development of theranostic radiopharmaceuticals.

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Section: Antibody Internalizationmentioning

confidence: 99%

Engineering of a Fully Human Anti-MUC-16 Antibody and Evaluation as a PET Imaging Agent

et al. 2022

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“…[ 89 Zr]Zr-oxalate in 1.0 M oxalic acid was purchased from the Saskatchewan Centre for Cyclotron Sciences (SCCS, operated by the Sylvia Fedoruk Canadian Centre for Nuclear Innovation) on the University of Saskatchewan campus. The apparent molar activity of the [ 89 Zr]Zr-oxalate produced routinely by the SCCS (as used for this study) was found to be in the range of 673–1161 MBq/μmol, by using a method from Holland et al , Purity was determined to be 99.99% . For radiolabeling of the immunoconjugates, the required amount of the [ 89 Zr]Zr 4+ (in 1.0 M oxalic acid) was neutralized to pH 6.8–7.2 by using sodium carbonate (1.0 M) and the reactions were performed in HEPES buffer (0.5 M, pH 7).…”

Section: Methodsmentioning

confidence: 98%

DFO-Km: A Modular Chelator as a New Chemical Tool for the Construction of Zirconium-89-Based Radiopharmaceuticals

Salih,

Dominguez Garcia,

Raheem

et al. 2023

Inorg. Chem.

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Zirconium-89-labeled monoclonal antibodies and other large macromolecules such as nanoparticles hold great promise as positron emission tomography imaging agents. In general, zirconium-89 is an ideal radionuclide for long-circulating vectors such as antibodies or nanoparticles. It is also a promising radionuclide for theranostic radiopharmaceuticals due to its suitable match in half-life with actinium-225, thorium-227, lutetium-177, and others. As such, demand for new and optimized bifunctional chelators for zirconium-89 continues to grow. Herein, we present the modular chelator DFO-Km, which is octadentate and features lysine as a modular amino acid linker. The modular amino acid linker can be changed to other natural or unnatural amino acids to access different bioconjugation chemistries, while the chelating portion is unchanged thus retaining identical metal ion coordination properties to DFO-Km. The epsilon-amine in the DFO-Km linker (lysine) was used to complete synthesis of a bifunctional derivative bearing a p-SCN-Ph moiety. The chelator DFO-Km includes a redesigned hydroxamic acid, which provides more flexibility for metal ion coordination relative to the monomer used in the previously published DFO-Em. Moreover, a set of comprehensive DFT calculations were performed to model and evaluate 16 geometric isomers of Zr-(DFO-Km), which suggested the complex would form the optimum cic–cis–trans–trans octadentate Zr(IV) coordination geometry with no aqua or hydroxide ligands present. The bifunctional derivative p-SCN-Ph-DFO-Km was compared directly with the commercially available p-SCN-Ph-DFO, and both underwent efficient conjugation to a nonspecific human serum antibody (IgG) to yield two model immunoconjugates. The behavior of [89Zr]Zr-DFO-Km-IgG was studied in healthy mice for 2 weeks and compared to an equivalent cohort injected with [89Zr]Zr-DFO-IgG as a clinical “gold standard” control. PET-CT and biodistribution results revealed higher stability of [89Zr]Zr-(DFO-Km)-IgG in vivo over [89Zr]Zr-DFO-IgG, as demonstrated by the significant reduction of zirconium-89 in the whole skeleton as visualized and quantified by PET-CT at 1, 3, 7, and 14 days post-injection. Using CT-gated regions of interest over these PET-CT images, the whole skeleton was selected and uptake values were measured at 14 days post-injection of 3.6 ± 0.9 (DFO) vs 1.9 ± 0.1 (DFO-Km) %ID/g (n = 4, * p = 0.02), which represents a ∼48% reduction in bone uptake with DFO-Km relative to DFO. Biodistribution experiments performed on these same mice following the 14 day imaging time point revealed bone (both tibia) uptake values of 3.7 ± 1.3 (DFO) vs 2.0 ± 0.6 (DFO-Km) %ID/g (n = 6, * p < 0.05), with the tibia uptake values in close agreement with whole-skeleton ROI PET-CT data. These results indicate that DFO-Km is an improved chelator for [89Zr]Zr4+ applications relative to DFO. The bifunctional chelator p-SCN-Ph-DFO-Km shows potential as a new chemical tool for creating bioconjugates using targeting vectors such as antibodies, peptides, an...

