Smac mimetics LCL161 and GDC-0152 inhibit osteosarcoma growth and metastasis in mice

Background Current therapies fail to cure over a third of osteosarcoma patients and around three quarters of those with metastatic disease. “Smac mimetics” (also known as “IAP antagonists”) are a new class of anti-cancer agents. Previous work revealed that cells from murine osteosarcomas were efficiently sensitized by physiologically achievable concentrations of some Smac mimetics (including GDC-0152 and LCL161) to killing by the inflammatory cytokine TNFα in vitro, but survived exposure to Smac mimetics as sole agents. Methods Nude mice were subcutaneously or intramuscularly implanted with luciferase-expressing murine 1029H or human KRIB osteosarcoma cells. The impacts of treatment with GDC-0152, LCL161 and/or doxorubicin were assessed by caliper measurements, bioluminescence, 18FDG-PET and MRI imaging, and by weighing resected tumors at the experimental endpoint. Metastatic burden was examined by quantitative PCR, through amplification of a region of the luciferase gene from lung DNA. ATP levels in treated and untreated osteosarcoma cells were compared to assess in vitro sensitivity. Immunophenotyping of cells within treated and untreated tumors was performed by flow cytometry, and TNFα levels in blood and tumors were measured using cytokine bead arrays. Results Treatment with GDC-0152 or LCL161 suppressed the growth of subcutaneously or intramuscularly implanted osteosarcomas. In both models, co-treatment with doxorubicin and Smac mimetics impeded average osteosarcoma growth to a greater extent than either drug alone, although these differences were not statistically significant. Co-treatments were also more toxic. Co-treatment with LCL161 and doxorubicin was particularly effective in the KRIB intramuscular model, impeding primary tumor growth and delaying or preventing metastasis. Although the Smac mimetics were effective in vivo, in vitro they only efficiently killed osteosarcoma cells when TNFα was supplied. Implanted tumors contained high levels of TNFα, produced by infiltrating immune cells. Spontaneous osteosarcomas that arose in genetically-engineered immunocompetent mice also contained abundant TNFα. Conclusions These data imply that Smac mimetics can cooperate with TNFα secreted by tumor-associated immune cells to kill osteosarcoma cells in vivo. Smac mimetics may therefore benefit osteosarcoma patients whose tumors contain Smac mimetic-responsive cancer cells and TNFα-producing infiltrating cells

18F Site-Specific Labelling of a Single-Chain Antibody against Activated Platelets for the Detection of Acute Thrombosis in Positron Emission Tomography

Positron emission tomography is the imaging modality of choice when it comes to the high sensitivity detection of key markers of thrombosis and inflammation, such as activated platelets. We, previously, generated a fluorine-18 labelled single-chain antibody (scFv) against ligand-induced binding sites (LIBS) on activated platelets, binding it to the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. We used a non-site-specific bio conjugation approach with N-succinimidyl-4-[18F]fluorobenzoate (S[18F]FB), leading to a mixture of products with reduced antigen binding. In the present study, we have developed and characterised a novel fluorine-18 PET radiotracer, based on this antibody, using site-specific bio conjugation to engineer cysteine residues with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). ScFvanti-LIBS and control antibody mut-scFv, with engineered C-terminal cysteine, were reduced, and then, they reacted with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). Radiolabelled scFv was injected into mice with FeCl3-induced thrombus in the left carotid artery. Clots were imaged in a PET MR imaging system, and the amount of radioactivity in major organs was measured using an ionisation chamber and image analysis. Assessment of vessel injury, as well as the biodistribution of the radiolabelled scFv, was studied. In the in vivo experiments, we found uptake of the targeted tracer in the injured vessel, compared with the non-injured vessel, as well as a high uptake of both tracers in the kidney, lung, and muscle. As expected, both tracers cleared rapidly via the kidney. Surprisingly, a large quantity of both tracers was taken up by organs with a high glutathione content, such as the muscle and lung, due to the instability of the maleimide cysteine bond in vivo, which warrants further investigations. This limits the ability of the novel antibody radiotracer 18F-scFvanti-LIBS to bind to the target in vivo and, therefore, as a useful agent for the sensitive detection of activated platelets. We describe the first fluorine-18 variant of the scFvanti-LIBS against activated platelets using site-specific bio conjugation

Effect of structural modifications to Glyoxal-bis(thiosemicarbazonato)copper(II) complexes on cellular copper uptake, copper-mediated ATP7A trafficking, and P-Glycoprotein mediated efflux

