Sophie Hernot

Comparison of the Biodistribution and Tumor Targeting of Two 99mTc-Labeled Anti-EGFR Nanobodies in Mice, Using Pinhole SPECT/Micro-CT

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, 99mTc-7C12 and 99mTc-7D12. Methods: Nanobodies were labeled via their hexahistidine tail with 99mTc-tricarbonyl (99mTc(CO)3) generated from a kit. Mice bearing subcutaneous A431 (EGFR-positive) and R1M (EGFR-negative) xenografts were intravenously injected with 99mTc-7C12 and 99mTc-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 57Co sources. Images were quantified, and tracer uptake was expressed as percentage injected activity per gram per cubic centimeter (%IA/cm3) of tissue. Ex vivo biodistribution of mice bearing A431 injected with either 99mTc-7C12 or 99mTc-7D12 was also assessed; activity in the tumor and organs was recorded and expressed as percentage injected activity per gram (%IA/g). Results: Binding of both tracers was receptor-specific. Image analysis showed high and similar tumor uptake values for both 99mTc-7C12 and 99mTc-7D12 (4.55 ± 0.24 %IA/cm3 and 4.62 ± 0.36 %IA/cm3, respectively) in A431 xenografts, whereas the uptake in the negative tumor (R1M) was low (1.16 ± 0.14 for 99mTc-7C12 and 1.49 ± 0.60 for 99mTc-7D12). 99mTc-7C12 showed significantly higher kidney uptake (63.48 ± 2.36 vs. 56.25 ± 2.46 %IA/cm3) and lower liver uptake (2.55 ± 0.26 vs. 4.88 ± 0.86 %IA/cm3) than did 99mTc-7D12. The ex vivo analysis confirmed the image quantification with high tumor-to-background ratio; however, 99mTc-7C12 showed higher tumor uptake (9.11 ± 1.12 %IA/g) than did 99mTc-7D12 (6.09 ± 0.77 %IA/g). 99mTc-7D12 demonstrated significantly higher blood activity than did 99mTc-7C12, but both showed short plasma half-lives (<10 min).Conclusion: 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, 99mTc-7C12 was selected for further studies.

Bone marrow stromal cell–derived exosomes as communicators in drug resistance in multiple myeloma cells

The interplay between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells performs a crucial role in MM pathogenesis by secreting growth factors, cytokines, and extracellular vesicles. Exosomes are membranous vesicles 40 to 100 nm in diameter constitutively released by almost all cell types, and they mediate local cell-to-cell communication by transferring mRNAs, miRNAs, and proteins. Although BMSC-induced growth and drug resistance of MM cells has been studied, the role of BMSC-derived exosomes in this action remains unclear. Here we investigate the effect of BMSC-derived exosomes on the viability, proliferation, survival, migration, and drug resistance of MM cells, using the murine 5T33MM model and human MM samples. BMSCs and MM cells could mutually exchange exosomes carrying certain cytokines. Both naive and 5T33 BMSC-derived exosomes increased MM cell growth and induced drug resistance to bortezomib. BMSC-derived exosomes also influenced the activation of several survival relevant pathways, including c-Jun N-terminal kinase, p38, p53, and Akt. Exosomes obtained from normal donor and MM patient BMSCs also induced survival and drug resistance of human MM cells. Taken together, our results demonstrate the involvement of exosome-mediated communication in BMSC-induced proliferation, migration, survival, and drug resistance of MM cells.

