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Purpose: TEM-1 (tumor endothelial marker-1) is a single-pass transmembrane cell surface glycoprotein expressed at high levels by tumor vasculature and malignant cells. We aimed to perform a preclinical investigation of a novel anti-TEM-1 scFv-Fc fusion antibody, 1C1m-Fc, which was radiolabeled with 177Lu for use in soft tissue sarcomas models. Methods: 1C1m-Fc was first conjugated to p-SCN-Bn-DOTA using different excess molar ratios and labeled with 177Lu. To determine radiolabeled antibody immunoreactivity, Lindmo assays were performed. The in vivo behavior of [177Lu]Lu-1C1m-Fc was characterized in mice bearing TEM-1 positive (SK-N-AS) and negative (HT-1080) tumors by biodistribution and single-photon emission SPECT/CT imaging studies. Estimated organ absorbed doses were obtained based on biodistribution results. Results: The DOTA conjugation and the labeling with 177Lu were successful with a radiochemical purity of up to 95%. Immunoreactivity after radiolabeling was 86% ± 4%. Biodistribution showed a specific uptake in TEM-1 positive tumor versus liver as critical non-specific healthy organ, and this specificity is correlated to the number of chelates per antibody. A 1.9-fold higher signal at 72 h was observed in SPECT/CT imaging in TEM-1 positive tumors versus control tumors. Conclusion: TEM-1 is a promising target that could allow a theranostic approach to soft-tissue sarcoma, and 1C1m-Fc appears to be a suitable targeting candidate. In this study, we observed the influence of the ratio DOTA/antibody on the biodistribution. The next step will be to investigate the best conjugation to achieve an optimal tumor-to-organ radioactivity ratio and to perform therapy in murine xenograft models as a prelude to future translation in patients. [ABSTRACT FROM AUTHOR]

Cysteine cathepsins are a family of proteases that are relevant therapeutic targets for the treatment of different cancers and other diseases. However, no clinically approved drugs for these proteins exist, as their systemic inhibition can induce deleterious side effects. To address this problem, we developed a modular antibody-based platform for targeted drug delivery by conjugating non-natural peptide inhibitors (NNPIs) to antibodies. NNPIs were functionalized with reactive warheads for covalent inhibition, optimized with deep saturation mutagenesis and conjugated to antibodies to enable cell-type-specific delivery. Our antibody-peptide inhibitor conjugates specifically blocked the activity of cathepsins in different cancer cells, as well as osteoclasts, and showed therapeutic efficacy in vitro and in vivo. Overall, our approach allows for the rapid design of selective cathepsin inhibitors and can be generalized to inhibit a broad class of proteases in cancer and other diseases., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Purpose: CXCR3 is expressed on activated T cells and plays a crucial role in T-cell recruitment to the tumor microenvironment (TME) during cell-based and immune checkpoint inhibitor (ICI) immunotherapy. This study utilized a 64 Cu-labeled NOTA-α-CXCR3 antibody to assess CXCR3 expression in the TME and validate it as a potential T cell activation biomarker in vivo., Procedures: CXCR3 + cells infiltrating MC38 tumors (B57BL/6 mice, untreated and treated with αPD-1/αCTLA-4 ICI) were quantified using fluorescence microscopy and flow cytometry. A commercial anti-mouse CXCR3 antibody (α-CXCR3) was site-specifically conjugated with 2,2,2-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid (NOTA) and radiolabeled with 64 Cu. Saturation binding of [ 64 Cu]Cu-NOTA-α-CXCR3 was investigated using CHO cells stably transfected with murine CXCR3. Biodistribution and PET imaging studies both at baseline and after 1 to 3 cycles of ICI, respectively, were carried out using different molar activities (10 GBq/µmol to 300 GBq/µmol) of [ 64 Cu]Cu-NOTA-α-CXCR3., Results: Flow cytometry analysis at baseline confirmed the presence of CXCR3 + T-cells in MC38 tumors, which was significantly increased at day five after ICI (treated 33.8 ± 17.4 vs. control 8.8 ± 6.2 CD3 + CXCR3 + cells/mg). These results were qualitatively and quantitatively confirmed by immunofluorescence of tumor cryoslices. In vivo PET imaging of MC38 tumor bearing mice before, during and after ICI using [ 64 Cu]Cu-NOTA-α-CXCR3 (Kd = 3.3 nM) revealed a strong dependence of CXCR3-specificity of tracer accumulation in secondary lymphoid organs on molar activity. At 300 GBq/µmol (1.5 µg of antibody/mouse), a specific signal was observed in lymph nodes (6.33 ± 1.25 control vs. 3.95 ± 1.23%IA/g blocking) and the spleen (6.04 ± 1.02 control vs. 3.84 ± 0.79%IA/g blocking) at 48 h p.i. Spleen-to-liver ratios indicated a time dependent systemic immune response showing a steady increase from 1.08 ± 0.19 (untreated control) to 1.54 ± 0.14 (three ICI cycles)., Conclusions: This study demonstrates the feasibility of in vivo imaging of CXCR3 upregulation under immunotherapy using antibodies. However, high molar activities and low antibody doses are essential for sensitive detection in lymph nodes and spleen. Detecting therapy-induced changes in CXCR3 + T cell numbers in tumors was challenging due to secondary antibody-related effects. Nonetheless, CXCR3 remains a promising target for imaging T cell activation, with anticipated improvements in sensitivity using alternative tracers with high affinities and favorable pharmacokinetics., (© 2024. The Author(s).)