A 1-month field campaign of ozone (O3) flux measurements along a five-level vertical profile above, inside and below the canopy was run in a mature broadleaf forest of the Po Valley, northern Italy. The study aimed to characterize O3 flux dynamics and their interactions with nitrogen oxides (NOx) fluxes from the forest soil and the atmosphere above the canopy. Ozone fluxes measured at the levels above the canopy were in good agreement, thus confirming the validity of the constant flux hypothesis, while below-canopy O3 fluxes were lower than above. However, at the upper canopy edge O3 fluxes were surprisingly higher than above during the morning hours. This was attributed to a chemical O3 sink due to a reaction with the nitric oxide (NO) emitted from soil and deposited from the atmosphere, thus converging at the top of the canopy. Moreover, this mechanism was favored by the morning coupling between the forest and the atmosphere, while in the afternoon the fluxes at the upper canopy edge became similar to those of the levels above as a consequence of the in-canopy stratification. Nearly 80 % of the O3 deposited to the forest ecosystem was removed by the canopy by stomatal deposition, dry deposition on physical surfaces and by ambient chemistry reactions (33.3 % by the upper canopy layer and 46.3 % by the lower canopy layer). Only a minor part of O3 was removed by the understorey vegetation and the soil surface (2 %), while the remaining 18.2 % was consumed by chemical reaction with NO emitted from soil. The collected data could be used to improve the O3 risk assessment for forests and to test the predicting capability of O3 deposition models. Moreover, these data could help multilayer canopy models to separate the influence of ambient chemistry vs. O3 dry deposition on the observed fluxes

Engineering anti-Lewis-Y hu3S193 antibodies with improved therapeutic ratio for radioimmunotherapy of epithelial cancers

BACKGROUND: The aim of the study was to explore Fc mutations of a humanised anti-Lewis-Y antibody (IgG1) hu3S193 as a strategy to improve therapeutic ratios for therapeutic payload delivery. METHODS: Four hu3S193 variants (I253A, H310A, H435A and I253A/H310A) were generated via site-directed mutagenesis and radiolabelled with diagnostic isotopes iodine-125 or indium-111. Biodistribution studies in Lewis-Y-positive tumour-bearing mice were used to calculate the dose in tumours and organs for therapeutic isotopes (iodine-131, yttrium-90 and lutetium-177). RESULTS: (111)In-labelled I253A and H435A showed similar slow kinetics (t(1/2β), 63.2 and 62.2 h, respectively) and a maximum tumour uptake of 33.11 ± 4.05 and 33.69 ± 3.77 percentage injected dose per gramme (%ID/g), respectively. (111)In-labelled I253A/H310A cleared fastest (t(1/2β), 9.1 h) with the lowest maximum tumour uptake (23.72 ± 0.85 %ID/g). The highest increase in tumour-to-blood area under the curve (AUC) ratio was observed with the metal-labelled mutants ((90)Y and (177)Lu). (177)Lu-CHX-A" DTPA-hu3S193 I253A/H310A (6:1) showed the highest tumour-to-blood AUC ratio compared to wild type (3:1) and other variants and doubling of calculated dose to tumour based on red marrow dose constraints. CONCLUSIONS: These results suggest that hu3S193 Fc can be engineered with improved therapeutic ratios for (90)Y- and (177)Lu-based therapy, with the best candidate being hu3S193 I253A/H310A for (177)Lu-based therapy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13550-016-0180-0) contains supplementary material, which is available to authorized users

18 F Site‐Specific Labelling of a Single‐Chain Antibody against Activated Platelets for the Detection of Acute Thrombosis in Positron Emission Tomography

