An immunologically relevant rodent model demonstrates safety of therapy using a tumour-specific IgE.

BACKGROUND: Designing biologically informative models for assessing the safety of novel agents, especially for cancer immunotherapy, carries substantial challenges. The choice of an in vivo system for studies on IgE antibodies represents a major impediment to their clinical translation, especially with respect to class-specific immunological functions and safety. Fcε receptor expression and structure are different in humans and mice, so that the murine system is not informative when studying human IgE biology. By contrast, FcεRI expression and cellular distribution in rats mirror that of humans. METHODS: We are developing MOv18 IgE, a human chimeric antibody recognizing the tumour-associated antigen folate receptor alpha. We created an immunologically congruent surrogate rat model likely to recapitulate human IgE-FcεR interactions and engineered a surrogate rat IgE equivalent to MOv18. Employing this model, we examined in vivo safety and efficacy of antitumour IgE antibodies. RESULTS: In immunocompetent rats, rodent IgE restricted growth of syngeneic tumours in the absence of clinical, histopathological or metabolic signs associated with obvious toxicity. No physiological or immunological evidence of a "cytokine storm" or allergic response was seen, even at 50 mg/kg weekly doses. IgE treatment was associated with elevated serum concentrations of TNFα, a mediator previously linked with IgE-mediated antitumour and antiparasitic functions, alongside evidence of substantially elevated tumoural immune cell infiltration and immunological pathway activation in tumour-bearing lungs. CONCLUSION: Our findings indicate safety of MOv18 IgE, in conjunction with efficacy and immune activation, supporting the translation of this therapeutic approach to the clinical arena.

Imaging tumour hypoxia with positron emission tomography.

Hypoxia, a hallmark of most solid tumours, is a negative prognostic factor due to its association with an aggressive tumour phenotype and therapeutic resistance. Given its prominent role in oncology, accurate detection of hypoxia is important, as it impacts on prognosis and could influence treatment planning. A variety of approaches have been explored over the years for detecting and monitoring changes in hypoxia in tumours, including biological markers and noninvasive imaging techniques. Positron emission tomography (PET) is the preferred method for imaging tumour hypoxia due to its high specificity and sensitivity to probe physiological processes in vivo, as well as the ability to provide information about intracellular oxygenation levels. This review provides an overview of imaging hypoxia with PET, with an emphasis on the advantages and limitations of the currently available hypoxia radiotracers.

PET and SPECT imaging of a radiolabeled minigastrin analogue conjugated with DOTA, NOTA, and NODAGA and labeled with (64)Cu, (68)Ga, and (111)In.

Cholecystokinin-2 (CCK-2) receptors, overexpressed in cancer types such as small cell lung cancers (SCLC) and medullary thyroid carcinomas (MTC), may serve as targets for peptide receptor radionuclide imaging. A variety of CCK and gastrin analogues has been developed, but a major drawback is metabolic instability or high kidney uptake. The minigastrin analogue PP-F11 has previously been shown to be a promising peptide for imaging of CCK-2 receptor positive tumors and was therefore further evaluated. The peptide was conjugated with one of the macrocyclic chelators DOTA, NOTA, or NODAGA. The peptide conjugates were then radiolabeled with either (68)Ga, (64)Cu, or (111)In. All (radio)labeled compounds were evaluated in vitro (IC50) and in vivo (biodistribution and PET/CT and SPECT/CT imaging). IC50 values were in the low nanomolar range for all compounds (0.79-1.51 nM). In the biodistribution studies, (68)Ga- and (111)In-labeled peptides showed higher tumor-to-background ratios than the (64)Cu-labeled compounds. All tested radiolabeled compounds clearly visualized the CCK2 receptor positive tumor in PET or SPECT imaging. The chelator did not seem to affect in vivo behavior of the peptide for (111)In- and (68)Ga-labeled peptides. In contrast, the biodistribution of the (64)Cu-labeled peptides showed high uptake in the liver and in other organs, most likely caused by high blood levels, probably due to dissociation of (64)Cu from the chelator and subsequent transchelation to proteins. Based on the present study, (68)Ga-DOTA-PP-F11 might be a promising radiopharmaceutical for PET/CT imaging of CCK2 receptor expressing tumors such as MTC and SCLC. Clinical studies are warranted to investigate the potential of this tracer.