Template-directed methods are emerging as some of the most effective means to conjugate payloads at selective sites of monoclonal antibodies (mAbs). We have previously reported a method based on an engineered Fc-III reactive peptide to conjugate a radionuclide chelator to K317 of antibodies with the concomitant release of the Fc-III peptide ligand. Here, our method was redesigned to target two lysines proximal to the Fc-III binding site, K248 and K439. Using energy minimization predictions and a semi-combinatorial synthesis approach, we sampled multiple Fc-III amino acid substituents of A3, H5, L6 and E8, which were then converted into Fc-III reactive conjugates. Middle-down MS/MS subunit analysis of the resulting trastuzumab conjugates revealed that K248 and K439 can be selectively targeted using the Fc-III reactive variants L6Dap, L6Orn, L6Y and A3K or A3hK, respectively. Across all variants tested, L6Orn-carbonate appeared to be the best candidate, yielding a degree and yield of conjugation of almost 2 and 100% for a broad array of payloads including radionuclide chelators, fluorescent dyes, click-chemistry reagents, pre-targeted imaging reagents, and some cytotoxic small molecules. Furthermore, L6Orn carbonate appeared to yield similar conjugation results across multiple IgG subtypes. In vivo proof of concept was achieved by conjugation of NODAGA to the PD1/PD-L1 immune checkpoint inhibitor antibody atezolizumab, followed by PET imaging of PD-L1 expression in mice bearing PD-L1 expressing tumor xenograft using radiolabeled [ 64 Cu]Cu-atezolizumab., Competing Interests: There are no conflicts to declare. V. P., L. M., F. L., P. G., J.-M. S., O. N. are coinventors of application WO 2022/078566 A1 filed on 12 October 2020, entitled “Reactive conjugates”., (This journal is © The Royal Society of Chemistry.)

Background: CXCR4-targeted radioligand therapy (RLT) with [ 177 Lu]Lu/[ 90 Y]Y-PentixaTher has recently evolved as a promising therapeutic option for patients with advanced hematological cancers. Given their advanced disease stage, most patients scheduled for PentixaTher RLT require concomitant or bridging chemotherapy to prevent intermittent tumor progression. These (mostly combination) therapies may cause significant downregulation of tumoral CXCR4 expression, challenging the applicability of PentixaTher RLT. This study therefore aimed at investigating the influence of corticosteroids, a central component of these chemotherapies, on CXCR4 regulation in diffuse large B cell lymphoma (DLBCL)., Methods: Different DLBCL cell lines (Daudi, OCI-LY1, SUDHL-4, -5-, -6 and -8) as well as the human T-cell lymphoma cell line Jurkat were incubated with Dexamethasone (Dex; 0.5 and 5 µM, respectively) and Prednisolone (Pred; 5 and 50 µM, respectively) for different time points (2 h, 24 h). Treatment-induced modulation of cellular CXCR4 surface expression was assessed via flow cytometry (FC) and compared to untreated cells. A radioligand binding assay with [ 125 I]CPCR4.3 was performed in parallel using the same cells. To quantify potential corticosteroid treatment effects on tumoral CXCR4 expression in vivo, OCI-LY1 bearing NSG mice were injected 50 µg Dex/mouse i.p. (daily for 6 days). Then, a biodistribution study (1 h p.i.) using [ 68 Ga]PentixaTher was performed, and tracer biodistribution in treated (n = 5) vs untreated mice (n = 5) was compared., Results: In the in vitro experiments, a strongly cell line-dependent upregulation of CXCR4 was observed for both Dex and Pred treatment, with negligible differences between the high and low dose. While in Jurkat, Daudi and SUDHL-8 cells, CXCR4 expression remained unchanged, a 1.5- to 3.5-fold increase in CXCR4 cell surface expression was observed for SUDHL-5 < SUDHL-4 /-6 < OCI-LY1 via FC compared to untreated cells. This increase in CXCR4 expression was also reflected in correspondingly enhanced [ 125 I]CPCR4.3 accumulation in treated cells, with a linear correlation between FC and radioligand binding data. In vivo, Dex treatment led to a general increase of [ 68 Ga]PentixaTher uptake in all organs compared to untreated animals, as a result of a higher tracer concentration in blood. However, we observed an overproportionally enhanced [ 68 Ga]PentixaTher uptake in the OCI-LY1 tumors in treated (21.0 ± 5.5%iD/g) vs untreated (9.2 ± 2.8%iD/g) mice, resulting in higher tumor-to-background ratios in the treatment group., Conclusion: Overall, corticosteroid treatment (Dex/Pred) consistently induced an upregulation of CXCR4 expression DBLCL cells in vitro, albeit in a very cell line-dependent manner. For the cell line with the most pronounced Dex-induced CXCR4 upregulation, OCI-LY1, the in vitro findings were corroborated by an in vivo biodistribution study. This confirms that at least the corticosteroid component of stabilizing chemotherapy regimens in DLBCL patients prior to [ 177 Lu]Lu-PentixaTher RLT does not lead to downregulation of the molecular target CXCR4 and may even have a beneficiary effect. However, further studies are needed to investigate if and to what extent the other commonly used chemotherapeutic agents affect CXCR4 expression on DLBCL to ensure the choice of an appropriate treatment regimen prior to [ 177 Lu]Lu/[ 90 Y]Y-PentixaTher RLT., (© 2023. The Author(s).)

