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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 attractive characteristics for tumor targeting in cancer diagnosis and therapy. A radiolabeled nanobody binding the prostate-specific membrane antigen (PSMA) could offer a noninvasive strategy to select prostate cancer patients eligible for PSMA-targeted therapies. We here describe the generation, production and in vivo evaluation of anti-PSMA nanobodies. Nanobodies were derived from heavy-chain-only antibodies, raised in immunized dromedaries. Binding characteristics were evaluated through ELISA and flow cytometry. Selected nanobodies were radiolabeled with 99mTc at their hexahistidine tail, after which cell binding capacity and internalization were evaluated on PSMApos LNCaP and PSMAneg PC3 cell lines. In vivo tumor targeting was analyzed in both LNCaP and PC3 xenografted mice through SPECT/microCT and tissue sampling. A panel of 72 generated clones scored positive on ELISA, all contributing to three nanobody groups, of which group 3 dominated with 70 clones. ELISA and FACS analysis led to the selection of two dominant nanobodies. 99mTc-labeled PSMA6 and PSMA30 both showed specific binding on LNCAP cells, but not on PC3 cells. 99mTc-PSMA30 internalized significantly more in LNCaP cells compared to 99mTc-PSMA6. Higher absolute tumor uptake and tumor-to-normal organ ratios were observed for 99mTc-PSMA30 compared with 99mTc-PSMA6 and a 99mTc-control nanobody in LNCaP but not in PC3 tumor-bearing mice. PSMA30 nanobody has improved targeting characteristics both in vitro as well as in vivo compared with PSMA6 and the control nanobody, and was therefore selected as our in-house-developed lead compound for PSMA targeting. Copyright © 2014 John Wiley & Sons, Ltd.

Absolute quantification using single photon emission computed tomography (SPECT) was demonstrated in vitro and in large immobile organs in vivo. To determine the feasibility of in vivo quantification of myocardial perfusion in pinhole gated SPECT, we added an ultrasound derived partial volume correction factor to attenuation and scatter corrections, in combination with gated acquisitions. In nine male Wistar rats, cardiac ultrasound was performed prior to SPECT/CT scans to determine the myocardial wall thickness. SPECT/CT scans were then performed 30 min after injection of 99mTc Tetrofosmin. Animals were killed and six midventricular segments of the left ventricle were excised and counted in a γ-well counter. Using AMIDE, regional myocardial activity was measured after combined scatter correction (SC) and attenuation correction (AC). These image derived activities were compared with the ex vivo counted activity. To correct for the partial volume effect, a recovery coefficient was determined from a phantom study, to determine the thickness specific partial volume effect. Combined AC and SC led to a significant underestimation of activity compared with ex vivo data (root mean squared error = 0.145 mCi g−1). The recovery coefficient calculated from the phantom study showed a linear relationship with object size from 1 to 6 mm, positioned in the vicinity of the center of the field of view (R2 = 0.98). Correction of nongated SPECT images with a recovery coefficient derived from the diastolic phase results in a global overestimation with root mean squared error = 0.04 mCi g−1. Nongated SPECT images corrected with a recovery coefficient with a weighted average ratio diastolic and systolic phase led to an improved root mean squared error of 0.03 mCi g−1. Combining attenuation correction with scatter correction and a gated partial volume correction yields the best correlation with ex vivo counting (root mean squared error = 0.021 mCi g−1 (systolic) and 0.025 mCi g−1 (diastolic). This study demonstrates a method for improved segmental myocardial perfusion quantification in pinhole gated SPECT, using combined attenuation-, scatter- and ultrasound-derived partial volume effect corrections. Copyright © 2012 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.

The aim of this study was the comparison of the tumour uptake and the long-term retention of [123I]-2-I-L-phenylalanine and [123I]-2-I-D-phenylalanine with those of [123I]-2-I-L-tyrosine and [123I]-2-I-D-tyrosine in R1M rhabdomyosarcoma tumour-bearing rats. The biodistribution of the radioactivity as a function of time in R1M tumour-bearing rats was measured by planar gamma camera imaging (dynamic and static). If dissection was applied, the activity in the tumours and tissues of interest was measured by gamma counting. [123I]-2-iodo-L-phenylalanine, [123I]-2-iodo-D-phenylalaine, [123I]-2-I-L-tyrosine showed a considerable tumour uptake reaching a maximum between 10 and 30 min. At 30 min p.i. the differential uptake ratio values of this uptake were, respectively, 2.1, 2.3, 2.5 and 1.7. The activity in the tumour was shown to be related to a tumour cell uptake and not to an increased blood pool activity. All the tracers showed a clearance from the blood to the bladder without renal retention. At longer times both L- and D- [123I]-2-I-tyrosine were cleared for a large part from the tumours and the body. [123I]-2-I-L-Phe and [123I]-2-I-D-Phe showed a considerable and equal retention in the tumours: as compared with 0.5 h, 91% at 24 h and 80% at 48 h. This was related to the longer retention of activity in the blood pool noticed for these compounds (81% at 24 h and 65% at 48 h). The tumour-to-background ratio increased with 25% at those longer times. At short times all the tracers were taken up to a considerable extent in the tumours. In the R1M-bearing Wag/Rij rat model only [123I]-2-I-L-phenylalanine and [123I]-2-I-D-phenylalanine showed an especially high retention at long times without any significant difference between the enantiomers. Copyright © 2007 John Wiley & Sons, Ltd.

Background 99mTc labeled radiotracers used in clinical practice lack the perfect characteristics for myocardial perfusion imaging. In particular, the high liver uptake can interfere in the interpretation of the inferior myocardial wall. Within the tricarbonyl approach, we used tris(pyrazolyl)methane 99mTc organometallic complexes as a lead structure. Herein we present the production, in vivo and in vitro metabolic studies in rats and the first in vivo biodistribution in rats for tri-methoxy-tris-pyrazolyl-99mTc-(CO)3 (99mTc-TMEOP), compared with 99mTc-sestamibi and 99mTc-tetrofosmin. Methods The chemical identity of 99mTc-TMEOP was characterized by RP-HPLC. The octanol–water partition coefficient was determined under physiological conditions. In vitro stability and protein binding were determined using RP-HPLC. In vivo stability was determined in blood, heart, liver and kidney homogenates, intestine and urine using RP-HPLC. In vivo biodistribution was determined using dynamic planar acquisitions. Pinhole gated SPECT images were performed in other animals. Cardiac, liver and lung uptake were expressed as differential uptake ratios by drawing regions of interest in the organs of interest and around the total body. Heart–liver and heart–lung ratios were derived. Cardiac uptake was also expressed as percentage of injected activity. SPECT images were processed to determine the heart–liver ratio on SPECT images, to compare functional parameters between different tracers and to visualize homogeneous intracardiac tracer distribution. Results 99mTc-TMEOP is a moderately lipophilic cation, is stable and does not undergo any transformation in vitro. 99mTc-TMEOP also shows a high in vivo stability. In vivo imaging shows liver kinetics faster than those of 99mTc-sestamibi and 99mTc-tetrofosmin. Cardiac uptake and functional analysis of pinhole gated SPECT data are comparable to those of 99mTc-sestamibi and 99mTc-tetrofosmin. Conclusion Although 99mTc-TMEOP shows a cardiac uptake between those of 99mTc-sestamibi and 99mTc-tetrofosmin, a better heart–liver ratio is obtained due to the faster liver washout. These results suggest possible faster cardiac perfusion imaging using 99mTc-TMEOP without liver activity interference. Copyright © 2010 John Wiley & Sons, Ltd.