PET imaging of cardiac hypoxia: opportunities and challenges.

Myocardial hypoxia is a major factor in the pathology of cardiac ischemia and myocardial infarction. Hypoxia also occurs in microvascular disease and cardiac hypertrophy, and is thought to be a prime determinant of the progression to heart failure, as well as the driving force for compensatory angiogenesis. The non-invasive delineation and quantification of hypoxia in cardiac tissue therefore has the potential to be an invaluable experimental, diagnostic and prognostic biomarker for applications in cardiology. However, at this time there are no validated methodologies sufficiently sensitive or reliable for clinical use. PET imaging provides real-time spatial information on the biodistribution of injected radiolabeled tracer molecules. Its inherent high sensitivity allows quantitative imaging of these tracers, even when injected at sub-pharmacological (≥pM) concentrations, allowing the non-invasive investigation of biological systems without perturbing them. PET is therefore an attractive approach for the delineation and quantification of cardiac hypoxia and ischemia. In this review we discuss the key concepts which must be considered when imaging hypoxia in the heart. We summarize the PET tracers which are currently available, and we look forward to the next generation of hypoxia-specific PET imaging agents currently being developed. We describe their potential advantages and shortcomings compared to existing imaging approaches, and what is needed in terms of validation and characterization before these agents can be exploited clinically.

SPECT/CT lymphoscintigraphy of heterotopic cardiac grafts reveals novel sites of lymphatic drainage and T cell priming.

The normal function of lymphatic vessels is to facilitate the trafficking of antigen presenting cells to draining lymph nodes where they evoke an immune response. Donor lymphatic vessels are not connected to that of recipients' during organ transplantation. The pathophysiology of this disruption has received little attention. Murine heterotopic cardiac transplantation has been used extensively in transplantation research. Following vascularized organ transplantation, the main site of allosensitization is thought to be in the spleen of the recipient as a result of migration of donor passenger leukocytes via blood. Here, using Single Photon Emission Computed Tomography/Computerized Tomography (SPECT/CT) lymphoscintigraphy, we studied the pattern of lymphatic flow from mouse heterotopic abdominal cardiac grafts and identified mediastinal lymph nodes as the draining nodes for the donor graft. Staining with HY tetramer after transplantation of HY mismatched heart grafts and ELISPOT following allogeneic grafts to detect donor specific T cells revealed them as important sites for allosensitization. Our data indicates that mediastinal lymph nodes play a crucial role in the alloimmune response in this model, and should be used for ex vivo and adoptive transfer studies after transplantation in addition to the spleen.

Harnessing engineered antibodies of the IgE class to combat malignancy: initial assessment of FcɛRI-mediated basophil activation by a tumour-specific IgE antibody to evaluate the risk of type I hypersensitivity.