Positron emission tomography is the imaging modality of choice when it comes to the high sensitivity detection of key markers of thrombosis and inflammation, such as activated platelets. We, previously, generated a fluorine‐18 labelled single‐chain antibody (scFv) against ligand‐induced binding sites (LIBS) on activated platelets, binding it to the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. We used a non‐site‐specific bio conjugation approach with N‐succinimidyl‐ 4‐[18F]fluorobenzoate (S[18F]FB), leading to a mixture of products with reduced antigen binding. In the present study, we have developed and characterised a novel fluorine‐18 PET radiotracer, based on this antibody, using site‐specific bio conjugation to engineer cysteine residues with N‐[2‐(4‐ [18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). ScFvanti‐LIBS and control antibody mut‐scFv, with engineered C‐terminal cysteine, were reduced, and then, they reacted with N‐[2‐(4‐ [18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). Radiolabelled scFv was injected into mice with FeCl3‐induced thrombus in the left carotid artery. Clots were imaged in a PET MR imaging system, and the amount of radioactivity in major organs was measured using an ionisation chamber and image analysis. Assessment of vessel injury, as well as the biodistribution of the radiolabelled scFv, was studied. In the in vivo experiments, we found uptake of the targeted tracer in the injured vessel, compared with the non‐injured vessel, as well as a high uptake of both tracers in the kidney, lung, and muscle. As expected, both tracers cleared rapidly via the kidney. Surprisingly, a large quantity of both tracers was taken up by organs with a high glutathione content, such as the muscle and lung, due to the instability of the maleimide cysteine bond in vivo, which warrants further investigations. This limits the ability of the novel antibody radiotracer18F‐scFvanti‐LIBS to bind to the target in vivo and, therefore, as a useful agent for the sensitive detection of activated platelets. We describe the first fluorine‐18 variant of the scFvanti‐LIBS against activated platelets using site‐specific bio conjugation

Additional file 1: of Engineering anti-Lewis-Y hu3S193 antibodies with improved therapeutic ratio for radioimmunotherapy of epithelial cancers

Figures S1-S3; Tables S1-S3. (DOC 1691 kb

Toward Hypoxia-Selective Rhenium and Technetium Tricarbonyl Complexes

With the aim of preparing hypoxia-selective imaging and therapeutic agents, technetium­(I) and rhenium­(I) tricarbonyl complexes with pyridylhydrazone, dipyridylamine, and pyridylaminocarboxylate ligands containing nitrobenzyl or nitroimidazole functional groups have been prepared. The rhenium tricarbonyl complexes were synthesized with short reaction times using microwave irradiation. Rhenium tricarbonyl complexes with deprotonated <i>p</i>-nitrophenyl pyridylhydrazone ligands are luminescent, and this has been used to track their uptake in HeLa cells using confocal fluorescent microscopy. Selected rhenium tricarbonyl complexes displayed higher uptake in hypoxic cells when compared to normoxic cells. A ^99mTc tricarbonyl complex with a dipyridylamine ligand bearing a nitroimidazole functional group is stable in human serum and was shown to localize in a human renal cell carcinoma (RCC; SK-RC-52) tumor in a mouse

l-Tyrosine Confers Residualizing Properties to a d-Amino Acid-Rich Residualizing Peptide for Radioiodination of Internalizing Antibodies

Purpose: The aims of the study were to develop and evaluate a novel residualizing peptide for labeling internalizing antibodies with 124I to support clinical development using immuno-positron emission tomography (PET). Methods: The anti-epidermal growth factor receptor antibody ch806 was radiolabeled directly or indirectly with isotopes and various residualizing peptides. Azido-derivatized radiolabeled peptides were conjugated to dibenzylcyclooctyne-derivatized ch806 antibody via click chemistry. The radiochemical purities, antigen-expressing U87MG.de2-7 human glioblastoma cell-binding properties, and targeting of xenografts at 72 hours post injection of all radioconjugates were compared. Biodistribution of 124I-PEG4-tptddYddtpt-ch806 and immuno-PET imaging were evaluated in tumor-bearing mice. Results: Biodistribution studies using xenografts at 72 hours post injection showed that 131I-PEG4-tptddYddtpt-ch806 tumor uptake was similar to 111In-CHX-A″-DTPA-ch806. 125I-PEG4-tptddyddtpt-ch806 showed a lower tumor uptake value but higher than directly labeled 125I-ch806. 124I-PEG4-tptddYddtpt-ch806 was produced at 23% labeling efficiency, 98% radiochemical purity, 25.9 MBq/mg specific activity, and 64% cell binding in the presence of antigen excess. Tumor uptake for 124I-PEG4-tptddYddtpt-ch806 was similar to 111In-CHX-A″-DTPA-ch806. High-resolution immuno-PET/magnetic resonance imaging of tumors showed good correlation with biodistribution data. Conclusions: The mixed d/l-enantiomeric peptide, dThr-dPro-dThr-dAsp-dAsp-Tyr-dAsp-dAsp-dThr-dPro-dThr, is suitable for radiolabeling antibodies with radiohalogens such as 124I for high-resolution immuno-PET imaging of tumors and for evaluation in early-phase clinical trials