Explore the vast range of titles available.

The successful in vivo implementation of gene expression modulation strategies relies on effective, non-immunogenic delivery vehicles. Lipid nanoparticles are one of the most advanced non-viral clinically approved nucleic-acid delivery systems. Yet lipid nanoparticles accumulate naturally in liver cells upon intravenous administration, and hence, there is an urgent need to enhance uptake by other cell types. Here we use a conformation-sensitive targeting strategy to achieve in vivo gene silencing in a selective subset of leukocytes and show potential therapeutic applications in a murine model of colitis. In particular, by targeting the high-affinity conformation of α4β7 integrin, which is a hallmark of inflammatory gut-homing leukocytes, we silenced interferon-γ in the gut, resulting in an improved therapeutic outcome in experimental colitis. The lipid nanoparticles did not induce adverse immune activation or liver toxicity. These results suggest that our lipid nanoparticle targeting strategy might be applied for selective delivery of payloads to other conformation-sensitive targets.While targeted lipid nanoparticles might allow partial delivery of genetic materials to non-hepatic cells, the selectivity of this approach is still unsatisfying. Here the authors functionalize their lipid nanoparticles with a targeting moiety that recognizes a protein conformation specific to gut-homing leukocytes, inducing gene silencing exclusively in this cellular subset and providing a potential therapeutic strategy for inflammatory bowel disease.

BackgroundA major challenge to the long-term success of neuroblastoma therapy is widespread metastases that survive initial therapy as minimal residual disease (MRD). The SSTR2 receptor is expressed by most neuroblastoma tumors making it an attractive target for molecularly targeted radionuclide therapy. SARTATE consists of octreotate, which targets the SSTR2 receptor, conjugated to MeCOSar, a bifunctional chelator with high affinity for copper. Cu-SARTATE offers the potential to both detect and treat neuroblastoma MRD by using [64Cu]Cu-SARTATE to detect and monitor the disease and [67Cu]Cu-SARTATE as the companion therapeutic agent. In the present study, we tested this theranostic pair in a preclinical model of neuroblastoma MRD. An intrahepatic model of metastatic neuroblastoma was established using IMR32 cells in nude mice. The biodistribution of [64Cu]Cu-SARTATE was measured using small-animal PET and ex vivo tissue analysis. Survival studies were carried out using the same model: mice (6–8 mice/group) were given single doses of saline, or 9.25 MBq (250 µCi), or 18.5 MBq (500 µCi) of [67Cu]Cu-SARTATE at either 2 or 4 weeks after tumor cell inoculation.ResultsPET imaging and ex vivo biodistribution confirmed tumor uptake of [64Cu]Cu-SARTATE and rapid clearance from other tissues. The major clearance tissues were the kidneys (15.6 ± 5.8% IA/g at 24 h post-injection, 11.5 ± 2.8% IA/g at 48 h, n = 3/4). Autoradiography and histological analysis confirmed [64Cu]Cu-SARTATE uptake in viable, SSTR2-positive tumor regions with mean tumor uptakes of 14.1–25.0% IA/g at 24 h. [67Cu]Cu-SARTATE therapy was effective when started 2 weeks after tumor cell inoculation, extending survival by an average of 13 days (30%) compared with the untreated group (mean survival of control group 43.0 ± 8.1 days vs. 55.6 ± 9.1 days for the treated group; p = 0.012). No significant therapeutic effect was observed when [67Cu]Cu-SARTATE was started 4 weeks after tumor cell inoculation, when the tumors would have been larger (control group 14.6 ± 8.5 days; 9.25 MBq group 9.5 ± 1.6 days; 18.5 MBq group 15.6 ± 4.1 days; p = 0.064).ConclusionsClinical experiences of peptide-receptor radionuclide therapy for metastatic disease have been encouraging. This study demonstrates the potential for a theranostic approach using [64/67Cu]Cu-SARTATE for the detection and treatment of SSTR2-positive neuroblastoma MRD.

The immune system’s ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition. SynTacs, when labeled with positron-emitting isotopes, can noninvasively image antigen-specific CD8 + T cells in vivo. Using radiolabeled synTacs loaded with the appropriate peptides, we imaged human papillomavirus-specific CD8 + T cells by positron emission tomography in mice bearing human papillomavirus-positive tumors, as well as influenza A virus–specific CD8 + T cells in the lungs of influenza A virus–infected mice. It is thus possible to visualize antigen-specific CD8 + T-cell populations in vivo, which may serve prognostic and diagnostic roles. Antigen-specific CD8 + T cells can be imaged by immunoPET with the help of synTacs, MHC-based tools that bind to relevant T-cell receptors.

