Human cytomegalovirus (HCMV) is a widely spread herpesvirus, suggested to play a role in tumor progression. US28, a chemokine receptor encoded by HCMV, binds a broad spectrum of chemokines and constitutively activates various pathways linked to proliferation. Our studies reveal that expression of US28 induces a proangiogenic and transformed phenotype by up-regulating the expression of vascular endothelial growth factor and enhancing cell growth and cell cycle progression. US28-expressing cells promote tumorigenesis when injected into nude mice. The G proteinuncoupled constitutively inactive mutant of US28, induces delayed and attenuated tumor formation, indicating the importance of constitutive receptor activity in the early onset of tumor development. Importantly, also in glioblastoma cells infected with the newly isolated clinical HCMV strain Titan, US28 was shown to be involved in the HCMV-induced angiogenic phenotype. Hence, the constitutively activated chemokine receptor US28 might act as a viral oncogene and enhance and͞or promote HCMV-associated tumor progression.cancer ͉ G protein-coupled receptor ͉ VEGF ͉ viral infection ͉ drug target

Selection of the right drug for the right patient is a promising approach to increase clinical benefit of targeted therapy with monoclonal antibodies (mAbs). Assessment of in vivo biodistribution and tumor targeting of mAbs to predict toxicity and efficacy is expected to guide individualized treatment and drug development. Molecular imaging with positron emission tomography (PET) using zirconium-89 (89Zr)-labeled monoclonal antibodies also known as 89Zr-immuno-PET, visualizes and quantifies uptake of radiolabeled mAbs. This technique provides a potential imaging biomarker to assess target expression, as well as tumor targeting of mAbs. In this review we summarize results from initial clinical trials with 89Zr-immuno-PET in oncology and discuss technical aspects of trial design. In clinical trials with 89Zr-immuno-PET two requirements should be met for each 89Zr-labeled mAb to realize its full potential. One requirement is that the biodistribution of the 89Zr-labeled mAb (imaging dose) reflects the biodistribution of the drug during treatment (therapeutic dose). Another requirement is that tumor uptake of 89Zr-mAb on PET is primarily driven by specific, antigen-mediated, tumor targeting. Initial trials have contributed toward the development of 89Zr-immuno-PET as an imaging biomarker by showing correlation between uptake of 89Zr-labeled mAbs on PET and target expression levels in biopsies. These results indicate that 89Zr-immuno-PET reflects specific, antigen-mediated binding. 89Zr-immuno-PET was shown to predict toxicity of RIT, but thus far results indicating that toxicity of mAbs or mAb-drug conjugate treatment can be predicted are lacking. So far, one study has shown that molecular imaging combined with early response assessment is able to predict response to treatment with the antibody-drug conjugate trastuzumab-emtansine, in patients with human epithelial growth factor-2 (HER2)-positive breast cancer. Future studies would benefit from a standardized criterion to define positive tumor uptake, possibly supported by quantitative analysis, and validated by linking imaging data with corresponding clinical outcome. Taken together, these results encourage further studies to develop 89Zr-immuno-PET as a predictive imaging biomarker to guide individualized treatment, as well as for potential application in drug development.

Brain cancer is a devastating disease affecting many people worldwide. Effective treatment with chemotherapeutics is limited due to the presence of the blood-brain barrier (BBB) that tightly regulates the diffusion of endogenous molecules but also xenobiotics. Glutathione pegylated liposomal doxorubicin (2B3-101) is being developed as a new treatment option for patients with brain cancer. It is based on already marketed pegylated liposomal doxorubicin (Doxil®/Caelyx®), with an additional glutathione coating that safely enhances drug delivery across the BBB.Uptake of 2B3-101 by human brain capillary endothelial cells in vitro was time-, concentration- and temperature-dependent, while pegylated liposomal doxorubicin mainly remained bound to the cells. In vivo, 2B3-101 and pegylated liposomal doxorubicin had a comparable plasma exposure in mice, yet brain retention 4 days after administration was higher for 2B3-101. 2B3-101 was overall well tolerated by athymic FVB mice with experimental human glioblastoma (luciferase transfected U87MG). In 2 independent experiments a strong inhibition of brain tumor growth was observed for 2B3-101 as measured by bioluminescence intensity. The effect of weekly administration of 5 mg/kg 2B3-101 was more pronounced compared to pegylated liposomal doxorubicin (p<0.05) and saline (p<0.01). Two out of 9 animals receiving 2B3-101 showed a complete tumor regression. Twice-weekly injections of 5 mg/kg 2B3-101 again had a significant effect in inhibiting brain tumor growth (p<0.001) compared to pegylated liposomal doxorubicin and saline, and a complete regression was observed in 1 animal treated with 2B3-101. In addition, twice-weekly dosing of 2B3-101 significantly increased the median survival time by 38.5% (p<0.001) and 16.1% (p<0.05) compared to saline and pegylated liposomal doxorubicin, respectively.Overall, these data demonstrate that glutathione pegylated liposomal doxorubicin enhances the effective delivery of doxorubicin to brain tumors and could become a promising new therapeutic option for the treatment of brain malignancies.