Nanobodies Targeting Mouse/Human VCAM1 for the Nuclear Imaging of Atherosclerotic Lesions

Rationale: A noninvasive tool allowing the detection of vulnerable atherosclerotic plaques is highly needed. By combining nanomolar affinities and fast blood clearance, nanobodies represent potential radiotracers for cardiovascular molecular imaging. Vascular cell adhesion molecule-1 (VCAM1) constitutes a relevant target for molecular imaging of atherosclerotic lesions. Objective: We aimed to generate, radiolabel, and evaluate anti-VCAM1 nanobodies for noninvasive detection of atherosclerotic lesions. Methods and Results: Ten anti-VCAM1 nanobodies were generated, radiolabeled with technetium-99m, and screened in vitro on mouse and human recombinant VCAM1 proteins and endothelial cells and in vivo in apolipoprotein E–deficient (ApoE−/−) mice. A nontargeting control nanobody was used in all experiments to demonstrate specificity. All nanobodies displayed nanomolar affinities for murine VCAM1. Flow cytometry analyses using human human umbilical vein endothelial cells indicated murine and human VCAM1 cross-reactivity for 6 of 10 nanobodies. The lead compound cAbVCAM1-5 was cross-reactive for human VCAM1 and exhibited high lesion-to-control (4.95±0.85), lesion-to-heart (8.30±1.11), and lesion-to-blood ratios (4.32±0.48) (P<0.05 versus control C57Bl/6J mice). Aortic arch atherosclerotic lesions of ApoE−/− mice were successfully identified by single-photon emission computed tomography imaging. 99mTc-cAbVCAM1-5 binding specificity was demonstrated by in vivo competition experiments. Autoradiography and immunohistochemistry further confirmed cAbVCAM1-5 uptake in VCAM1-positive lesions. Conclusions: The 99mTc-labeled, anti-VCAM1 nanobody cAbVCAM1-5 allowed noninvasive detection of VCAM1 expression and displayed mouse and human cross-reactivity. Therefore, this study demonstrates the potential of nanobodies as a new class of radiotracers for cardiovascular applications. The nanobody technology might evolve into an important research tool for targeted imaging of atherosclerotic lesions and has the potential for fast clinical translation.

Immuno-imaging using nanobodies

Immuno-imaging is a developing technology that aims at studying disease in patients using imaging techniques such as positron emission tomography in combination with radiolabeled immunoglobulin derived targeting probes. Nanobodies are the smallest antigen-binding antibody-fragments and show fast and specific targeting in vivo. These probes are currently under investigation as therapeutics but preclinical studies indicate that nanobodies could also become the next generation of magic bullets for immuno-imaging. Initial data show that imaging can be performed as early as 1 hour post-injection enabling the use of short-lived radio-isotopes. These unique properties should enable patient friendly and safe imaging protocols. This review focuses on the current status of radiolabeled nanobodies as targeting probes for immuno-imaging.

Nanobody-coupled microbubbles as novel molecular tracer

Camelid-derived single-domain antibody-fragments (~15kDa), called nanobodies, are a new class of molecular tracers that are routinely identified with nanomolar affinity for their target and that are easily tailored for molecular imaging and drug delivery applications. We hypothesized that they are well-suited for the design of targeted microbubbles (μBs) and aimed to develop and characterize eGFP- and VCAM-1-targeted μBs. Anti-eGFP (cAbGFP4) and anti-VCAM-1 (cAbVCAM1-5) nanobodies were site-specifically biotinylated in bacteria. This metabolic biotinylation method yielded functional nanobodies with one biotin located at a distant site of the antigen-binding region of the molecule. The biotinylated nanobodies were coupled to biotinylated lipid μBs via streptavidin-biotin bridging. The ability of μB-cAbGFP4 to recognize eGFP was tested as proof-of-principle by fluorescent microscopy and confirmed the specific binding of eGFP to μB-cAbGFP4. Dynamic flow chamber studies demonstrated the ability of μB-cAbVCAM1-5 to bind VCAM-1 in fast flow (up to 5 dynes/cm(2)). In vivo targeting studies were performed in MC38 tumor-bearing mice (n=4). μB-cAbVCAM1-5 or control μB-cAbGFP4 were injected intravenously and imaged using a contrast-specific ultrasound imaging mode. The echo intensity in the tumor was measured 10min post-injection. μB-cAbVCAM1-5 showed an enhanced signal compared to control μBs (p<0.05). Using metabolic and site-specific biotinylation of nanobodies, a method to develop nanobody-coupled μBs was described. The application of VCAM-1-targeted μBs as novel molecular ultrasound contrast agent was demonstrated both in vitro and in vivo.