Positron emission tomography combined with a specific probe presents the ability to noninvasively assess inflammatory bowel disease. We previously reported increased intestinal uptake of a 64Cu-labeled anti-β7 integrin antibody (clone FIB504.64) in colitic mice. Here, we evaluated an anti-α4β7 integrin antibody (clone DATK32), and the F(ab′)2 and Fab fragments of the anti-β7 antibody, which should have faster blood clearance than the intact antibody, as imaging probes for the detection of colitis in a mouse model.MethodsThe immunoproteins were labeled with 64Cu, injected into mice with dextran sodium sulphate-induced colitis. Positron emission tomography data were collected between 1 and 48 hours postinjection.ResultsFocal uptake of the anti-β7 fragments was observed in the gut as early as 1 hour postinjection, and they cleared more rapidly from normal tissues than the whole antibody. For example, the blood concentrations at 24 hours postinjection were 23.3 ± 3.0% ID/g for 64Cu-labeled DATK32, 12.9 ± 2.1% ID/g for FIB504.64, 4.1 ± 0.4% ID/g for FIB504.64-F(ab′)2, and 0.62 ± 0.2% ID/g for FIB504.64-Fab (P < 0.0001, analysis of variance). The ratio of uptake of DATK32 between the colitis and control groups in the large intestine (1.38) was lower than for the FIB504.64 fragments (3.15 for F(ab′)2, 1.84 for Fab) or intact FIB504.64 (1.78).ConclusionsThe lower intestinal uptake ratio of the 64Cu-labeled anti-α4β7 antibody (DATK32) compared with the anti-β7 immunoproteins suggests that targeting all β7-expressing lymphocytes, not just those expressing α4β7, is a more promising route to the development of an inflammatory bowel disease imaging agent. The FIB504.64-F(ab′)2 fragment demonstrated the greatest differential between colitis and control groups, and is therefore the most promising lead molecule for the development of an inflammatory bowel disease-specific imaging agent.

The well-known perfusion tracer CuPTSM, labelled with 62Cu or 64Cu, is believed to be trapped in cells non-selectively by a bioreductive mechanism. It is proposed that by modifying the ligand to increase its electron donor strength (for example by adding alkyl functionality or replacing sulphur ligands with oxygen ligands), the copper complexes will become less easily reduced and tracers with selectivity for hypoxic tissues could thus be developed. The aim of this work was to prepare 64Cu-labelled complexes of two series of ligands, based on the bis(thiosemicarbazone) (13 ligands) and bis(salicylaldimine) (3 ligands) skeletons, and to evaluate the hypoxia dependence of their uptake in cells. The complexes were incubated with Chinese hamster ovary cells under normoxic and hypoxic conditions, and the cells isolated by centrifugation to determine radioactivity uptake at various time points up to 90 min. Several members of both series demonstrated significant (P<0.05) or highly significant (P<0.01) hypoxia selectivity, indicating that both series of complexes offer a basis for development of hypoxia-targeting radiopharmaceuticals for positron emission tomography (60Cu, 61Cu, 62Cu, 64Cu) and targeted radiotherapy (64Cu, 67Cu).

Fluorine-18 fluoro-2-deoxyglucose ((18)FDG) and carbon-14 2-deoxyglucose ((14)C-2-DG) are both widely used tracers of myocardial glucose uptake and phosphorylation. We have recently shown, using positron emission tomography (PET) and nuclear magnetic resonance, that ischaemia-reperfusion (I-R) causes differential changes in their uptake. We describe here the novel application of an autoradiographic technique allowing the investigation of this phenomenon at high resolution, using tracer concentrations of both analogues in the dual-perfused isolated rat heart. We also investigate the importance of glucose transporter (GLUT 1 and GLUT 4) distribution in governing the observed phosphorylated analogue accumulation. Hearts ( n=5) were perfused with Krebs buffer for 40 min, made regionally zero-flow ischaemic for 40 min and reperfused for 60 min with Krebs containing tracer (18)FDG (200 MBq) and tracer (14)C-2-DG (0.37 MBq). Hearts were then frozen and five sections (10 micro m) were cut per heart, fixed and exposed on phosphor storage plates for 18 h (for (18)FDG) and then for a further 9 days (for (14)C-2-DG). Quantitative digital images of tracer accumulation were obtained using a phosphor plate reader. The protocol was repeated in a second group of hearts and GLUT 1 and GLUT 4 distribution analysed. Post-ischaemic accumulation of (18)FDG-6-P was inhibited by 38.2%+/-1.7% and (14)C-DG-6-P by 19.0%+/-2.2%, compared with control ( P<0.05). After placing seven \"lines of interrogation\" across each heart section and analysing the phosphorylated tracer accumulation along them, a transmural gradient of both tracers was observed; this was highest at the endocardium and lowest at the epicardium. GLUT 4 translocated to the sarcolemma in the ischaemic/reperfused region (from 24%+/-3% to 59%+/-5%), while there was no cellular redistribution of GLUT 1. We conclude that since decreased phosphorylated tracer accumulation occurs after ischaemia-reperfusion, despite greater externalisation of GLUT 4, hexokinase or the affinities of the GLUT transporters are changed under these conditions.

