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Article

Improved Characteristics of RANKL Immuno-PET Imaging Using Radiolabeled Antibody Fab Fragments

1
Molecular Imaging Center Antwerp (MICA), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Universiteitsplein 1, Wilrijk, B-2610 Antwerpen, Belgium
2
ICNAS-Produção Unipessoal Lda., Pólo das Ciências da Saúde, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal
3
Laboratory of Cell Biology and Histology, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, Wilrijk, B-2610 Antwerpen, Belgium
4
Department of Nuclear Medicine, Antwerp University Hospital, Drie Eikenstraat 655, B-2650 Edegem, Belgium
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Institute for Nuclear Sciences Applied to Health (ICNAS/CIBIT), Pólo das Ciências da Saúde, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal
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Multidisciplinary Oncologic Centre Antwerp (MOCA), Integrated Personalized and Precision Oncology Network (IPPON), Antwerp University Hospital, Drie Eikenstraat 655, B-2650 Edegem, Belgium
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Academic Editors: Frederik Cleeren and Guy Bormans
Pharmaceutics 2022, 14(5), 939; https://doi.org/10.3390/pharmaceutics14050939
Received: 10 March 2022 / Revised: 15 April 2022 / Accepted: 20 April 2022 / Published: 26 April 2022
(This article belongs to the Special Issue Radiopharmaceuticals for Cancer Imaging and Therapy)
Purpose: RANKL expression in the tumor microenvironment has been identified as a biomarker of immune suppression, negating the effect of some cancer immunotherapies. Previously we had developed a radiotracer based on the FDA-approved RANKL-specific antibody denosumab, [89Zr]Zr-DFO-denosumab, enabling successful immuno-PET imaging. Radiolabeled denosumab, however, showed long blood circulation and delayed tumor uptake, potentially limiting its applications. Here we aimed to develop a smaller radiolabeled denosumab fragment, [64Cu]Cu-NOTA-denos-Fab, that would ideally show faster tumor accumulation and better diffusion into the tumor for the visualization of RANKL. Experimental design: Fab fragments were prepared from denosumab using papain and conjugated to a NOTA chelator for radiolabeling with 64Cu. The bioconjugates were characterized in vitro using SDS-PAGE analysis, and the binding affinity was assessed using a radiotracer cell binding assay. Small animal PET imaging evaluated tumor targeting and biodistribution in transduced RANKL-ME-180 xenografts. Results: The radiolabeling yield of [64Cu]Cu-NOTA-denos-Fab was 58 ± 9.2%, with a specific activity of 0.79 ± 0.11 MBq/µg (n = 3). A radiotracer binding assay proved specific targeting of RANKL in vitro. PET imaging showed fast blood clearance and high tumor accumulation as early as 1 h p.i. (2.14 ± 0.21% ID/mL), which peaked at 5 h p.i. (2.72 ± 0.61% ID/mL). In contrast, [64Cu]Cu-NOTA-denosumab reached its highest tumor uptake at 24 h p.i. (6.88 ± 1.12% ID/mL). [64Cu]Cu-NOTA-denos-Fab specifically targeted human RANKL in transduced ME-180 xenografts compared with the blocking group and negative ME-180 xenograft model. Histological analysis confirmed RANKL expression in RANKL-ME-180 xenografts. Conclusions: Here, we report on a novel RANKL PET imaging agent, [64Cu]Cu-NOTA-denos-Fab, that allows for fast tumor imaging with improved imaging contrast when compared with its antibody counterpart, showing promise as a potential PET RANKL imaging tool for future clinical applications. View Full-Text
Keywords: RANKL; antibody; Fab fragment; tumor imaging; immuno-PET RANKL; antibody; Fab fragment; tumor imaging; immuno-PET
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MDPI and ACS Style

Dewulf, J.; Hrynchak, I.; Geudens, S.; Pintelon, I.; Vangestel, C.; Sereno, J.; van Dam, P.A.; Abrunhosa, A.J.; Elvas, F.; Van den Wyngaert, T. Improved Characteristics of RANKL Immuno-PET Imaging Using Radiolabeled Antibody Fab Fragments. Pharmaceutics 2022, 14, 939. https://doi.org/10.3390/pharmaceutics14050939

AMA Style

Dewulf J, Hrynchak I, Geudens S, Pintelon I, Vangestel C, Sereno J, van Dam PA, Abrunhosa AJ, Elvas F, Van den Wyngaert T. Improved Characteristics of RANKL Immuno-PET Imaging Using Radiolabeled Antibody Fab Fragments. Pharmaceutics. 2022; 14(5):939. https://doi.org/10.3390/pharmaceutics14050939

Chicago/Turabian Style

Dewulf, Jonatan, Ivanna Hrynchak, Sarah Geudens, Isabel Pintelon, Christel Vangestel, José Sereno, Peter A. van Dam, Antero J. Abrunhosa, Filipe Elvas, and Tim Van den Wyngaert. 2022. "Improved Characteristics of RANKL Immuno-PET Imaging Using Radiolabeled Antibody Fab Fragments" Pharmaceutics 14, no. 5: 939. https://doi.org/10.3390/pharmaceutics14050939

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