BACKGROUND: IgE antibodies, sequestered into tissues and retained locally by the high-affinity IgE receptor, FcɛRI, on powerful effector cells such as mast cells, macrophages and eosinophils, may offer improvements in the therapy of solid tumours. The chimeric antibody, MOv18 IgE, against the human ovarian carcinoma antigen, folate receptor α (FRα), is more effective than its IgG1 counterpart in xenograft models of ovarian cancer. Although MOv18 IgE binds to a single epitope on FRα and cannot cross-link IgE receptors on basophils, there remains a risk that components in the circulation of ovarian cancer patients might cross-link FRα-MOv18-IgE-receptor-FcɛRI complexes on basophils to cause type I hypersensitivity. OBJECTIVE: To assess the propensity for MOv18 used in a therapeutic setting to cause FcɛRI-mediated type I hypersensitivity. METHODS: As validated readouts of the potential for MOv18 to cause FcɛRI-mediated type I hypersensitivity we measured release of a granule-stored mediator from a rat basophilic leukaemia cell line RBL SX-38 stably transfected with human tetrameric (αßγ2) FcɛRI, and induction of CD63 on blood basophils from patients with ovarian carcinoma and healthy controls ex vivo. RESULTS: Serum FRα levels were increased in ovarian cancer patients compared with healthy controls. MOv18 IgE alone, or in the presence of its antigen recombinant human FRα, or of healthy volunteer (n=14) or ovarian carcinoma patient (n=32) sera, did not induce RBL SX-38 cell degranulation. Exposure to FRα-expressing ovarian tumour cells at target-to-effector ratios expected within tumours induced degranulation. MOv18 IgE did not induce expression of CD63 in blood basophils from either healthy volunteers (n=6), or cancer patients, despite detectable levels of circulating FRα (n=5). CONCLUSION AND CLINICAL RELEVANCE: These encouraging data are compatible with the hypothesis that, when ovarian carcinoma patients are treated with MOv18, FcɛRI-mediated activation of effector cells occurs within the tumour mass but not in the circulation mandating, with due caution, further pre-clinical studies.

[18F]tetrafluoroborate-PET/CT enables sensitive tumor and metastasis in vivo imaging in a sodium iodide symporter-expressing tumor model.

Cancer cell metastasis is responsible for most cancer deaths. Non-invasive in vivo cancer cell tracking in spontaneously metastasizing tumor models still poses a challenge requiring highest sensitivity and excellent contrast. The goal of this study was to evaluate if the recently introduced PET radiotracer [18F]tetrafluoroborate ([18F]BF4-) is useful for sensitive and specific metastasis detection in an orthotopic xenograft breast cancer model expressing the human sodium iodide symporter (NIS) as a reporter. In vivo imaging was complemented by ex vivo fluorescence microscopy and γ-counting of harvested tissues. Radionuclide imaging with [18F]BF4- (PET/CT) was compared to the conventional tracer [123I]iodide (sequential SPECT/CT). We found that [18F]BF4- was superior due to better pharmacokinetics, i.e. faster tumor uptake and faster and more complete clearance from circulation. [18F]BF4--PET was also highly specific as in all detected tissues cancer cell presence was confirmed microscopically. Undetected comparable tissues were similarly found to be free of metastasis. Metastasis detection by routine metabolic imaging with [18F]FDG-PET failed due to low standard uptake values and low contrast caused by adjacent metabolically active organs in this model. [18F]BF4--PET combined with NIS expressing disease models is particularly useful whenever preclinical in vivo cell tracking is of interest.

Al(OH)3 facilitated synthesis of water-soluble, magnetic, radiolabelled and fluorescent hydroxyapatite nanoparticles.

Magnetic and fluorescent hydroxyapatite nanoparticles were synthesised using Al(OH)3-stabilised MnFe2O4 or Fe3O4 nanoparticles as precursors. They were readily and efficiently radiolabelled with (18)F. Bisphosphonate polyethylene glycol polymers were utilised to endow the nanoparticles with excellent colloidal stability in water and to incorporate cyclam for high affinity labelling with (64)Cu.

Low-doses of ionising radiation induce melanoma metastases and trigger the immune system--adrenal axis feedback loop.