Cyproheptadine prevents pergolide-induced valvulopathy in rats: an echocardiographic and histopathological study

Serotonergic drugs, such as pergolide, have been associated with the development of cardiac valvular myxoid thickening and regurgitation in humans and more recently in rats. These effects are potentially mediated by the 5-hydroxytryptamine (5-HT)(2B) receptor (5-HT(2B)R). Therefore, we sought to determine whether cyproheptadine, a 5-HT(2B)R antagonist, might prevent toxic valvulopathy in an animal model of pergolide-induced valvular heart disease. For this purpose, 50 male Wistar rats received daily intraperitoneal injections of pergolide (0.5 mg/kg, n = 14), pergolide (0.5 mg/kg) combined with cyproheptadine (10 mg/kg, n = 12), cyproheptadine (10 mg/kg, n = 12), or no injections (control, n = 12) for 20 wk. Echocardiography was performed blindly at baseline and at 10 and 20 wk followed by pathology. At baseline, no differences between groups were found with echocardiography. At 20 wk, aortic regurgitation was present in all pergolide-treated animals, whereas it was less frequently observed in the other groups (P < 0.0001). For the other valves, this difference was less pronounced. On histopathology, not only aortic but also mitral valves were thicker, myxoid, and exhibited more 5-HT(2B)R-positive cells in pergolide-treated animals compared with the other groups. Moreover, regurgitant aortic and mitral valves were thicker than nonregurgitant aortic and mitral valves. In conclusion, we found that cyproheptadine prevented pergolide-induced valvulopathy in rats, which was associated with a reduced number of 5-HT(2B)R-positive valvular cells. This may have important clinical implications for the prevention of serotonergic drug-induced valvular heart disease.

Effect of streptozotocin-induced diabetes on myocardial blood flow reserve assessed by myocardial contrast echocardiography in rats

AbstractThe role of structural and functional abnormalities of small vessels in diabetes cardiomyopathy remains unclear. Myocardial contrast echocardiography allows the quantification of myocardial blood flow at rest and during dipyridamole infusion. The aim of the study was to determine the myocardial blood flow reserve in normal rats compared with Streptozotocin-induced diabetic rats using contrast echocardiography.MethodsWe prospectively studied 40 Wistar rats. Diabetes was induced by intravenous streptozotocin in 20 rats. All rats underwent baseline and stress (dipyridamole: 20 mg/kg) high power intermittent imaging in short axis view under anaesthesia baseline and after six months. Myocardial blood flow was determined and compared at rest and after dipyridamole in both populations. The myocardial blood flow reserve was calculated and compared in the 2 groups. Parameters of left ventricular function were determined from the M-mode tracings and histological examination was performed in all rats at the end of the study.ResultsAt six months, myocardial blood flow reserve was significantly lower in diabetic rats compared to controls (3.09 ± 0.98 vs. 1.28 ± 0.67 ml min-1 g-1; p < 0.05). There were also a significant decrease in left ventricular function and a decreased capillary surface area and diameter at histology in the diabetic group.ConclusionIn this animal study, diabetes induced a functional alteration of the coronary microcirculation, as demonstrated by contrast echocardiography, a decrease in capillary density and of the cardiac systolic function. These findings may offer new insights into the underlying mechanisms of diabetes cardiomyopathy.

Site-Specific Labeling of His-Tagged Nanobodies with 99m Tc: A Practical Guide

99mTc-tricarbonyl chemistry provides an elegant technology to site-specifically radiolabel histidine-tagged biomolecules. Considering their unique biochemical properties, this straightforward technology is particularly suited for Nanobodies. This chapter gives a detailed guide to generate highly specific Nanobody-derived radiotracers for both in vitro binding studies and in vivo molecular imaging.