Metabolic syndrome (MetS) refers to cardiometabolic risk factors, such as visceral obesity, dyslipidemia, hyperglycemia/insulin resistance, arterial hypertension and non-alcoholic fatty liver disease (NAFLD). Individuals born after intrauterine growth restriction (IUGR) are particularly at risk of developing metabolic/hepatic disorders later in life. Oxidative stress and cellular senescence have been associated with MetS and are observed in infants born following IUGR. However, whether these mechanisms could be particularly associated with the development of NAFLD in these individuals is still unknown. IUGR was induced in rats by a maternal low-protein diet during gestation versus. a control (CTRL) diet. In six-month-old offspring, we observed an increased visceral fat mass, glucose intolerance, and hepatic alterations (increased transaminase levels, triglyceride and neutral lipid deposit) in male rats with induced IUGR compared with the CTRL males; no differences were found in females. In IUGR male livers, we identified some markers of stress-induced premature senescence (SIPS) (lipofuscin deposit, increased protein expression of p21 WAF , p16 INK4a and Acp53, but decreased pRb/Rb ratio, foxo-1 and sirtuin-1 protein and mRNA expression) associated with oxidative stress (higher superoxide anion levels, DNA damages, decreased Cu/Zn SOD, increased catalase protein expression, increased nfe2 and decreased keap1 mRNA expression). Impaired lipogenesis pathways (decreased pAMPK/AMPK ratio, increased pAKT/AKT ratio, SREBP1 and PPARγ protein expression) were also observed in IUGR male livers. At birth, no differences were observed in liver histology, markers of SIPS and oxidative stress between CTRL and IUGR males. These data demonstrate that the livers of IUGR males at adulthood display SIPS and impaired liver structure and function related to oxidative stress and allow the identification of specific therapeutic strategies to limit or prevent adverse consequences of IUGR, particularly metabolic and hepatic disorders.

Background: Extrapolation of human absorbed doses (ADs) from biodistribution experiments on laboratory animals is used to predict the efficacy and toxicity profiles of new radiopharmaceuticals. Comparative studies between available animal-to-human dosimetry extrapolation methods are missing. We compared five computational methods for mice-to-human AD extrapolations, using two different radiopharmaceuticals, namely [ 111 In]CHX-DTPA-scFv78-Fc and [ 68 Ga]NODAGA-RGDyK. Human organ-specific time-integrated activity coefficients (TIACs) were derived from biodistribution studies previously conducted in our centre. The five computational methods adopted are based on simple direct application of mice TIACs to human organs (M1), relative mass scaling (M2), metabolic time scaling (M3), combined mass and time scaling (M4), and organ-specific allometric scaling (M5), respectively. For [ 68 Ga]NODAGA-RGDyK, these methods for mice-to-human extrapolations were tested against the ADs obtained on patients, previously published by our group. Lastly, an average [ 68 Ga]NODAGA-RGDyK-specific allometric parameter α new was calculated from the organ-specific biological half-lives in mouse and humans and retrospectively applied to M3 and M4 to assess differences in human AD predictions with the α = 0.25 recommended by previous studies., Results: For both radiopharmaceuticals, the five extrapolation methods showed significantly different AD results (p < 0.0001). In general, organ ADs obtained with M3 were higher than those obtained with the other methods. For [ 68 Ga]NODAGA-RGDyK, no significant differences were found between ADs calculated with M3 and those obtained directly on human subjects (H) (p = 0.99; average M3/H AD ratio = 1.03). All other methods for dose extrapolations resulted in ADs significantly different from those calculated directly on humans (all p ≤ 0.0001). Organ-specific allometric parameters calculated using combined experimental [ 68 Ga]NODAGA-RGDyK mice and human biodistribution data varied significantly. ADs calculated with M3 and M4 after the application of α new  = 0.17 were significantly different from those obtained by the application of α = 0.25 (both p < 0.001)., Conclusions: Available methods for mouse-to-human dosimetry extrapolations provided significantly different results in two different experimental models. For [ 68 Ga]NODAGA-RGDyK, the best approximation of human dosimetry was shown by M3, applying a metabolic scaling to the mouse organ TIACs. The accuracy of more refined extrapolation algorithms adopting model-specific metabolic scaling parameters should be further investigated., (© 2022. The Author(s).)

Antibodies are an attractive therapeutic modality for cancer treatment as they allow the increase of the treatment response rate and avoid the severe side effects of chemotherapy. Notwithstanding the strong benefit of antibodies, the efficacy of anti-cancer antibodies can dramatically vary among patients and ultimately result in no response to the treatment. Here, we have developed a novel means to regioselectively label the Fc domain of any therapeutic antibody with a radionuclide chelator in a single step chemistry, with the aim to study by SPECT/CT imaging if the radiolabeled antibody is capable of targeting cancer cells in vivo . A Fc-III peptide was used as bait to bring a carbonate electrophilic site linked to a metal chelator and to a carboxyphenyl leaving group in close proximity with an antibody Fc nucleophile amino acid (K317), thereby triggering the covalent linkage of the chelator to the antibody lysine, with the concomitant release of the carboxyphenyl Fc-III ligand. Using CHX-A''-DTPA, we radiolabeled trastuzumab with indium-111 and showed in biodistribution and imaging experiments that the antibody accumulated successfully in the SK-OV-3 xenograft tumour implanted in mice. We found that our methodology leads to homogeneous conjugation of CHX-A''-DTPA to the antibody, and confirmed that the Fc domain can be selectively labeled at K317, with a minor level of unspecific labeling on the Fab domain. The present method can be developed as a clinical diagnostic tool to predict the success of the therapy. Furthermore, our Fc-III one step chemistry concept paves the way to a broad array of other applications in antibody bioengineering., Competing Interests: There are no conflicts to declare. V. P, L. M, F. L, P. G, J.-M. S, and O. N are coinventors of application WO2021110860 A1 filed on 3 December 2019, entitled “Reactive conjugates”., (This journal is © The Royal Society of Chemistry.)