Low-doses of ionising radiation are frequently implicated in triggering and/or accelerating the growth of skin and other malignancies. It seemed probable that the radiation at similar dose levels might initiate metastasis from already existing tumours. Highly pigmented human melanoma xenograft that had lost its ability for a spontaneous metastasising and grown subcutaneously in athymic mice was exposed to very low and well-defined doses of ionising radiation to determine whether low linear energy transfer radiation can restore metastatic potential of the tumour. To ensure that all effects derived from radiation-activated neoplastic cells only, I was delivered selectively to the cutaneous melanoma instead of using the external beam. The direct response of these tumours to radiation was monitored by determining the growth rate of the lesions. Histopathological methods were employed to detect metastases. The lowest radiation dose of approximately 6 cGy deposited in the tumours initiated metastatic spread in all animals. Gradual increase of the radiation doses diminished both the frequency of the appearance of metastases and their distance from the primary lesions. There were no metastases from non-irradiated melanomas. The highest dose used (60 cGy) did not affect significantly the growth of cutaneous (primary) tumours, but lower doses that enhanced inflammatory infiltration of the lesions reduced tumour growth. Such radiation-stimulated immune responses were accompanied by increased pigmentation in cutaneous lesions and activation of the adrenal cortex indicating that the immune system-adrenal axis feedback loop had been triggered. The results demonstrate that very low-doses of ionising radiation induce melanoma metastases. The phenomenon is accompanied by the stimulation of the immune system-adrenal axis feedback loop that regulates eicosanoid synthesis, thereby suggesting an involvement of these molecules in the process. Radiation doses approaching the therapeutic level do not initiate melanoma dissemination.

Frozen section microautoradiography in the study of radionuclide targeting: application to indium-111-oxine-labeled leukocytes.

UNLABELLED: The microscopic biodistribution of radioactivity in tissues is important in determining microdosimetry. This study addresses the use of frozen section microautoradiography in studying the subcellular distribution of 111In in leukocytes labeled with 111In-oxine. METHODS: In conjunction with frozen section microautoradiography, computer image analysis methods were applied to the analysis and quantification of leukocyte sections and superimposed autoradiographs. Rapid cell fractionation was used to confirm the results. RESULTS: The emulsion (Ilford K2) response was linear over the concentration range investigated (0-33 MBq ml-1). Resolution of radionuclide distribution was better than 2 microns. The autoradiographs showed no dependence of radiolabel uptake on cell type. Classification of all cells into intervals according to grain density suggests an exponential rather than normal distribution, with approximately 50% of cells having little or no radiolabel. In any one sample, cells which were heavily labeled were approximately 10 times more likely to be found in aggregates (60% found in aggregates, mostly neutrophils) than cells which were not heavily labeled (6% found in aggregates); and the grain densities were at least twofold higher over nuclei than over cytoplasm. The last observation was confirmed by the rapid cell fractionation method which showed that approximately 57% of the total radioactivity was bound to nuclei. CONCLUSION: Frozen section microautoradiography is a practical and reliable approach to determining sub-cellular distribution of 111In. The radiolabeling process causes aggregation of neutrophils. Uptake is not significantly dependent on cell type, but only a fraction of cells are appreciably labeled. The radioactive concentration in cell nuclei is at least two-fold higher than in cytoplasm. Microautoradiography can be used to provide distribution data as input into computer models for sub-cellular dosimetry.

The chemical identity of pentavalent technetium-99m-dimercaptosuccinic acid.

The tumor-targeting radiopharmaceutical known as pentavalent technetium-99m-meso dimercaptosuccinic acid (DMSA) has been studied by a variety of techniques in order to elucidate its structure and chemical behavior. The radiopharmaceutical is identical with a chemically characterized sample of [99TcO(DMSA)2]- when studied by mobility methods including thin-layer chromatography, reversed-phase high-performance liquid chromatography, gel filtration, and electrophoresis. The technetium is pentavalent and coordinated by an oxo-ligand and four thiolate sulfurs of two DMSA ligands. No-carrier-added preparations consist of mixtures of three stereoisomers of the square pyramidal, mononuclear complex. The isomers arise from differing orientations of the carboxylate groups in the DMSA ligands and may be designated syn-endo, syn-exo, and anti. All three isomers are significant components of the radiopharmaceutical, raising the question of which are tumor-specific. The carboxylate groups in the complex are almost completely ionized at pH 7, thus the average charge on the complex at this pH approaches -5.

