The most widely cited procedures for radiolabeling antibodies with zirconium-89 for immuno-PET require multi-milligram amounts of antibody which can be cost-prohibitive, particularly during the research and development process. We therefore sought to develop a reliable (89)Zr-radiolabeling procedure that provides high radiochemical yields at the microgram scale.
\n \n\n \n \nPURPOSE: The monoclonal antibody (mAb) 14C5 is a murine IgG1 directed against a yet undefined molecule involved in cell substrate adhesion found on the surface of malignant breast cancer tissue. mAb 14C5 is able to inhibit cell substrate adhesion and invasion of breast cancer cells in vitro. In normal tissues as well as in the stroma surrounding in situ carcinomas of the breast, no expression of the antigen 14C5 occurs. The aim of this study was to investigate the in vitro and in vivo targeting properties of 123I- and 131I-labeled mAb 14C5 as a novel agent for radioimmunodetection and radioimmunotherapy. EXPERIMENTAL DESIGN: Internalization of mAb 14C5 was investigated with 125I-labeled mAb 14C5 and by confocal laser scanning microscopy. Biodistribution studies of 131I-labeled mAb 14C5 and planar gamma imaging were done in nude mice bearing an A549 (non-small cell lung carcinoma) or a LoVo (colon carcinoma) tumor. RESULTS: Internalization studies with both A549 and LoVo cells showed that 125I-labeled mAb 14C5 is slowly internalized with approximately 30% of the initially bound mAb 14C5 internalized after 2 hours at 37 degrees C. Internalization of mAb 14C5 could be visualized with confocal laser scanning microscopy. In vivo, radioisotope uptake peaked at 24 hours for both tumor models (n = 5) with no significant difference in percentage of injected dose/g tissue (A549 10.4 +/- 0.8 and LoVo 9.3 +/- 0.8). Via planar gamma camera imaging, A549 lung tumors as well as LoVo colon tumors could be clearly visualized. CONCLUSIONS: The in vitro and in vivo targeting properties of 123I- and 131I-labeled mAb 14C5 are promising and could provide a new antibody-based agent for radioimmunodetection and radioimmunotherapy of patients bearing antigen 14C5-expressing tumors.
\n \n\n \n \nThe tumor suppressor p53 is widely dysregulated in cancer and represents an attractive target for immunotherapy. Because of its intracellular localization, p53 is inaccessible to classical therapeutic monoclonal antibodies, an increasingly successful class of anticancer drugs. However, peptides derived from intracellular antigens are presented on the cell surface in the context of MHC I and can be bound by T-cell receptors (TCR). Here, we report the development of a novel antibody, T1-116C, that acts as a TCR mimic to recognize an HLA-A*0201-presented wild-type p53 T-cell epitope, p5365-73(RMPEAAPPV). The antibody recognizes a wide range of cancers, does not bind normal peripheral blood mononuclear cells, and can activate immune effector functions to kill cancer cells in vitroIn vivo, the antibody targets p5365-73 peptide-expressing breast cancer xenografts, significantly inhibiting tumor growth. This represents a promising new agent for future cancer immunotherapy. Cancer Res; 77(10); 2699-711. \u00a92017 AACR.
\n \n\n \n \nOver the last couple of years, the number of original papers and reviews discussing various applications of cell penetrating peptides (CPPs) has grown exponentially. This is not remarkable since CPPs are capable of transporting the most varying cargo across cell membranes which is one of the biggest problems in drug delivery and targeted therapy. In this review, we focus on the use of CPPs and related peptides for delivery of imaging contrast agents and radionuclides to cells and tissues with the ultimate goal of in vivo molecular imaging and molecular radiotherapy of intracellular and even intranuclear targets.
\n \n\n \n \nA pretargeted imaging strategy based on the HaloTag dehalogenase enzyme is described. Here, a HaloTag-Trastuzumab conjugate has been used as the primary agent targeting HER2 expression, and three new radiolabelled HaloTag ligands have been used as secondary agents, two of which offer dual-modality (SPECT/optical) imaging capability.
\n \n\n \n \n\u00a9 2015, Springer-Verlag Berlin Heidelberg. Purpose: The efficacy of most anticancer treatments, including radiotherapy, depends on an ability to cause DNA double-strand breaks (DSBs). Very early during the DNA damage signalling process, the histone isoform H2AX is phosphorylated to form \u03b3H2AX. With the aim of positron emission tomography (PET) imaging of DSBs, we synthesized a89Zr-labelled anti-\u03b3H2AX antibody, modified with the cell-penetrating peptide, TAT, which includes a nuclear localization sequence. Methods:89Zr-anti-\u03b3H2AX-TAT was synthesized using EDC/NHS chemistry for TAT peptide linkage. Desferrioxamine conjugation allowed labelling with89Zr. Uptake and retention of89Zr-anti-\u03b3H2AX-TAT was evaluated in the breast adenocarcinoma cell line MDA-MB-468 in vitro or as xenografts in athymic mice. External beam irradiation was used to induce DSBs and expression of \u03b3H2AX. Since89Zr emits ionizing radiation, detailed radiobiological measurements were included to ensure89Zr-anti-\u03b3H2AX-TAT itself does not cause any additional DSBs. Results: Uptake of89Zr-anti-\u03b3H2AX-TAT was similar to previous results using111In-anti-\u03b3H2AX-TAT. Retention of89Zr-anti-\u03b3H2AX-TAT was eightfold higher at 1\u00a0h post irradiation, in cells expressing \u03b3H2AX, compared to non-irradiated cells or to non-specific IgG control. PET imaging of mice showed higher uptake of89Zr-anti-\u03b3H2AX-TAT in irradiated xenografts, compared to non-irradiated or non-specific controls (12.1 \u00b1 1.6 vs 5.2 \u00b1 1.9 and 5.1 \u00b1 0.8\u00a0%ID/g, respectively; p < 0.0001). The mean absorbed dose to the nucleus of cells taking up89Zr-anti-\u03b3H2AX-TAT was twofold lower compared to111In-anti-\u03b3H2AX-TAT. Additional exposure of neither irradiated nor non-irradiated cells nor tissues to89Zr-anti-\u03b3H2AX-TAT resulted in any significant changes in the number of observable DNA DSBs, \u03b3H2AX foci or clonogenic survival. Conclusion:89Zr-anti-\u03b3H2AX-TAT allows PET imaging of DNA DSBs in a tumour xenograft mouse model.