Introduction: Evaluation of glomerular filtration rate is very important in both preclinical and clinical setting, especially in the context of chronic kidney disease. It is typically performed using 51 Cr-EDTA or by imaging with 123 I-Hippuran scintigraphy, which has a significantly lower resolution and sensitivity as compared to PET. 68 Ga-EDTA represents a valid alternative due to its quick availability using a 68 Ge/ 68 Ga generator, while PET/CT enables both imaging of renal function and accurate quantitation of clearance of activity from both plasma and urine. Therefore, we aimed at investigating the use of 68 Ga-EDTA as a preclinical tracer for determining renal function in a knock-in rat model known to present progressive decline of renal function., Methods: 68 Ga-EDTA was injected in 23 rats, either wild type (n=10) or knock-in (n=13). By applying a unidirectional, two-compartment model and Rutland-Patlak Plot linear regression analysis, split renal function was determined from the age of 6 weeks to 12 months., Results: Glomerular filtration ranged from 0.025±0.01 ml/min at 6 weeks to 0.049±0.05 ml/min at 6 months in wild type rats. Glomerular filtration was significantly lower in knock-in rats at 6 and 12 months (P<0.01). No significant difference was observed in renal volumes between knock-in and wild type animals, based on imaging-derived volume calculations., Conclusions: 68 Ga-EDTA turned out to be a very promising PET/CT tracer for the evaluation of split renal function. This method allowed detection of progressive renal impairment in a knock-in rat model. Additional validation in a human cohort is warranted to further assess clinical utility in both, healthy individuals and patients with renal impairment., Competing Interests: None., (AJNMMI Copyright © 2021.)

1C1m-Fc, a promising anti-TEM-1 DOTA conjugate, was labeled with 64 Cu to target cancer cells for PET imaging and predicting the efficacy and safety of a previously studied [ 177 Lu]Lu-1C1m-Fc companion therapy. DOTA-conjugated 1C1m-Fc was characterized by mass spectrometry, thin layer chromatography and immunoreactivity assessment. PET/CT and biodistribution studies were performed in human neuroblastoma xenografted mice. Absorbed doses were assessed from biodistribution results and extrapolated to 177 Lu based on the [ 64 Cu]Cu-1C1m-Fc data. The immunoreactivity was ≥ 70% after 48 h of incubation in serum, and the specificity of [ 64 Cu]Cu-1C1m-Fc for the target was validated. High-resolution PET/CT images were obtained, with the best tumor-to-organ ratios reached at 24 or 48 h and correlated with results of the biodistribution study. Healthy organs receiving the highest doses were the liver, the kidneys and the uterus. [ 64 Cu]Cu-1C1m-Fc could be of interest to give an indication of 177 Lu dosimetry for parenchymal organs. In the uterus and the tumor, characterized by specific TEM-1 expression, the 177 Lu-extrapolated absorbed doses are overestimated because of the lack of later measurement time points. Nevertheless, 1C1m-Fc radiolabeled with 64 Cu for imaging would appear as an interesting radionuclide companion for therapeutic application with [ 177 Lu]Lu-1C1m-Fc.

The use of radioactivity in medicine has been developed over a century. The discovery of radioisotopes and their interactions with living cells and tissue has led to the emergence of new diagnostic and therapeutic modalities. The CERN-MEDICIS infrastructure, recently inaugurated at the European Center for Nuclear Research (CERN), provides a wide range of radioisotopes of interest for diagnosis and treatment in oncology. Our objective is to draw attention to the progress made in nuclear medicine in collaboration with CERN and potential future applications, in particular for the treatment of aggressive tumors such as pancreatic adenocarcinoma, through an extensive review of literature. Fifty seven out of two hundred and ten articles, published between 1997 and 2020, were selected based on relevancy. Meetings were held with a multi-disciplinary team, including specialists in physics, biological engineering, chemistry, oncology and surgery, all actively involved in the CERN-MEDICIS project. In summary, new diagnostic, and therapeutic modalities are emerging for the treatment of pancreatic adenocarcinoma. Targeted radiotherapy or brachytherapy could be combined with existing therapies to improve the quality of life and survival of these patients. Many studies are still in the pre-clinical stage but open new paths for patients with poor prognosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Burkhardt, Bühler, Viertl and Stora.)

1C1m-Fc, an anti-tumor endothelial marker 1 (TEM-1) scFv-Fc fusion protein antibody, was previously successfully radiolabeled with 177 Lu. TEM-1 specific tumor uptake was observed together with a non-saturation dependent liver uptake that could be related to the number of dodecane tetraacetic acid (DOTA) chelator per 1C1m-Fc. The objective of this study was to verify this hypothesis and to find the best DOTA per 1C1m-Fc ratio for theranostic applications. 1C1m-Fc was conjugated with six concentrations of DOTA. High-pressure liquid chromatography, mass spectrometry, immunoreactivity assessment, and biodistribution studies in mice bearing TEM-1 positive tumors were performed. A multi-compartment pharmacokinetic model was used to fit the data and a global pharmacokinetic model was developed to illustrate the effect of liver capture and immunoreactivity loss. Organ absorbed doses in mice were calculated from biodistribution results. A loss of immunoreactivity was observed with the highest DOTA per 1C1m-Fc ratio. Except for the spleen and bone, an increase of DOTA per 1C1m-Fc ratio resulted in an increase of liver uptake and absorbed dose and a decrease of uptake in tumor and other tissues. Pharmacokinetic models correlated these results. The number of DOTA per antibody played a determining role in tumor targeting. One DOTA per 1C1m-Fc gave the best pharmacokinetic behavior for a future translation of [ 177 Lu]Lu-1C1m-Fc in patients.

The tumour endothelial marker 1 (TEM1/endosialin/CD248) is a receptor overexpressed in several human solid tumours and silenced in normal adult tissues, representing a suitable and potentially safe target for radioimmunotherapy of sarcoma. To develop new tools with improved TEM1 targeting properties, a new panel of antibody fragments was for the first time evaluated preclinically following 125 I radiolabelling. The antibody fragment 1C1m-Fc, with the highest human/murine TEM1 binding affinity, was extensively characterized in vitro and in vivo in a Ewing's sarcoma human xenograft mouse model. In silico studies were also performed to elucidate the influence of a single amino acid mutation in the complementarity-determining region (CDR3) of the heavy chain, upon affinity maturation of the parental clone 1C1-Fc. From this study, 1C1m-Fc emerged as a promising candidate for the development of TEM1-targeted radioimmunoconjugates, namely to be further explored for theranostic applications with other suitable medical radionuclides., (Copyright © 2020. Published by Elsevier B.V.)