Cyclic and acyclic polyamines as chelators of rhenium-186 and rhenium-188 for therapeutic use.

Several polyamine ligands L1-L7 were assessed as chelators for rhenium-188 (and by analogy, rhenium-186) for incorporation into the design of radiopharmaceuticals for targeted radiotherapy. Both ease of synthesis of the complexes and their kinetic stability in human serum were examined. Chelation of Re-188 by stannous reduction of perrhenate in the presence of acyclic ligands such as L1 and L2 (L1 = ethylenediamine, L2 = 1,4,8,11-tetraazaundecane) proceeded in acceptable yield (50-90%) under aqueous conditions (pH 11; 20-100 degrees C, 30 min) in a single step. In contrast, synthesis of complexes of the cyclic ligands such as L6 (L6 = 1,4,8,11-tetraazacyclotetradecane, cyclam) in acceptable yield (> 50%) required more involved procedures including use of nonaqueous solvents. The chelates were unambiguously identified as the cationic trans-dioxorhenium(V) tetrakis(amino) complexes, by chromatographic comparison with spectroscopically characterised nonradioactive samples. The complexes of tetradentate ligands L2 and L6 showed no evidence of degradation on incubation for up to 24 h in human serum. The complex of L1 degraded by less than 3% under these conditions. These preliminary studies indicate that the acyclic tetradentate ligands offer an appropriate compromise between biological stability and ease of synthesis, and they have potential as chelators for rhenium in radiopharmaceuticals.

Radioiodinated methylene blue for melanoma targeting: chemical characterisation and tumour selectivity of labelled components.

Radioiodinated methylene blue contains a mixture of components showing selective uptake in human pigmented melanoma, and it has potential for imaging and therapy. Nuclear magnetic resonance and mass spectroscopic studies show that the majority of the radioactivity (85%) is in the form of monoiodinated methylene blue, 4-iodo-3-methylamino-7-dimethylaminophenaza thionium chloride. The amino group ortho-to iodine has become demethylated to a mono-methylamino group. The remainder (15%) of the mixture is the doubly labelled 4,5-diiodo-3,7-bis(methylamino) phenazathionium chloride. The separated components show similar tumour selectivity in athymic mice bearing human pigmented melanomas.

Copper radionuclides and radiopharmaceuticals in nuclear medicine.

The chemistry, radiochemistry, radiobiology, and radiopharmacology of radiopharmaceuticals containing copper radionuclides are reviewed. Copper radionuclides offer application in positron emission tomography, targeted radiotherapy, and single photon imaging. The chemistry of copper is relatively simple and well-suited to radiopharmaceutical application. Current radiopharmaceuticals include biomolecules labelled via bifunctional chelators primarily based on cyclic polyaminocarboxylates and polyamines, and pyruvaldehyde-bis(N4-methylthiosemicarbazone) (PTSM) and its analogues. The chemistry of copper, of which only a fraction has yet been exploited, is likely to be applied more fully in the future.

Disposition of intravenous 123iodopentamidine in man.

This study compared the disposition of the radiopharmaceutical [123I]iodopentamidine with that of pentamidine after intravenous infusion by measuring plasma concentrations of each using scintilation counting and high-performance liquid chromatography (HPLC), respectively. There was rapid hepatic uptake and biliary excretion of the 123I label. Distribution kinetics of the 123I label were similar to those of pentamidine, but its elimination half-life (41 +/- 27 h) was longer than that of pentamidine measured by HPLC (11 +/- 8 h). [123I]iodopentamidine distribution reflects that of pentamidine, but elimination of the radiopharmaceutical appears slower.

Towards new transition metal-based hypoxic selective agents for therapy and imaging.

