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Anneleen Blykers 1 , Catarina Xavier 1 , Ilse Vaneycken 2 , Damya Laoui 3 , Nick Devoogdt 1 , Matthias D’huyvetter 1 , Tony Lahoutte 1,2 , Jo A. Van Ginderachter 3 and Vicky Caveliers 1,2 1 In vivo Cellular and Molecular Imaging (ICMI), Vrije Universiteit Brussel, Belgium; 2 Nuclear Medicine, UZBrussel, Belgium, 3 Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium. Summary Tumor-associated macrophages (TAMs) are an important component of the tumor stroma. Macrophages can mainly be divided in two subtypes: the M1 and M2 macrophages. This latest subtype of macrophages, having a more malignant phenotype, express the Macrophage Mannose Receptor (MMR). Previously in our University it was proven that anti-MMR nanobodies (single domain antibodies) targeting MMR + TAMs accumulated in hypoxic regions 1 . As reduced oxygen levels in a tumor influences the clinical outcome, the development of anti-MMR nanobody probes for PET imaging of tumor hypoxia is very interesting. The pharmacokinetic profile of nanobodies is optimal for imaging with short-lived radioisotopes such as 18 F. Here we present the development of 18 F-labeled anti-MMR nanobodies using the 18 F-SFB method. Methods Synthesis of 18 F-SFB The automated synthesis and purification of 18 F-SFB was optimized on the SynthERA platform comprising two-coupled synthesis modules (v1 and v2). The 18 F-SFB prosthetic group is produced using the original disposable 18 F-FDG cassette (IFP TM nucleophilic) and editing the existing 18 F-FDG-script. Conjugation between 18 F-SFB and nanobody After incubation of the nanobody with 18 F-SFB for 30 min at room temperature and pH 8.5, the radiolabeled nanobody was purified with a Sephadex G25 disposable column (PD-10). Assessment of radiochemical purity was performed by RP-HPLC. Stability of 18 F-Nanobody was evaluated by incubation in PBS or human serum for up to 2 h and analyzed by size exclusion chromatography (SEC). 30’ RT pH 8,5 Ex-vivo biodistribution and tumor targeting C57Bl/6-mice have been subcutaneously inoculated with 3 10 6 3LL-R cells on the left hindlimb. After tumor growth, the tracer was injected and 1 and 3 hours post injection the animals were dissected and the activity distribution within the different organs and tissues measured ex vivo. Tumors grown in MMR-deficient (Knock-out) mice were used to prove specificity. Conclusions We showed for the first time the successful labeling of anti-MMR targeting Nanobodies with 18 F, using the Synthera module. This PET-probe targets the MMR-receptor in-vivo, enabling his use in the clinic. 18 F-Anti-MMR-nanobodies for PET imaging of tumor-associated macrophage subtypes as surrogate markers for tumor hypoxia. Figure 2: Conjugation of 18 F-SFB with Nb Figure 1: The synthesis of 18 F-SFB N O O F O O F O O F O O N O O 5’ 110°C 5’ 95°C 15’ 110°C 18 F - Acknowledgments: Onderzoeksproject gerealiseerd met de steun van de Vlaamse Liga tegen Kanker - Stichting van Openbaar Nut. This work was supported by a grant from the Scientific Fund W. Gepts UZ Brussel. O O 18 F N O O O 18 F H N VHH VHH H 2 N + Results Synthesis and conjugation 18 F-SFB can be succesfully synthesized using the Synthera platform with a yield of about 40%. The radiochemical purity was >99% after AFFINIMIP ® purification. The formed 18 F-SFB is coupled to the Nb with a yield of 10% using 1mg/ml Nb at pH 8,5 reacting 30’ at RT. 18 F-anti-MMR nanobody was obtained with > 97% radiochemical purity. The labelled Nanobody was stable in PBS and human serum and keeps his functionality after labeling with fluorine. figure 3: purified 18 F-SFB figure 4: purified 18 F-SFB-Nb Ex-vivo biodistribution and tumor targeting The biodistribution profile (1h and 3h p.i.) showed specific uptake in liver, spleen, tumor and clearance via the kidneys. Specificity has been confirmed by comparison with MMR-deficient mice. The degree of kidney uptake of fluorinated Nanobody was remarkably 80% lower compared to 99m Tc analogs. Figure 5: Increased tumor targeting and –specificity was observed for 18 F-SFB anti-MMR- Nb in wild–type (WT) compared to MMR/KO tumor bearing mice (3h p.i.). Minutes 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 mAU -25 0 25 50 75 100 125 150 175 200 225 250 mV 0 200 400 600 800 1000 1200 DAD-324 nm 5/02/2013 10:55:57SFB_purified Retention Time Area Percent γ γ l u n gs heart l iver righ t ki d n ey left ki d ney sple en m uscle blood bo ne in t estine t umor 0 5 10 15 20 25 tissue %iA/g 18F -MMR 10_4 WT mice KO mice
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