Purpose: Endosialin/tumor endothelial marker-1 (TEM1) is an attractive theranostic target expressed by the microenvironment of a wide range of tumors, as well as by sarcoma and neuroblastoma cells. We report on the radiolabeling and preclinical evaluation of the scFv78-Fc, a fully human TEM1-targeting antibody fragment cross-reactive with mouse TEM1., Procedures: The scFv78-Fc was conjugated with the chelator p-SCN-Bn-CHX-A"-DTPA, followed by labeling with indium-111. The number of chelators per molecule was estimated by mass spectrometry. A conventional saturation assay, extrapolated to infinite antigen concentration, was used to determine the immunoreactive fraction of the radioimmunoconjugate. The radiopharmaceutical biodistribution was assessed in immunodeficient mice grafted with Ewing's sarcoma RD-ES and neuroblastoma SK-N-AS human TEM1-positive tumors. The full biodistribution studies were preceded by a dose-escalation experiment based on the simultaneous administration of the radiopharmaceutical with increasing amounts of unlabeled scFv78-Fc. Radiation dosimetry extrapolations to human adults were obtained from mouse biodistribution data according to established methodologies and additional assumptions concerning the impact of the tumor antigenic sink in the cross-species translation., Results: [ 111 In]CHX-DTPA-scFv78-Fc was obtained with a radiochemical purity > 98 % after 1 h incubation at 42 °C and ultrafiltration. It showed good stability in human serum and > 70 % immunoreactive fraction. Biodistribution data acquired in tumor-bearing mice confirmed fast blood clearance and specific tumor targeting in both xenograft models. The radiopharmaceutical off-target uptake was predominantly abdominal. After a theoretical injection of [ 111 In]CHX-DTPA-scFv78-Fc to the reference person, the organs receiving the highest absorbed dose would be the spleen (0.876 mGy/MBq), the liver (0.570 mGy/MBq) and the kidneys (0.298 mGy/MBq). The total body dose and the effective dose would be 0.058 mGy/MBq and 0.116 mSv/MBq, respectively., Conclusions: [ 111 In]CHX-DTPA-scFv78-Fc binds specifically to endosialin/TEM1 in vitro and in vivo. Dosimetry estimates are in the range of other monoclonal antibodies radiolabeled with indium-111. [ 111 In]CHX-DTPA-scFv78-Fc could be potentially translated into clinic.

Background: Biodistribution studies based on organ harvesting represent the gold standard pre-clinical technique for dose extrapolations. However, sequential imaging is becoming increasingly popular as it allows the extraction of longitudinal data from single animals, and a direct correlation with deterministic radiation effects. We assessed the feasibility of mouse-specific, microPET-based dosimetry of an antibody fragment labeled with the positron emitter 152 Tb [(T 1/2  = 17.5 h, Eβ + mean = 1140 keV (20.3%)]. Image-based absorbed dose estimates were compared with those obtained from the extrapolation to 152 Tb of a classical biodistribution experiment using the same antibody fragment labeled with 111 In. 152 Tb was produced by proton-induced spallation in a tantalum target, followed by mass separation and cation exchange chromatography. The endosialin-targeting scFv78-Fc fusion protein was conjugated with the chelator p-SCN-Bn-CHX-A"-DTPA, followed by labeling with either 152 Tb or 111 In. Micro-PET images of four immunodeficient female mice bearing RD-ES tumor xenografts were acquired 4, 24, and 48 h after the i.v. injection of 152 Tb-CHX-DTPA-scFv78-Fc. After count/activity camera calibration, time-integrated activity coefficients (TIACs) were obtained for the following compartments: heart, lungs, liver, kidneys, intestines, tumor, and whole body, manually segmented on CT. For comparison, radiation dose estimates of 152 Tb-CHX-DTPA-scFv78-Fc were extrapolated from mice dissected 4, 24, 48, and 96 h after the injection of 111 In-CHX-DTPA-scFv78-Fc (3-5 mice per group). Imaging-derived and biodistribution-derived organ TIACs were used as input in the 25 g mouse model of OLINDA/EXM® 2.0, after appropriate mass rescaling. Tumor absorbed doses were obtained using the OLINDA2 sphere model. Finally, the relative percent difference (RD%) between absorbed doses obtained from imaging and biodistribution were calculated., Results: RD% between microPET-based dosimetry and biodistribution-based dose extrapolations were + 12, - 14, and + 17 for the liver, the kidneys, and the tumors, respectively. Compared to biodistribution, the imaging method significantly overestimates the absorbed doses to the heart and the lungs (+ 89 and + 117% dose difference, respectively)., Conclusions: MicroPET-based dosimetry of 152 Tb is feasible, and the comparison with organ harvesting resulted in acceptable dose discrepancies for body districts that can be segmented on CT. These encouraging results warrant additional validation using radiolabeled biomolecules with a different biodistribution pattern.