\n \n\n \n \nA novel pretargeted SPECT imaging strategy based on the HaloTag enzyme has been evaluated for the first time in a living system. To determine the efficacy of this approach, two clinically relevant cancer biomarkers, HER2 and TAG-72, were selected to represent models of internalizing and noninternalizing antigens, respectively. In MDA-MB-231/H2N (HER2-expressing) and LS174T (TAG-72-expressing) xenograft tumors in mice, pretargeting experiments were performed in which HaloTag-conjugated derivatives of the antibodies trastuzumab (anti-HER2) or CC49 (anti-TAG-72) were utilized as primary agents, and the small molecule HaloTag ligands 111In-HTL-1, -2, and -3 were evaluated as secondary agents. While this approach was not sufficiently sensitive to detect the internalizing HER2 antigen, pretargeting experiments involving the most optimal secondary agent, 111In-HTL-3, were successful in detecting the noninternalizing antigen TAG-72 and provided high-contrast SPECT images at 4 and 24 h postinjection.
\n \n\n \n \nPARP inhibitors are increasingly being studied as cancer drugs, as single agents or as a part of combination therapies. Imaging of PARP using a radiolabeled inhibitor has been proposed for patient selection, outcome prediction, dose optimization, genotoxic therapy evaluation, and target engagement imaging of novel PARP-targeting agents. Here, via the copper-mediated 18F-radiofluorination of aryl boronic esters, we accessed, for the first time, the 18F-radiolabeled isotopologue of the Food and Drug Administration-approved PARP inhibitor olaparib. The use of the 18F-labeled equivalent of olaparib allows direct prediction of the distribution of olaparib, given its exact structural likeness to the native, non-radiolabeled drug. [18F]Olaparib was taken up selectively in vitro in PARP-1-expressing cells. Irradiation increased PARP-1 expression and [18F]olaparib uptake in a radiation-dose-dependent fashion. PET imaging in mice showed specific uptake of [18F]olaparib in tumors expressing PARP-1 (3.2\u00b10.36%ID/g in PSN-1 xenografts), correlating linearly with PARP-1 expression. Two hours after irradiation of the tumor (10 Gy), uptake of [18F]olaparib increased by 70% (P = 0.025). Taken together, we show that [18F]olaparib has great potential for non-invasive tumor imaging and monitoring of radiation damage.
\n \n\n \n \nSchizophrenia is a devastating mental disorder characterized by relapsing psychotic episodes accompanied with emotional, professional and social decline. The classical dopamine hypothesis of schizophrenia postulates that hyperactivity of dopaminergic neurotransmission is responsible for the positive symptoms of the disorder, more exactly hyperactivity of the dopamine D2-like receptors. One of these receptors is the D4 receptor which is thought to be involved in the motor side-effects caused antipsychotics. However, research into the specific role of this receptor has been hampered by the lack of specific ligands. Therefore, a new 123I-labelled compound was developed which may allow in vivo visualization of the D4 receptor by SPECT. [123I]-3-(4-iodobenzyl)-1,2,3,4 -tetrahydro-8- hydroxychromeno[3,4-c]pyridin-5-one was prepared by electrophillic aromatic substitution of the tributylstannyl derivative. The radiochemical yield was 68 \u00b1 3 % (n = 5) and the specific activity was > 2.96Ci/\u03bcmol. Copyright \u00a9 2005 John Wiley & Sons, Ltd.
\n \n\n \n \nShort hairpin RNA (shRNA)-pooled screening is a valuable and cost-effective tool for assaying the contribution of individual genes to cell viability and proliferation on a genomic scale. Here we describe the key considerations for the design and execution of a pooled shRNA screen to identify determinants of radiosensitivity.
\n \n\n \n \nSUMMARY: BACKGROUND: Trastuzumab is used as adjuvant treatment in patients with HER2-positive breast cancers and has been shown to reduce the chance of recurrence by up to 50%. However, experience with it given with radiotherapy is limited and there is in vitro evidence of a radiosensiti-sation effect. We describe the first case of trastuzumab-associated radiation-induced myelitis. CASE REPORT: This patient received a calculated dose of 28 Gy to the spinal cord when receiving adjuvant radiotherapy to the chest wall and supraclavicular and axillary lymph nodes. This is well below the accepted radiation tolerance of the spinal cord (50-60 Gy) but she developed radiation-induced myelitis of her spinal cord with characteristic magnetic resonance imaging changes. We postulate that trastuzu-mab given concurrently with radiation may have acted as a radiosensitiser and that normal repair mechanisms in the acute stage were affected by trastuzumab blockage of epidermal growth factor receptors, resulting in demy-elination at a lower dose of radiation than normally seen. CONCLUSIONS: Concomitant radiotherapy and adjuvant trastuzumab treatment should be given with caution and consideration made of delaying trastuzumab until after radiotherapy has been completed. As longer-term data become available for patients who received trastuzumab and radiation, it will become clearer whether there is a significant interaction on organs such as the heart and spinal cord in the radiation field.
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