The advancement of positron emission tomography (PET) depends on the development of new radiotracers that will complement ¹⁸F-FDG. Copper-64 (⁶⁴Cu) is a promising PET radionuclide, particularly for antibody-targeted imaging, but the high in vivo lability of conventional chelates has limited its clinical application. The objective of this work was to evaluate the novel chelating agent SarAr (1-N-(4-aminobenzyl)-3, 6,10,13,16,19-hexaazabicyclo[6.6.6] eicosane-1,8-diamine) for use in developing a new class of tumor-specific ⁶⁴Cu radiopharmaceuticals for imaging neuroblastoma and melanoma. The anti-GD2 monoclonal antibody (mAb) 14.G2a, and its chimeric derivative, ch14.18, target disialogangliosides that are overexpressed on neuroblastoma and melanoma. Both mAbs were conjugated to SarAr using carbodiimide coupling. Radiolabeling with ⁶⁴Cu resulted in >95% of the ⁶⁴Cu being chelated by the immunoconjugate. Specific activities of at least 10 μCi/μg (1 Ci = 37 GBq) were routinely achieved, and no additional purification was required after ⁶⁴Cu labeling. Solid-phase radioimmunoassays and intact cell-binding assays confirmed retention of bioactivity. Biodistribution studies in athymic nude mice bearing s.c. neuroblastoma (IMR-6, NMB-7) and melanoma (M21) xenografts showed that 15-20% of the injected dose per gram accumulated in the tumor at 24 hours after injection, and only 5-10% of the injected dose accumulated in the liver, a lower value than typically seen with other chelators. Uptake by a GD2-negative tumor xenograft was significantly lower (<5% injected dose per gram). MicroPET imaging confirmed significant uptake of the tracer in GD-2-positive tumors, with minimal uptake in GD-2-negative tumors and nontarget tissues such as liver. The ⁶⁴Cu-SarAr-mAb system described here is potentially applicable to ⁶⁴Cu-PET imaging with a broad range of antibody or peptide-based imaging agents.

Experience with imaging of the multi-drug resistance (MDR) phenotype in tumours using technetium-99m sestamibi, a substrate of the P-glycoprotein (Pgp) transporter, suggests that better quantification of images and separation of MDR from other variables affecting tracer uptake in tumours are required. One approach to these problems is the development of short half-life positron-emitting tracers which are substrates of Pgp. Several lipophilic cationic copper(I) bis(diphosphine) complexes labelled with copper-64 have been synthesised and evaluated in vitro as substrates for Pgp. The synthesis is rapid and efficient with no need for purification steps. The chemistry is suitable for use with very short half-life radionuclides such as copper-62 (9.7 min) and copper-60 (23.7 min). Incubation of the complexes with human serum in vitro showed that they are sufficiently stable in serum to support clinical imaging, and the more lipophilic members of the series are taken up rapidly by cells (Chinese hamster ovary and human ovarian carcinoma) in vitro with great avidity. Uptake in human ovarian carcinoma cells is significantly reduced after several months of conditioning in the presence of doxorubicin, which induces increased Pgp expression. Uptake in hooded rat sarcoma (HSN) cells, which express Pgp, is significantly increased in the presence of the MDR modulator cyclosporin A. Biodistribution studies in hooded rats show rapid blood clearance, excretion through both kidneys and liver, and low uptake in other tissues. The one complex investigated in HSN tumour-bearing rats showed uptake in tumour increasing up to 30 min p.i. while it was decreasing in other tissues. We conclude that diphosphine ligands offer a good basis for development of radiopharmaceuticals containing copper radionuclides, and that this series of complexes should undergo further evaluation in vivo as positron emission tomography imaging agents for MDR.

