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| 2. |
- Sundberg, Åsa Liljegren, et al.
(författare)
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[111In]Bz-DTPA-hEGF : Preparation and in vitro characterization of a potential anti-glioblastoma targeting agent
- 2003
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Ingår i: Cancer Biotherapy and Radiopharmaceuticals. - : Mary Ann Liebert Inc. - 1084-9785 .- 1557-8852. ; 18:4, s. 643-54
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Tidskriftsartikel (refereegranskat)abstract
- The overexpression of epidermal growth factor receptors, EGFR, in glioblastomas is well documented. Hence, the EGFR can be used as target structure for a specific targeting of glioblastomas. Both radiolabeled anti-EGFR antibodies and the natural ligand EGF are candidate agents for targeting. However, EGF, which has a rather low molecular weight (6 kDa), might have better tissue penetration properties through both normal tissue and tumors in comparison with anti-EGF antibodies and their fragments. The aim of this study was to prepare and evaluate in vitro an EGF-based antiglioma conjugate with residualizing label. Human recombinant EGF (hEGF) was coupled to isothiocyanate-benzyl-DTPA. The conjugate was purified from unreacted chelator using solid-phase extraction and labeled with (111)In. The labeling yield was 87% +/- 7%. The label was reasonably stable; the transchelation of (111)In to serum proteins was about 5% after incubation at 37 degrees C during 24 hours. The obtained [(111)In]benzyl-DTPA-hEGF conjugate was characterized in vitro using the EGFR expressing glioma cell line U343MGaCl2:6. The binding affinity, internalization, and retention of the conjugate were studied. The conjugate had receptor specific binding and the radioactivity was quickly internalized. The intracellular retention of radioactivity after interrupted incubation with conjugate was 71% +/- 1% and 59% +/- 1.5% at 24 and 45 hours, respectively. The dissociation constant was estimated to 2.0 nM. The results indicate that [(111)In]benzyl-DTPA-hEGF is a potential candidate for targeting glioblastoma cells, possibly using locoregional injection.
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| 3. |
- Sundberg, Åsa Liljegren, et al.
(författare)
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[177Lu]Bz-DTPA-EGF : Preclinical characterization of a potential radionuclide targeting agent against glioma
- 2004
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Ingår i: Cancer Biotherapy and Radiopharmaceuticals. - : Mary Ann Liebert Inc. - 1084-9785 .- 1557-8852. ; 19:2, s. 195-204
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Tidskriftsartikel (refereegranskat)abstract
- Patients with glioblastoma multiforme have a poor prognosis due to recurrences originating from spread cells. The use of radionuclide targeting might increase the chance of inactivating single tumor cells with minimal damage to surrounding healthy tissue. As a target, overexpressed epidermal growth factor receptors (EGFR) may be used. A natural ligand to EGFR, the epidermal growth factor (EGF) is an attractive targeting agent due to its low molecular weight (6 kDa) and high affinity for EGFR. 177Lu (T(1/2) = 6.7 days) is a radionuclide well suited for treatment of small tumor cell clusters, since it emits relatively low-energy beta particles. The goal of this study was to prepare and preclinically evaluate both in vitro and in vivo the [177Lu]Bz-DTPA-EGF conjugate. The conjugate was characterized in vitro for its cell-binding properties, and in vivo for its pharmacokinetics and ability to target EGFR. [177Lu]Bz-DTPA-EGF bound to cultured U343 glioblastoma cells with an affinity of 1.9 nM. Interaction with EGFR led to rapid internalization, and more than 70% of the cell-associated radioactivity was internalized after 30 minutes of incubation. The retention of radioactivity was good, with more than 65% of the 177Lu still cell-associated after 2 days. Biodistribution studies of i.v. injected [177Lu]Bz-DTPA-EGF in NMRI mice demonstrated a rapid blood clearance. Most of the radioactivity was found in the liver and kidneys. The liver uptake was receptor-mediated, since it could be significantly reduced by preinjection of unlabeled EGF. In conclusion, [177Lu]Bz-DTPA-EGF seems to be a promising candidate for locoregional treatment of glioblastoma due to its high binding affinity, low molecular weight, and ability to target EGFR in vivo.
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| 4. |
- Sundberg, Åsa Liljegren, et al.
