Improved Characteristics of RANKL Immuno-PET Imaging Using Radiolabeled Antibody Fab Fragments
Jonatan Dewulf,
Ivanna Hrynchak,
Sarah Geudens,
Isabel Pintelon,
Christel Vangestel,
José Sereno,
Peter A. van Dam,
Antero J. Abrunhosa,
Filipe Elvas,
Tim Van den Wyngaert
Affiliations
Jonatan Dewulf
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
Ivanna Hrynchak
ICNAS-Produção Unipessoal Lda., Pólo das Ciências da Saúde, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal
Sarah Geudens
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
Isabel Pintelon
Laboratory of Cell Biology and Histology, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, Wilrijk, B-2610 Antwerpen, Belgium
Christel Vangestel
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
José Sereno
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
Peter A. van Dam
Multidisciplinary Oncologic Centre Antwerp (MOCA), Integrated Personalized and Precision Oncology Network (IPPON), Antwerp University Hospital, Drie Eikenstraat 655, B-2650 Edegem, Belgium
Antero J. Abrunhosa
ICNAS-Produção Unipessoal Lda., Pólo das Ciências da Saúde, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal
Filipe Elvas
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
Tim Van den Wyngaert
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
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.