Ionizing radiation constitutes a health risk to imaging scientists and study animals. Both PET and CT produce ionizing radiation. CT doses in pre-clinical in vivo imaging typically range from 50 to 1,000 mGy and biological effects in mice at this dose range have been previously described. [ 18 F]FDG body doses in mice have been estimated to be in the range of 100 mGy for [ 18 F]FDG. Yearly, the average whole body doses due to handling of activity by PET technologists are reported to be 3-8 mSv. A preclinical PET/CT system is presented with design features which make it suitable for small animal low-dose imaging. The CT subsystem uses a X-source power that is optimized for small animal imaging. The system design incorporates a spatial beam shaper coupled with a highly sensitive flat-panel detector and very fast acquisition (<10 s) which allows for whole body scans with doses as low as 3 mGy. The mouse total-body PET subsystem uses a detector architecture based on continuous crystals, coupled to SiPM arrays and a readout based in rows and columns. The PET field of view is 150 mm axial and 80 mm transaxial. The high solid-angle coverage of the sample and the use of continuous crystals achieve a sensitivity of 9% (NEMA) that can be leveraged for use of low tracer doses and/or performing rapid scans. The low-dose imaging capabilities of the total-body PET subsystem were tested with NEMA phantoms, in tumor models, a mouse bone metabolism scan and a rat heart dynamic scan. The CT imaging capabilities were tested in mice and in a low contrast phantom. The PET low-dose phantom and animal experiments provide evidence that image quality suitable for preclinical PET studies is achieved. Furthermore, CT image contrast using low dose scan settings was suitable as a reference for PET scans. Total-body mouse PET/CT studies could be completed with total doses of <10 mGy.

Background: Integrin-targeting radiopharmaceuticals have potential broad applications, spanning from cancer theranostics to cardiovascular diseases. We have previously reported preclinical dosimetry results of 68 Ga-NODAGA-RGDyK in mice. This study presents the first human dosimetry of 68 Ga-NODAGA-RGDyK in the five consecutive patients included in a clinical imaging protocol of carotid atherosclerotic plaques. Five male patients underwent whole-body time-of-flight (TOF) PET/CT scans 10, 60 and 120 min after tracer injection (200 MBq). Quantification of 68 Ga activity concentration was first validated by a phantom study. To be used as input in OLINDA/EXM, time-activity curves were derived from manually drawn regions of interest over the following organs: brain, thyroid, lungs, heart, liver, spleen, stomach, kidneys, red marrow, pancreas, small intestine, colon, urinary bladder and whole body. A separate dosimetric analysis was performed for the choroid plexuses. Female dosimetry was extrapolated from male data. Effective doses (EDs) were estimated according to both ICRP60 and ICRP103 assuming 30-min and 1-h voiding cycles., Results: The body regions receiving the highest dose were urinary bladder, kidneys and choroid plexuses. For a 30-min voiding cycle, the EDs were 15.7 and 16.5 μSv/MBq according to ICRP60 and ICRP103, respectively. The extrapolation to female dosimetry resulted in organ absorbed doses 17% higher than those of male patients, on average. The 1-h voiding cycle extrapolation resulted in EDs of 19.3 and 19.8 μSv/MBq according to ICRP60 and ICRP103, respectively. A comparison is made with previous mouse dosimetry and with other human studies employing different RGD-based radiopharmaceuticals., Conclusions: According to ICRP60/ICRP103 recommendations, an injection of 200 MBq 68 Ga-NODAGA-RGDyK leads to an ED in man of 3.86/3.92 mSv. For future therapeutic applications, specific attention should be directed to delivered dose to kidneys and potentially also to the choroid plexuses., Trial Registration: Clinical trial.gov, NCT01608516.

The synthetic peptide TAT-RasGAP 317-326 has been shown to potentiate the efficacy of anti-cancer drugs. In this study, we explored the action of TAT-RasGAP 317-326 when combined with radiation by investigating its radiosensitizing activity in vitro and in vivo. To investigate the modulation of intrinsic radiosensitivity induced by TAT-RasGAP 317-326 , clonogenic assays were performed using four human cancer cell lines, HCT116 p53 +/+ (ATCC: CCL-247), HCT116 p53 -/- , PANC-1 (ATCC: CRL-1469) and HeLa (ATCC: CCL-2), as well as one nontumor cell line, HaCaT (CLS: 300493). Next, to investigate tumor growth delay after irradiation, HCT116 cell lines were selected and xenografted onto nude mice that were then treated with TAT-RasGAP 317-326 alone or in combination with radiation or cisplatin. Afterwards, cell cycle and death modulation were investigated by quantification of micronuclei and apoptosis-related protein array. TAT-RasGAP 317-326 radiosensitized all four human carcinoma cell lines tested but displayed no effect on normal cells. It also displayed no effect when administered as monotherapy. This radiosensitizing effect was confirmed in vivo in both p53-positive and p53-negative HCT116 xenografts. TAT-RasGAP 317-326 combined with radiation enhanced the number of cells in S phase and subsequently delayed cell death, but had almost no effect on major apoptosis-related proteins. TAT-RasGAP 317-326 is a radiosensitizing agent that acts on carcinoma cells and its radiosensitizing effect might be mediated, at least in part, by the enhancement of mitotic cell death.

The interest around small-animal cardiac radionuclide imaging is growing as rodent models can be manipulated to allow the simulation of human diseases. In addition to new radiopharmaceuticals testing, often researchers apply well-established probes to animal models, to follow the evolution of the target disease. This reverse translation of standard radiopharmaceuticals to rodent models is complicated by technical shortcomings and by obvious differences between human and rodent cardiac physiology. In addition, radionuclide studies involving small animals are affected by several extrinsic variables, such as the choice of anesthetic. In this paper, we review the major cardiac features that can be studied with classical single-photon and positron-emitting radiopharmaceuticals, namely, cardiac function, perfusion and metabolism, as well as the results and pitfalls of small-animal radionuclide imaging techniques. In addition, we provide a concise guide to the understanding of the most frequently used anesthetics such as ketamine/xylazine, isoflurane, and pentobarbital. We address in particular their mechanisms of action and the potential effects on radionuclide imaging. Indeed, cardiac function, perfusion, and metabolism can all be significantly affected by varying anesthetics and animal handling conditions.