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“…[ 89 Zr]Zr-oxalate was obtained from Saskatchewan Centre for Cyclotron Sciences (SCCS) on the University of Saskatchewan campus and was delivered purified in a minimum volume of 1 M oxalic acid. The apparent molar activity of the [ 89 Zr]Zr-oxalate produced routinely in SCCS (as used for this study), was found to be in the range of 673−1161 MBq/μmol, using a method adapted from Holland et al, 45,46 and radionuclidic purity was determined to be 99.99%. 47 Warning statement: zirconium-89 is a radionuclide that emits potentially hazardous ionizing radiation (gamma rays, positrons) and requires substantial infrastructure (e.g., lead shielding, detectors, radioactive waste storage), radiation safety training, and radiochemistry training to safely handle.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Design, Synthesis, and Evaluation of DFO-Em: A Modular Chelator with Octadentate Chelation for Optimal Zirconium-89 Radiochemistry

et al. 2022

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Zirconium-89 has quickly become a favorite radionuclide among academics and clinicians for nuclear imaging. This radiometal has a relatively long half-life, which matches the biological half-life of most antibodies, suitable decay properties for positron emission tomography (PET), and efficient and affordable cyclotron production and purification. The "gold standard" chelator for [ 89 Zr]Zr 4+ is desferrioxamine B (DFO), and although it has been used both preclinically and clinically for immunoPET with great success, it has revealed its suboptimal stability in vivo. DFO can only bind to [ 89 Zr]Zr 4+ through its six available coordination sites made up by three hydroxamic acid (HA) moieties, which is not sufficient to saturate the coordination sphere . In this study, we have designed, synthesized, and characterized a new octadentate chelator we have called DFO-Em, which is an improved derivative of our previously published dodecadentate chelator DFO2. This octadentate DFO-Em chelator is smaller than DFO2 but still satisfies the coordination sphere of zirconium-89 and forms a highly stable radiometal−chelator complex. DFO-Em was synthesized by tethering a hydroxamic acid monomer to commercially available DFO using glutamic acid as a linker, providing an octadentate chelator built on a modular amino acid-based synthesis platform. Radiolabeling performance and radiochemical stability of DFO-Em were assessed in vitro by serum stability, ethylenediamine tetraacetic acid (EDTA), and hydroxyapatite challenges. Furthermore, [ 89 Zr]Zr-(DFO-Em) and [ 89 Zr]Zr-DFO were injected in healthy mice and measured in vivo by PET/CT imaging and ex vivo biodistribution. Additionally, the coordination of DFO-Em with Zr(IV) and its isomers was studied using density functional theory (DFT) calculations. The radiolabeling studies revealed that DFO-Em has a comparable radiolabeling profile to the gold standard chelator DFO. The in vitro stability evaluation showed that [ 89 Zr]Zr-(DFO-Em) was significantly more stable than [ 89 Zr]Zr-DFO, and in vivo both had similar clearance in healthy mice with a small decrease in tissue retention for [ 89 Zr]Zr-(DFO-Em) at 24 h post injection. The DFT calculations also confirmed that Zr-(DFO-Em) can adopt highly stable 8-coordinate geometries, which along with NMR characterization suggest no fluxional behavior and the presence of a single isomer. The modular design of DFO-Em means that any natural or unnatural amino acid can be utilized as a linker to gain access to different chemistries (e.g., thiol, amine, carboxylic acid, azide) while retaining an identical coordination sphere to DFO-Em.

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89Zr-Labeled Domain II-Specific scFv-Fc ImmunoPET Probe for Imaging Epidermal Growth Factor Receptor In Vivo

Cited by 7 publications

References 28 publications

Engineering of a Fully Human Anti-MUC-16 Antibody and Evaluation as a PET Imaging Agent

Engineering of a Fully Human Anti-MUC-16 Antibody and Evaluation as a PET Imaging Agent

DFO-Km: A Modular Chelator as a New Chemical Tool for the Construction of Zirconium-89-Based Radiopharmaceuticals

Design, Synthesis, and Evaluation of DFO-Em: A Modular Chelator with Octadentate Chelation for Optimal Zirconium-89 Radiochemistry

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