The greater lability of Co(II) relative to Co(III) can potentially be used to achieve selective delivery of nitrogen mustard type molecules to hypoxic cells. Attempts to improve the stability of the Co(II) state by utilising tripodal tetradentate ligands are described, together with the results of DF calculations. Rhenium has two beta-emitting isotopes (186)Re and (188)Re that have potential for use to treat cancer if the complexes can be targeted with sufficient specificity. We describe some new rapid low temperature routes using hydrazines to labile Re(V) and Re(III) species which provide potential convenient access to a wide range of oxo- and diazenido-complexes. The synthesis of new Re(V) and Re(III) thiosemicarbazone complexes is presented in the context of obtaining hypoxic selective species. Copper(II) bis(thiosemicarbazone) complexes are known to be hypoxic selective and spectroscopic, cyclic voltammetric and computational studies of the mechanism are presented, together with the synthesis of new Cu(II) complexes directed towards the hypoxic selective delivery of nitrogen mustard type molecules.

Extending the life of a 99Tcm generator: a simple and convenient method for concentrating generator eluate for clinical use.

99Tcm-pertechnetate can conveniently be concentrated 5-20-fold by passing up to 20 ml through a sequence of two (or three) disposable columns, one (or two) to remove chloride and one to concentrate the pertechnetate for subsequent elution with a small volume of saline. This procedure is useful for utilizing eluates with low radioactive concentration in applications that require high radioactive concentration.

Rapid preparation of 123I-labelled methylene blue and toluidine blue: potential new agents for parathyroid scintigraphy.

Methylene blue and toluidine blue are phenothiazinium dyes used to localize parathyroid glands visually during surgery. For the purpose of assessing the potential of radiolabelled analogues for scintigraphic localization of parathyroids, a rapid and convenient method has been developed for covalently labelling the dyes with iodine-123. Treatment of aqueous methylene blue or toluidine blue with 123I-sodium iodide in the presence of potassium iodate and hydrochloric acid at 100 degrees C for 1 h results in incorporation of 80-90% of the radioiodine into the dye. The chemical and radiochemical impurities are readily removed by passage through a disposable sample preparation column. Using the optimized protocol, a sterile, pyrogen-free multi-dose solution of radioiodinated dye (pH 7, specific activity greater than or equal to 37 MBq mg-1, radioactivity concentration congruent to 37 MBq ml-1, radiochemical purity greater than 99%, overall activity yield available for injection 60-70%) can be prepared within 1.5 h.

123I-methylene blue: an unsatisfactory parathyroid imaging agent.

This study was designed to examine the possible use of 123I-labelled methylene blue as a parathyroid imaging agent. Five patients were studied, all of whom had parathyroid adenomas which were successfully localized preoperatively with 201Tl and 99Tcm-sestamibi, and which were subsequently proven at surgery. The 123I-labelled methylene blue localized only one of these adenomas, was negative in two and had equivocal uptake in the remaining two patients. It is therefore concluded that compared to other radionuclide methods for localizing parathyroid adenomas this agent is unsatisfactory.

Studies on the preparation and isomeric composition of 186Re- and 188Re-pentavalent rhenium dimercaptosuccinic acid complex.

The preparative conditions for 186Re(V)DMSA and 188Re(V)DMSA (DMSA = meso-dimercaptosuccinic acid), beta-emitting radiopharmaceuticals that have been shown to localize in medullary thyroid carcinoma, require modification depending on the amount of carrier rhenium and the chemical form and medium in which the rhenium is supplied. Preparative conditions are described for use with carrier-free 188ReO4- in saline, and for use with 186ReO4- in saline, sodium hydroxide or nitric acid. Preparation of 186Re(V)DMSA (carrier present up to 2 mg per 2.5 ml reaction volume) requires a DMSA:SnCl2:Re ratio of 10:5:1 at 100 degrees C for 30 min. Addition of excess nitric acid or hydrochloric acid up to a concentration of 155 mM does not reduce the yield from 100%. A commercial DMSA kit vial (e.g. Amerscan DMSA) can be used for preparation of 188Re(V)DMSA (carrier free) provided the required activity is in a volume of less than 1 ml per vial. A convenient method of concentrating the 188Re generator eluate to the required volume is described.