Background and Purpose: Second mitochondria-derived activator of caspase (SMAC)-mimetics are a new class of targeted drugs that specifically induce apoptotic cancer cell death and block pro-survival signaling by antagonizing selected members of the inhibitor of apoptosis protein (IAP) family., Material and Methods: The present study was designed to investigate the radiosensitizing effect and optimal sequence of administration of the novel SMAC-mimetic Debio 1143 in vitro and in vivo. Apoptosis, alteration of DNA damage repair (DDR), and tumor necrosis factor-alpha (TNF-α) signaling were examined., Results: In vitro, Debio 1143 displayed anti-proliferative activity and enhanced intrinsic radiation sensitivity in 5/6 head and neck squamous cell carcinoma (HNSCC) cell lines in a synergistic manner. In vivo, Debio 1143 dose-dependently radio-sensitized FaDu and SQ20B xenografts, resulting in complete tumor regression in 8/10 FaDu-xenografted mice at the high dose level. At the molecular level, Debio 1143 combined with radiotherapy (RT) induced enhancement of caspase-3 activity, increase in Annexin V-positive cells and karyopyknosis, and increase in TNF-α mRNA levels. Finally, in a neutralization experiment using a TNF-α-blocking antibody and a caspase inhibitor, it was shown that the radiosensitizing effect of Debio 1143 is mediated by caspases and TNF-α., Conclusions: These results demonstrate that the novel SMAC-mimetic Debio 1143 is a radiosensitizing agent that is worthy of further investigation in clinical trials in combination with radiotherapy., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

CERN-MEDICIS is a facility dedicated to research and development in life science and medical applications. The research platform was inaugurated in October 2014 and will produce an increasing range of innovative isotopes using the proton beam of ISOLDE for fundamental studies in cancer research, for new imaging and therapy protocols in cell and animal models and for preclinical trials, possibly extended to specific early phase clinical studies (phase 0) up to phase I trials. CERN, the University Hospital of Geneva (HUG), the University Hospital of Lausanne (CHUV), the Swiss Institute for Experimental Cancer (ISREC) at Swiss Federal Institutes of Technology (EPFL) that currently support the project will benefit of the initial production that will then be extended to other centers.

The p120 RasGAP protein negatively regulates Ras via its GAP domain. RasGAP carries several other domains that modulate several signaling molecules such as Rho. RasGAP is also a caspase-3 substrate. One of the caspase-3-generated RasGAP fragments, corresponding to amino acids 158-455 and called fragment N2, was previously reported to specifically sensitize cancer cells to death induced by various anticancer agents. Here, we show that fragment N2 inhibits migration in vitro and that it impairs metastatic progression of breast cancer to the lung. Hence, stress-activated caspase-3 might contribute to the suppression of metastasis through the generation of fragment N2. These results indicate that the activity borne by fragment N2 has a potential therapeutic relevance to counteract the metastatic process., (© 2013 UICC.)

Aim: 5-fluoro-2'-deoxyuridine (FdUrd) depletes the endogenous 5'-deoxythymidine triphosphate (dTTP) pool. We hypothesized whether uptake of exogenous dThd analogues could be favoured through a feedback enhanced salvage pathway and studied the FdUrd effect on cellular uptake of 3'-deoxy-3'-18F-fluorothymidine (18F-FLT) and 5-125I-iodo-2'-deoxyuridine (125I-IdUrd) in different cancer cell lines in parallel., Methods: Cell uptake of 18F-FLT and 125I-IdUrd was studied in 2 human breast, 2 colon cancer and 2 glioblastoma lines. Cells were incubated with/without 1 µmol/l FdUrd for 1 h and, after washing, with 1.2 MBq 18F-FLT or 125I-IdUrd for 0.3 to 2 h. Cell bound 18F-FLT and 125I-IdUrd was counted and expressed in % incubated activity (%IA). Kinetics of 18F-FLT cell uptake and release were studied with/without FdUrd modulation. 2'-3H-methyl-fluorothymidine (2'-3H-FLT) uptake with/without FdUrd pretreatment was tested on U87 spheroids and monolayer cells., Results: Basal uptake at 2 h of 18F-FLT and 125I-IdUrd was in the range of 0.8-1.0 and 0.4-0.6 Bq/cell, respectively. FdUrd pretreatment enhanced 18F-FLT and 125I-IdUrd uptake 1.2-2.1 and 1.7-4.4 fold, respectively, while co-incubation with excess thymidine abrogated all 18F-FLT uptake. FdUrd enhanced 18F-FLT cellular inflow in 2 breast cancer lines by factors of 1.8 and 1.6, respectively, while outflow persisted at a slightly lower rate. 2'-3H-FLT basal uptake was very low while uptake increase after FdUrd was similar in U87 monolayer cells and spheroids., Conclusions: Basal uptake of 18F-FLT was frequently higher than that of 125I-IdUrd but FdUrd induced uptake enhancement was stronger for 125I-IdUrd in five of six cell lines. 18F-FLT outflow from cells might be an explanation for the observed difference with 125I-IdUrd.

Background: The increasing availability of different monoclonal antibodies (mAbs) opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL) respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells., Results: The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to longer mouse survival, as compared to single mAb injection, up to complete inhibition of tumour growth in 100% mice treated with both anti-HLA-DR and anti-CD5., Conclusions: Altogether these data suggest that the combined use of two mAbs, such as anti-HLA-DR and anti-CD5, may significantly enhance their therapeutic potential.

