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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.

Potential confounding factors in bioluminescence imaging (BLI)-quantification need to be identified to improve its accuracy and repeatability.The aim of this study was to study the degree to which plasma protein binding of BLI substrates influences signal intensity and quantification, both in vitro and in vivo.Protein binding was assessed using ultrafiltration and spectrophotometry. Effects of increasing serum protein concentrations were studied in vitro, in intact cells expressing Firefly or Renilla luciferase, and in vivo, in a doxorubicin-induced hypoalbuminemia mouse model.Increasing concentrations of serum proteins showed an important negative effect on BLI signal intensity, both for D-luciferin and coelenterazine-dependent reactions. This was due to a decrease in the free fraction of the substrate in the presence of serum proteins of up to 88%. In vivo, hypoalbuminemia was associated with higher BLI signal intensity, although this was only significant in animals with a major decrease in albumin (2 g/dL). Important kinetic differences, including earlier peak luminescence and a more rapid decline in luminescence, were observed for coelenterazine-dependent BLI under hypoalbuminemic conditions. Changes in protein concentrations can significantly influence BLI quantification and its presence decreases sensitivity in vitro. In vivo, effects on signal intensity and kinetics are only noted at severe hypoalbuminemia and can be neglected in most experimental designs. These findings again emphasize the need for careful interpretation of BLI quantification data.

Nanobodies represent an interesting class of probes for the generic development of molecular imaging agents. We studied the relationship between tumor uptake of the epidermal growth factor receptor (EGFR)-specific nanobody (99m)Tc-7C12 and tumor burden and evaluated the possibility of using this probe to monitor tumor response to erlotinib.The specificity and affinity of (99m)Tc-7C12 was determined on A431 cells. Cells expressing firefly luciferase were used to evaluate tumor burden using bioluminescence imaging. We evaluated the effect of erlotinib on tumor burden and (99m)Tc-7C12 uptake in vitro as well as in vivo. In vivo bioluminescence imaging was performed followed by pinhole single-photon emission computed tomography/micro-computed tomography.(99m)Tc-7C12 binds specifically to the receptor with high affinity (3.67 ± 0.59 nM). Erlotinib reduced tumor uptake and cell viability in a concentration-dependent manner. Tumor uptake of (99m)Tc-7C12 showed good correlation with tumor burden. Erlotinib treatment resulted in a progressive reduction of tumor burden and tumor uptake of (99m)Tc-7C12.(99m)Tc-7C12 binds to EGFR with high affinity and specificity. Tumor uptake is correlated with tumor burden. Quantification of (99m)Tc-7C12 uptake is promising for monitoring therapy response of EGFR-expressing tumors.

Purpose : Overexpression of the epidermal growth factor receptor (EGFR) occurs with high incidence in various carcinomas. The oncogenic expression of the receptor has been exploited for immunoglobulin-based diagnositcs and therapeutics. We describe the use of a llama single-domain antibody fragment, termed Nanobody, for the in vivo radioimmunodetection of EGFR overexpressing tumors using single photon emission computed tomography (SPECT) in mice. Methods : Fluorescence-activated cell sorting (FAC) analysis was performed to evaluate the specificity and selectivity of 8B6 Nanobody to bind EGFR on EGFR overexpressing cells. The nanobody was then labeled with 99mTc via its C-terminal histidine tail. Uptake in normal organs and tissues was assessed by ex vivo analysis. In vivo tumor targeting of 99mTc-8B6 Nanobody was evaluated via pinhole SPECT in mice bearing xenografts of tumor cells with either high (A431) or moderate (DU 145) overexpression of EGFR. Results : FACS analysis indicated that the 8B6 Nanobody only recognizes cells overexpressing EGFR. In vivo blood clearance of 99mTc-8B6 Nanobody is relatively fast (half-life, 1.5h) and mainly via the kidneys. At 3h postinjection, total kidney accumulation is high(46.6+0.9%IA) compared to total liver uptake (18.9+0.6IA). Pinhole SPECT imaging of mice bearing A431 xenografts showed higher average tumor uptake (5.2+0.5%IA/cm3) of 99mTc-8B6 Nanobody compared to DU145 xenografts (1.8+0.3%IA/cm3, pConclusion : The EGFR-binding Nanobody investigated in this study shows high specificity and selectivity towards EGFR overexpressing cells. Pinhole SPECT analysis with 99mTc-8B6 Nanobody enabled in vivo discrimination between tumors with high and moderate EGFR overexpression. The favorable biodistribution further corroborates the suitability of Nanobodies for in vivo tumor imaging.

