RT Journal Article SR Electronic T1 Immuno-PET imaging of receptor activator of the nuclear factor k B ligand (RANKL) in the tumour microenvironment using [68Ga]Ga-NOTA-denosumab JF Journal of Nuclear Medicine JO J Nucl Med FD Society of Nuclear Medicine SP 159 OP 159 VO 61 IS supplement 1 A1 Dewulf, Jonatan A1 Vangestel, Christel A1 Elvas, Filipe A1 Van Den Wyngaert, Tim YR 2020 UL http://jnm.snmjournals.org/content/61/supplement_1/159.abstract AB 159Objectives: RANKL is a protein available in both soluble and transmembrane form that is expressed on many tissues (bone, thymus, immune cells, lymph nodes), and has an essential role in bone remodelling. This ligand is a known promotor of tumour growth and proliferation, and has recently emerged as an important target in tumour immunology. Expression of RANKL in the tumour microenvironment (TME) is associated with poor prognosis and more aggressive disease phenotype. Currently, non-invasive biomarkers of RANKL are not available for imaging the TME. Therefore, in this study we aim to investigate the ability to image RANKL in the TME using 68Ga-labeled denosumab. This proof of concept study using a 68Ga-labeled antibody will support the future translation of this work using a longer-lived isotope such as 89Zr. Methods: The radiolabelling of NOTA-denosumab (350µg) with 68Ga (314 MBq) was performed in 0.5M NH4OAC pH 6.5 buffer using metal free conditions. The resulting complex was purified using a PD-10 column, and characterized by iTLC and SEC-HPLC. Stability of [68Ga]Ga-NOTA-denosumab was evaluated by iTLC in vitro in the final formulation, mouse and human plasma (5h follow up), in vivo and ex vivo biodistribution at 30min, 1h, 2h and 3h post radiotracer injection (21±2.5µg; n=4) in healthy CD-1 nude mice. HCT116 human colon cancer (4 million cells) and SCC22b human hypopharyngeal squamous cell carcinoma (2.5 million cells) cells were injected subcutaneously in the hind leg of female CD-1 nude mice. When the tumours reached 0.2 cm3 [68Ga]Ga-NOTA-denosumab (34±1.5µg) PET-CT scans were performed 2h post radiotracer injection (n=5). Radiotracer uptake was obtained by γ-counting of the tumours (%ID/g). RANKL expression in the tumours will be determined by immunohistochemistry. Results: Conjugation of denosumab with p-SCN-NOTA bifunctional chelator resulted in an average of 1 NOTA group per antibody, as determined by mass spectrometry. A binding assay showed little change in binding affinity (Kd) for NOTA-denosumab when compared with native denosumab (0.16 vs. 0.13 nM Kd). [68Ga]Ga-NOTA-denosumab was obtained in a non-decay corrected yield of 66±5.5%, >99% radiochemical purity and a specific activity of 740 MBq/mg after PD10 purification. Radiotracer remained intact up to 5h in vitro, and up to 3h post radiotracer injection in vivo. Biodistribution experiment showed no non-specific uptake of the radiotracer, with high remaining circulating radiotracer in the blood. At different timepoints hepatic and renal clearance could be observed, however clearance was still limited. Biodistribution between healthy CD-1 nude mice and xenograft CD-1 nude mice was comparable (Figure 1A). A significantly increased ex vivo [68Ga]Ga-NOTA-denosumab uptake was detected in SCC22b compared to HCT116 tumours (4.0±0.1 vs. 2.5±0.3%ID/g; p=0.0001) (Figure 1B). This observation is in accordance with the known RANKL expression profiles of the SCC22b (high) and HCT116 (low) cell lines. Radiotracer uptake will be correlated to histology results. Conclusions: A new immuno-PET imaging agent [68Ga]Ga-NOTA-denosumab was successfully synthesized and radiolabelled in good radiochemical yield and showed good stability in vitro and in vivo. We demonstrated that [68Ga]Ga-NOTA-denosumab specifically accumulates in high RANKL expressing tumours, making this radiotracer potentially useful for imaging of the TME. Figure 1: A) Ex vivo comparison [68Ga]Ga-NOTA-denosumab bioD and xenograft models HCT116 & SCC22b B) PET scans 2h p.i. HCT116 & SCC22b xenograft models References: David L. Lacey et al. Bench to bedside: elucidation of the OPG-RANK-RANKL pathway and the development of denosumab. Nat Rev Drug Discov 2012; 11; 401-419 Xavier C et al. Synthesis, preclinical validation, dosimetry, and toxicity of 68Ga-NOTA-anti-HER2 Nanobodies for iPET imaging of HER2 receptor expression in cancer. J Nucl Med 2013; 54(5):776-84