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A generic site-specific conjugation method that generates a homogeneous product is of utmost importance in tracer development for molecular imaging and therapy. We explored the protein-ligation capacity of the enzyme Sortase A to label camelid single-domain antibody-fragments, also known as nanobodies. The versatility of the approach was demonstrated by conjugating independently three different imaging probes: the chelating agents CHX-A"-DTPA and NOTA for single-photon emission computed tomography (SPECT) with indium-111 and positron emission tomography (PET) with gallium-68, respectively, and the fluorescent dye Cy5 for fluorescence reflectance imaging (FRI). After a straightforward purification process, homogeneous single-conjugated tracer populations were obtained in high yield (30-50%). The enzymatic conjugation did not affect the affinity of the tracers, nor the radiolabeling efficiency or spectral characteristics. In vivo, the tracers enabled the visualization of human epidermal growth factor receptor 2 (HER2) expressing BT474M1-tumors with high contrast and specificity as soon as 1 h post injection in all three imaging modalities. These data demonstrate Sortase A-mediated conjugation as a valuable strategy for the development of site-specifically labeled camelid single-domain antibody-fragments for use in multiple molecular imaging modalities. Copyright © 2016 John Wiley & Sons, Ltd.

Nanobodies show favourable pharmacokinetic characteristics for tumor targeting, including high tumor-to-background-ratios. Labelled with a therapeutic radionuclide, nanobodies could be used as an adjuvant treatment option for HER2-overexpressing minimal residual disease. The therapeutic radionuclide Lutetium-177 is linked to the nanobody using a bifunctional chelator. The choice of the bifunctional chelator could affect the in vivo behaviour of the radiolabeled nanobody. Consequently, we compared four different bifunctional chelators - p-SCN-Bn-DOTA, DOTA-NHS-ester, CHX-A”-DTPA or 1B4M-DTPA - in order to select the optimal chemical link between Lutetium-177 and a HER2 targeting nanobody. MS results revealed different degrees of chelator-conjugation. High stability in time was observed, together with nanomolar affinities on HER2-expressing tumor cells. Ex vivo biodistributions as well as SPECT/micro-CT analyses showed high activities in tumors expressing medium HER2 levels with low background activity except for the kidneys. The 1B4M-DTPA-coupled conjugate was further evaluated in a high HER2-expressing tumor model. Here, tumor uptake values of 5.99 ± 0.63, 5.12 ± 0.17, 2.83 ± 0.36 and 2.47 ± 0.38 %IA/g were obtained at 1, 3, 24 and 48h p.i., which coincided with exceptionally low background values, except for the kidneys, and unprecedented tumor-to-background ratios. No specific binding was observed in a HER2-negative model. In conclusion, the in-house developed anti-HER2 nanobody 2Rs15dHIS can be successfully labeled with 177Lu using different bifunctional chelators. Both macrocyclic and acyclic chelators show high stability in time. High specific tumor uptake combined with the lowest background uptake was measured using the 1B4M-DTPA-based conjugate. Copyright © 2012 John Wiley & Sons, Ltd.

Contains fulltext : 96063.pdf (Publisher’s version ) (Open Access) BACKGROUND: Nanobodies are single-domain antigen binding fragments derived from functional heavy-chain antibodies elicited in Camelidae. They are powerful probes for radioimmunoimaging, but their renal uptake is relatively high. In this study we have evaluated the role of megalin on the renal uptake of anti-EGFR (99m)Tc-7C12 nanobody and the potency of gelofusine and/or lysine to reduce renal uptake of (99m)Tc-7C12. METHODS: First we compared the renal uptake of (99m)Tc-7C12 in megalin-deficient and megalin-wild-type mice using pinhole SPECT/microCT and ex vivo analysis. The effect of gelofusine and lysine administration on renal accumulation of (99m)Tc-7C12 was analyzed in CD-1 mice divided into lysine preload at 30 min before tracer injection (LysPreload), LysPreload + gelofusine coadministration (LysPreload + GeloCoad), lysine coadministration (LysCoad), gelofusine coadministration (GeloCoad) and LysCoad + GeloCoad. The combined effect of gelofusine and lysine on tumor uptake was tested in mice xenografts. RESULTS: Renal uptake of (99m)Tc-7C12 was 44.22 +/- 3.46% lower in megalin-deficient compared with megalin-wild-type mice. In CD-1 mice, lysine preload had no effect on the renal retention whereas coinjection of lysine or gelofusine with the tracer resulted in 25.12 +/- 2.99 and 36.22 +/- 3.07% reduction, respectively. The combined effect of gelofusine and lysine was the most effective, namely a reduction of renal retention of 45.24 +/- 2.09%. Gelofusine and lysine coadministration improved tumor uptake. CONCLUSION: Megalin contributes to the renal accumulation of (99m)Tc-7C12. Gelofusine and lysine coinjection with the tracer reduces the renal uptake while tumor uptake is improved. Although this methodology allows for optimization of imaging protocol using nanobodies, further improvements are needed before using these molecules for radionuclide therapy.