Hypoxia has been shown to be an important microenvironmental parameter influencing tumor progression and treatment efficacy. Patient guidance for hypoxia-targeted therapy requires evaluation of tumor oxygenation, preferably in a noninvasive manner. The aim of this study was to evaluate and validate the uptake of [ 18 F] HX4, a novel developed hypoxia marker for PET imaging. A heterogeneous accumulation of [ 18 F]HX4 was found within rat rhabdomyosarcoma tumors that was significantly (P < 0.0001) higher compared with the surrounding tissues, with temporal increasing tumor-to-blood ratios reaching a plateau of 7.638 ± 0.926 and optimal imaging properties 4 h after injection. [ 18 F]HX4 retention in normal tissues was found to be short-lived, homogeneous and characterized by a fast progressive temporal clearance. Heterogeneity in [ 18 F]HX4 tumor uptake was analyzed based on 16 regions within the tumor according to the different orthogonal planes at the largest diameter. Validation of heterogeneous [ 18 F]HX4 tumor uptake was shown by a strong and significant relationship (r = 0.722; P < 0.0001) with the hypoxic fraction as calculated by the percentage pimonidazole-positive pixels. Furthermore, a causal relationship with tumor oxygenation was established, because combination treatment of nicotinamide and carbogen resulted in a 40% reduction (P < 0.001) in [ 18 F]HX4 tumor accumulation whereas treatment with 7% oxygen breathing resulted in a 30% increased uptake (P < 0.05). [ 18 F]HX4 is therefore a promising candidate for noninvasive detection and evaluation of tumor hypoxia at a macroscopic level.cancer | nuclear medicine | experimental research T he presence of hypoxic regions due to abnormalities in tumor vasculature, heterogeneously spread within solid tumors influences clinical outcome; as it is an independent predictor of poor prognosis-free survival in several types of cancer (1). In contrast, this unique tumor characteristic makes it an attractive target for novel drugs to increase the therapeutic effect of conventional cancer treatment modalities. Another approach is the use of intensity-modulated radiotherapy to give a higher dose to hypoxic areas while sparing the surrounding normal tissue (2, 3). Although treatments to counteract the negative effect of intratumoral hypoxia are under investigation, not all patients will benefit from such selective treatments. Therefore, to guide hypoxiadirected therapies in individual patients, it is important to evaluate tumor oxygenation using a reliable noninvasive method.To date, a variety of methods are available for assessment of tumor oxygenation in solid tumors, of which polarographic oxygen electrodes and immunohistological assays remain the gold standard (4). These standard invasive modalities have not yielded reliable 3D images of the whole tumor for clinical use, and therefore research has been focused on noninvasive imaging techniques, such as positron-emission tomography (PET) using nitroimidazoles. The 2-nitroimidazole derivative fluoromisonidazole (FMISO)...

PurposeThe ∼15 kDa variable domains of camelid heavy-chain-only antibodies (called Nanobodies®) have the flexibility to be formatted as monovalent, monospecific, multivalent or multispecific single chain proteins with either fast or slow pharmacokinetics. We report the evaluation of the fast kinetic anti-epidermal growth factor receptor (EGFR) Nanobody 7D12, labelled with 68Ga via the novel bifunctional chelate (BFC) p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS). Df-Bz-NCS has recently been introduced as the chelate of choice for 89Zr immuno-positron emission tomography (PET).MethodsNanobody 7D12 was premodified with Df-Bz-NCS at pH 9. Radiolabelling with purified 68Ga was performed at pH 5.0–6.5 for 5 min at room temperature. For in vitro stability measurements in storage buffer (0.25 M NaOAc with 5 mg ml−1 gentisic acid, pH 5.5) at 4°C or in human serum at 37°C, a mixture of 67Ga and 68Ga was used. Biodistribution and immuno-PET studies of 68Ga-Df-Bz-NCS-7D12 were performed in nude mice bearing A431 xenografts using 89Zr-Df-Bz-NCS-7D12 as the reference conjugate.ResultsThe Df-Bz-NCS chelate was conjugated to Nanobody 7D12 with a chelate to Nanobody molar substitution ratio of 0.2:1. The overall 68Ga radiochemical yield was 55–70% (not corrected for decay); specific activity was 100–500 MBq/mg. Radiochemical purity of the conjugate was >96%, while the integrity and immunoreactivity were preserved. 68/67Ga-Df-Bz-NCS-7D12 was stable in storage buffer as well as in human serum during a 5-h incubation period (<2% radioactivity loss). In biodistribution studies the 68Ga-labelled Nanobody 7D12 showed high uptake in A431 tumours (ranging from 6.1 ± 1.3 to 7.2 ± 1.5%ID/g at 1–3 h after injection) and high tumour to blood ratios, which increased from 8.2 to 14.4 and 25.7 at 1, 2 and 3 h after injection, respectively. High uptake was also observed in the kidneys. Biodistribution was similar to that of the reference conjugate 89Zr-Df-Bz-NCS-7D12. Tumours were clearly visualized in a PET imaging study.ConclusionVia a rapid procedure under mild conditions a 68Ga-Nanobody was obtained that exhibited high tumour uptake and tumour to normal tissue ratios in nude mice bearing A431 xenografts. Fast kinetic 68Ga-Nanobody conjugates can be promising tools for tumour detection and imaging of target expression.