Purpose: 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT), a cell proliferation positron emission tomography (PET) tracer, has been shown in numerous tumors to be more specific than 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) but less sensitive. We studied the capacity of a nontoxic concentration of 5-fluoro-2'-deoxyuridine (FdUrd), a thymidine synthesis inhibitor, to increase uptake of [(18)F]FLT in tumor xenografts., Methods: The duration of the FdUrd effect in vivo on tumor cell cycling and thymidine analogue uptake was studied by varying FdUrd pretreatment timing and holding constant the timing of subsequent flow cytometry and 5-[(125)I]iodo-2'-deoxyuridine biodistribution measurements. In [(18)F]FLT studies, FdUrd pretreatment was generally performed 1 h before radiotracer injection. [(18)F]FLT biodistributions were measured 1 to 3 h after radiotracer injection of mice grafted with five different human tumors and pretreated or not with FdUrd and compared with [(18)F]FDG tumor uptake. Using microPET, the dynamic distribution of [(18)F]FLT was followed for 1.5 h in FdUrd pretreated mice. High-field T2-weighted magnetic resonance imaging (MRI) and histology were used comparatively in assessing tumor viability and proliferation., Results: FdUrd induced an immediate increase in tumor uptake of 5-[(125)I]iodo-2'-deoxyuridine, that vanished after 6 h, as also confirmed by flow cytometry. Biodistribution measurements showed that FdUrd pretreatment increased [(18)F]FLT uptake in all tumors by factors of 3.2 to 7.8 compared with controls, while [(18)F]FDG tumor uptake was about fourfold and sixfold lower in breast cancers and lymphoma. Dynamic PET in FdUrd pretreated mice showed that [(18)F]FLT uptake in all tumors increased steadily up to 1.5 h. MRI showed a well-vascularized homogenous lymphoma with high [(18)F]FLT uptake, while in breast cancer, a central necrosis shown by MRI was inactive in PET, consistent with the histomorphological analysis., Conclusion: We showed a reliable and significant uptake increase of [(18)F]FLT in different tumor xenografts after low-dose FdUrd pretreatment. These results show promise for a clinical application of FdUrd aimed at increasing the sensitivity of [(18)F]FLT PET.

Human cytosolic thymidine kinase (hTK1) has proven to be a suitable target for the noninvasive imaging of cancer cell proliferation using radiolabeled thymidine analogues such as [(18)F]3'-fluoro-3'-deoxythymidine ([(18)F]FLT). A thymidine analogue for single photon emission computed tomography (SPECT), which incorporates the readily available and inexpensive nuclide technetium-99m, would be of considerable practical interest. hTK1 is known to accommodate modification of the structure of the natural substrate thymidine at the positions N3 and C3' and, to a lesser extent, C5. In this work, we used the copper-catalyzed azide-alkyne cycloaddition to synthesize two series of derivatives in which thymidine is functionalized at either the C3' or N3 position with chelating systems suitable for the M(CO)(3) core (M = (99m)Tc, Re). The click chemistry approach enabled complexes with different structures and overall charges to be synthesized from a common precursor. Using this strategy, the first organometallic hTK1 substrates in which thymidine is modified at the C3' position were identified. Phosphorylation of the organometallic derivatives was measured relative to thymidine. We have shown that the influence of the overall charge of the derivatives is dependent on the position of functionalization. In the case of the C3'-functionalized derivatives, neutral and anionic substrates were most readily phosphorylated (20-28% of the value for the parent ligand thymidine), whereas for the N3-functionalized derivatives, cationic and neutral complexes were apparently better substrates for the enzyme (14-18%) than anionic derivatives (9%).

Amyloid-beta (Abeta) peptides play a key role in the pathogenesis of Alzheimer's disease and exert various toxic effects on neurons; however, relatively little is known about their influence on glial cells. Astrocytes play a pivotal role in brain homeostasis, contributing to the regulation of local energy metabolism and oxidative stress defense, two aspects of importance for neuronal viability and function. In the present study, we explored the effects of Abeta peptides on glucose metabolism in cultured astrocytes. Following Abeta(25-35) exposure, we observed an increase in glucose uptake and its various metabolic fates, i.e., glycolysis (coupled to lactate release), tricarboxylic acid cycle, pentose phosphate pathway, and incorporation into glycogen. Abeta increased hydrogen peroxide production as well as glutathione release into the extracellular space without affecting intracellular glutathione content. A causal link between the effects of Abeta on glucose metabolism and its aggregation and internalization into astrocytes through binding to members of the class A scavenger receptor family could be demonstrated. Using astrocyte-neuron cocultures, we observed that the overall modifications of astrocyte metabolism induced by Abeta impair neuronal viability. The effects of the Abeta(25-35) fragment were reproduced by Abeta(1-42) but not by Abeta(1-40). Finally, the phosphoinositide 3-kinase (PI3-kinase) pathway appears to be crucial in these events since both the changes in glucose utilization and the decrease in neuronal viability are prevented by LY294002, a PI3-kinase inhibitor. This set of observations indicates that Abeta aggregation and internalization into astrocytes profoundly alter their metabolic phenotype with deleterious consequences for neuronal viability.

The key pathogenic event in the onset of Alzheimer's disease (AD) is the aggregation of beta-amyloid (Abeta) peptides into toxic aggregates. Molecules that interfere with this process might act as therapeutic agents for the treatment of AD. The amino acid residues 16-20 (KLVFF) are known to be essential for the aggregation of Abeta. In this study, we have used a first-generation dendrimer as a scaffold for the multivalent display of the KLVFF peptide. The effect of four KLVFF peptides attached to the dendrimer (K(4)) on Abeta aggregation was compared to the effect of monomeric KLVFF (K(1)). Our data show that K(4) very effectively inhibits the aggregation of low-molecular-weight and protofibrillar Abeta(1-42) into fibrils, in a concentration-dependent manner, and much more potently than K(1). Moreover, we show that K(4) can lead to the disassembly of existing aggregates. Our data lead us to propose that conjugates that bear multiple copies of KLVFF might be useful as therapeutic agents for the treatment of Alzheimer's disease.