Camelidae possess an unusual class of antibodies devoid of light chains. Nanobodies are intact antigen-binding fragments that are stable, easily generated against different targets, and fully functional. Their rapid clearance from the blood circulation favors their use as imaging agents. We compared the in vivo tumor uptake and biodistribution of 2 anti-epidermal growth factor receptor (anti-EGFR) Nanobodies, (99m)Tc-7C12 and (99m)Tc-7D12.Nanobodies were labeled via their hexahistidine tail with (99m)Tc-tricarbonyl ((99m)Tc(CO)(3)) generated from a kit. Mice bearing subcutaneous A431 (EGFR-positive) and R1M (EGFR-negative) xenografts were intravenously injected with (99m)Tc-7C12 and (99m)Tc-7D12 on separate days. Pinhole SPECT/micro-CT images were acquired at 1 h after administration to assess noninvasively the biodistribution and tumor targeting of the labeled compounds. Pinhole SPECT and micro-CT images from the same mouse were automatically fused on the basis of a mathematic rigid-body-transformation algorithm using six (57)Co sources. Images were quantified, and tracer uptake was expressed as percentage injected activity per gram per cubic centimeter (%IA/cm(3)) of tissue. Ex vivo biodistribution of mice bearing A431 injected with either (99m)Tc-7C12 or (99m)Tc-7D12 was also assessed; activity in the tumor and organs was recorded and expressed as percentage injected activity per gram (%IA/g).Binding of both tracers was receptor-specific. Image analysis showed high and similar tumor uptake values for both (99m)Tc-7C12 and (99m)Tc-7D12 (4.55+/-0.24 %IA/cm(3) and 4.62+/-0.36 %IA/cm(3), respectively) in A431 xenografts, whereas the uptake in the negative tumor (R1M) was low (1.16+/-0.14 for (99m)Tc-7C12 and 1.49+/-0.60 for (99m)Tc-7D12). (99m)Tc-7C12 showed significantly higher kidney uptake (63.48+/-2.36 vs. 56.25+/-2.46 %IA/cm(3)) and lower liver uptake (2.55+/-0.26 vs. 4.88+/-0.86 %IA/cm(3)) than did (99m)Tc-7D12. The ex vivo analysis confirmed the image quantification with high tumor-to-background ratio; however, (99m)Tc-7C12 showed higher tumor uptake (9.11+/-1.12 %IA/g) than did (99m)Tc-7D12 (6.09+/-0.77 %IA/g). (99m)Tc-7D12 demonstrated significantly higher blood activity than did (99m)Tc-7C12, but both showed short plasma half-lives (10 min).The Nanobody fragments used here show high tumor uptake, low liver uptake, and rapid blood clearance. Nanobodies are promising probes for noninvasive radioimmunodetection of specific targets early after administration. On the basis of its favorable biodistribution, (99m)Tc-7C12 was selected for further studies.

INTRODUCTION: In vivo bioluminescence imaging (BLI) is a promising technique for non-invasive tumour imaging. D: -luciferin can be administrated intraperitonealy or intravenously. This will influence its availability and, therefore, the bioluminescent signal. The aim of this study is to compare the repeatability of BLI measurement after IV versus IP administration of D: -luciferin and assess the correlation between photon emission and histological cell count both in vitro and in vivo. MATERIALS AND METHODS: Fluc-positive R1M cells were subcutaneously inoculated in nu/nu mice. Dynamic BLI was performed after IV or IP administration of D: -luciferin. Maximal photon emission (PE(max)) was calculated. For repeatability assessment, every acquisition was repeated after 4 h and analysed using Bland-Altman method. A second group of animals was serially imaged, alternating IV and IP administration up to 21 days. When mice were killed, PE(max) after IV administration was correlated with histological cell number. RESULTS: The coefficients of repeatability were 80.2% (IV) versus 95.0% (IP). Time-to-peak is shorter, and its variance lower for IV (p < 0.0001). PE(max) was 5.6 times higher for IV. A trend was observed towards lower photon emission per cell in larger tumours. CONCLUSION: IV administration offers better repeatability and better sensitivity when compared to IP. In larger tumours, multiple factors may contribute to underestimation of tumour burden. It might, therefore, be beneficial to test novel therapeutics on small tumours to enable an accurate evaluation of tumour burden.