99mTc-Labeled Nanobodies: A New Type of Targeted Probes for Imaging Antigen Expression

Introduction: The development of specific radiolabeled probes towards molecular markers in vivo has gained interest as targeted imaging allows for a more accurate detection of diseases. We investigate the feasibility of targeted imaging of cancer antigens using the variable domain of single chain camelid antibodies (Nanobodies®) labeled with 99mTechnetium. Nanobodies against carcinoembryonic antigen (CEA) were used as a model. Methods: His6-CEA1 Nanobodies were generated and labeled with 99mTc at their His-tag using Tc(I)-tricarbonyl (Isolink, Mallinckrodt, B.V., Petten, The Netherlands). The normal biodistribution was assessed in healthy athymic mice by ex vivo analysis at 1 and 3 h. In vivo targeting was evaluated in the same mouse model bearing the CEA-positive LS174T tumour or a CEA-negative A431 (human skin carcinoma) control tumour. Pinhole SPECT imaging was performed at 3 hours after intravenous injection of 90 MBq 99mTc-His6-CEA1 using a dual-headed gamma camera equipped with pinhole collimators. Results: Radiolabeling efficiency was > 95%. General biodistribution showed intense renal uptake and marked liver accumulation. Using pinhole-SPECT, the average uptake of 99mTc- His6-CEA1 in LS174T (CEA positive) was significantly higher compared to the A431 (CEA negative) control tumour: respectively 3.2 ± 0.6 %IA/cm3 and 1.1 ± 0.2 %IA/cm3 (p < 0.05). Conclusion: This study presents effective labeling of Nanobodies with 99mTc using Tc(I)-carbonyl chemistry and shows their potential as a new type of specific probes for imaging antigen expression.

Comparison of contrast enhanced three dimensional echocardiography with MIBI gated SPECT for the evaluation of left ventricular function

BackgroundIn clinical practice and in clinical trials, echocardiography and scintigraphy are used the most for the evaluation of global left ejection fraction (LVEF) and left ventricular (LV) volumes. Actually, poor quality imaging and geometrical assumptions are the main limitations of LVEF measured by echocardiography. Contrast agents and 3D echocardiography are new methods that may alleviate these potential limitations.MethodsTherefore we sought to examine the accuracy of contrast 3D echocardiography for the evaluation of LV volumes and LVEF relative to MIBI gated SPECT as an independent reference. In 43 patients addressed for chest pain, contrast 3D echocardiography (RT3DE) and MIBI gated SPECT were prospectively performed on the same day. The accuracy and the variability of LV volumes and LVEF measurements were evaluated.ResultsDue to good endocardial delineation, LV volumes and LVEF measurements by contrast RT3DE were feasible in 99% of the patients. The mean LV end-diastolic volume (LVEDV) of the group by scintigraphy was 143 ± 65 mL and was underestimated by triplane contrast RT3DE (128 ± 60 mL; p < 0.001) and less by full-volume contrast RT3DE (132 ± 62 mL; p < 0.001). Limits of agreement with scintigraphy were similar for triplane andfull-volume, modalities with the best results for full-volume. Results were similar for calculation of LV end-systolic volume (LVESV). The mean LVEF was 44 ± 16% with scintigraphy and was not significantly different with both triplane contrast RT3DE (45 ± 15%) and full-volume contrast RT3DE (45 ± 15%). There was an excellent correlation between two different observers for LVEDV, LVESV and LVEF measurements and inter observer agreement was also good for both contrast RT3DE techniques.ConclusionContrast RT3DE allows an accurate assessment of LVEF compared to the LVEF measured by SPECT, and shows low variability between observers. Although RT3DE triplane provides accurate evaluation of left ventricular function, RT3DE full-volume is superior to triplane modality in patients with suspected coronary artery disease.