Flynn, A. A., Pedley, R. B., Green, A. J., Dearling, J. L., El-Emir, E., Boxer, G. M., Boden, R. and Begent, R. H. J. The Nonuniformity of Antibody Distribution in the Kidney and its Influence on Dosimetry. Radiat. Res. 159, 182–189 (2003). The therapeutic efficacy of radiolabeled antibody fragments can be limited by nephrotoxicity, particularly when the kidney is the major route of extraction from the circulation. Conventional dose estimates in kidney assume uniform dose deposition, but we have shown increased antibody localization in the cortex after glomerular filtration. The purpose of this study was to measure the radioactivity in cortex relative to medulla for a range of antibodies and to assess the validity of the assumption of uniformity of dose deposition in the whole kidney and in the cortex for these antibodies with a range of radionuclides. Storage phosphor plate technology (radioluminography) was used to acquire images of the distributions of a range of antibodies of various sizes, labeled with 125I, in kidney sections. This allowed the calculation of the antibody concentration in the cortex relative to the medulla. Beta-particle point dose kernels were then used to generate the dose-rate distributions from 14C, 131I, 186Re, 32P and 90Y. The correlation between the actual dose-rate distribution and the corresponding distribution calculated assuming uniform antibody distribution throughout the kidney was used to test the validity of estimating dose by assuming uniformity in the kidney and in the cortex. There was a strong inverse relationship between the ratio of the radioactivity in the cortex relative to that in the medulla and the antibody size. The nonuniformity of dose deposition was greatest with the smallest antibody fragments but became more uniform as the range of the emissions from the radionuclide increased. Furthermore, there was a strong correlation between the actual dose-rate distribution and the distribution when assuming a uniform source in the kidney for intact antibodies along with medium- to long-range radionuclides, but there was no correlation for small antibody fragments with any radioisotope or for short-range radionuclides with any antibody. However, when the cortex was separated from the whole kidney, the correlation between the actual dose-rate distribution and the assumed dose-rate distribution, if the source was uniform, increased significantly. During radioimmunotherapy, the extent of nonuniformity of dose deposition in the kidney depends on the properties of the antibody and radionuclide. For dosimetry estimates, the cortex should be taken as a separate source region when the radiopharmaceutical is small enough to be filtered by the glomerulus.

The hu14.18K322A variant of the GD2-targeting antibody hu14.18 has been shown to elicit a level of antibody-dependent cell-mediated cytotoxicity toward human neuroblastoma cells similar to that of the parent antibody. However, hu14.18K322A exhibited a decreased complement activation and associated pain, the dose-limiting toxicity in neuroblastoma immunotherapy. PET with a radiolabeled analog of the same antibody used in treatment will provide insight into the ability of hu14.18K322A to reach its target, as well as nontarget uptake that may cause side effects. Such antibody radiotracers might also provide a method for measuring GD2 expression in tumors, thus enabling the prediction of response to anti-GD2 therapy for individual patients. Methods: The conjugation of hu14.18K322A with p-NH^sub 2^-Bn-DOTA was accomplished using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide with subsequent ^sup 64^Cu radiolabeling at 37°C for 30 min. Immunoreactivity of the conjugate was assessed by a dose-escalation blocking experiment measuring binding to purified GD2 versus GD1b as a negative control. Cell uptake and biodistribution studies in M21 (GD2-positive) and PC-3 (GD2-negative) tumor models were performed, as was small-animal PET/CT of M21 and PC-3 tumor-bearing mice. Results: The labeling of ^sup 64^Cu-p-NH^sub 2^-Bn-DOTA-hu14.18K322A was achieved at more than 95% radiochemical purity and a specific activity of 127-370 MBq/mg (3.4-10 mCi/mg) after chromatographic purification. Preliminary in vitro data demonstrated a greater than 6-fold selectivity of binding to GD2 versus GD1b and dose-dependent inhibition of binding by unmodified hu14.8K322A. In vivo data, including small-animal PET/CT, showed significant GD2-positive tumor-targeting ability, with a persistent 2-fold-higher uptake of radiotracer than in GD2-negative tumors. Conclusion: ^sup 64^Cu-p-NH^sub 2^-Bn-DOTA-hu14.18K322A represents a novel PET radiotracer to facilitate clinical investigations of anti-GD2 immunotherapies and to complement other imaging modalities in the staging and treatment of neuroblastoma. [PUBLICATION ABSTRACT]