(författare)
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Cellular retention of radioactivity and increased radiation dose : Modelexperiments with EGF-dextran
- 2003
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Ingår i: Nuclear Medicine and Biology. - 0969-8051 .- 1872-9614. ; 30:3, s. 303-315
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Tidskriftsartikel (refereegranskat)abstract
- Targeting of tumor cells with radiolabeled biomolecules is a possible approach to inactivate disseminated tumor cells. However, rapid degradation of the biomolecules after cellular internalization and subsequent excretion of the radioactivity is a problem. We studied the possibility of using dextran as a carrier of radionuclides to improve the intracellular retention. An EGF-dextran conjugate, aimed for targeting of tumor cells overexpressing the EGF-receptor, was used as model. Retention tests were performed with (125)I on different parts: [(125)I]-EGF-dextran-[(125)I], [(125)I]-EGF-dextran and EGF-dextran-[(125)I]. Comparisons were made with [(125)I]-EGF. The radiolabeled compounds were incubated with cultured glioma cells for different times. The cellular retention of radioactivity was then measured for up to 24 h. Expected radiation doses at the cellular level were calculated assuming that (131)I, instead of (125)I, was coupled to EGF and EGF-dextran. The results indicated that the EGF-part of the conjugate was degraded and the EGF-attached radioactivity was rapidly excreted, whereas radioactivity on dextran was retained intracellularly to a high degree, i.e. 70-80% of the radioactivity bound to dextran was still cell-associated after 24 h. The retention after 24 h was significantly higher (p < 0.001) when the radioactivity was on the dextran instead of the EGF-part. The radiolabeled EGF-dextran had a notably high specific radioactivity; up to 11 MBq/microg. There was potential for at least hundred times increased radiation dose per receptor interaction when the radioactivity was on the dextran part. The advantage with radioactivity on the dextran part was the high cellular retention and the high specific radioactivity (higher than previously reported for other residualizing labels) without severe loss of receptor specific binding. Thus, dextran seems suitable as a carrier of radionuclides aimed for therapy and gives potential for a highly increased radiation dose.
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| 5. |
- Sundberg, Åsa Liljegren, et al.
(författare)
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Combined effect of gefitinib ('Iressa', ZD1839) and targeted radiotherapy with 211At-EGF
- 2003
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Ingår i: European Journal of Nuclear Medicine and Molecular Imaging. - : Springer Science and Business Media LLC. - 1619-7070 .- 1619-7089. ; 30:10, s. 1348-56
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Tidskriftsartikel (refereegranskat)abstract
- The EGFR-TKI (epidermal growth factor receptor tyrosine kinase inhibitor) gefitinib ['Iressa' (trademark of the AstraZeneca group of companies), ZD1839] increases the cellular uptake of radiolabelled epidermal growth factor (EGF). We investigated gefitinib treatment combined with astatine-211 EGF targeting in vitro using two cell lines expressing high levels of EGFR: A431 (sensitive to gefitinib) and U343MGaCl2:1 (resistant to gefitinib). In both cell lines, the uptake of 211At-EGF was markedly increased by concomitant treatment with gefitinib. Survival was investigated using both a clonogenic survival assay and a cell growth assay. Combined gefitinib and 211At-EGF treatment reduced the survival of U343 cells 3.5-fold compared with 211At-EGF alone. In A431 cells, 211At-EGF treatment resulted in very low survival, but combined treatment with gefitinib increased the survival by about 20-fold. These results indicate that combined treatment with gefitinib might increase the effect of ligand-mediated radionuclide therapy in gefitinib-resistant tumours and decrease the effect of such therapy in gefitinib-sensitive tumours.
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| 6. |
- Sundberg, Åsa Liljegren, et al.
(författare)
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Enhancing the effect of radionuclide tumor targeting, using lysosomotropic weak bases
- 2007
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Ingår i: International Journal of Radiation Oncology, Biology, Physics. - : Elsevier BV. - 0360-3016 .- 1879-355X. ; 67:1, s. 279-287
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: The aim of the present study was to investigate if treatment with lysosomotropic weak bases could increase the intracellular retention of radiohalogens and thereby increase the therapeutic effect of radionuclide tumor targeting. METHODS AND MATERIALS: Four different lysosomotropic bases, chloroquine, ammonium chloride, amantadine, and thioridazine, were investigated for their ability to increase radiohalogen retention in vitro. The two most promising substances, chloroquine and ammonium chloride, were studied in several cell lines (A431, U343MGaCl2:6, SKOV-3, and SKBR-3) in combination with radiolabeled epidermal growth factor (EGF) or the HER2 binding affibody (Z(HER2:4))(2). RESULTS: The uptake and retention of radionuclides was found to be substantially increased by simultaneous treatment with the lysosomotropic bases. The effect was, however, more pronounced in the epidermal growth factor:epidermal growth factor receptor (EGF:EGFR) system than in the (Z(HER2:4))(2):HER2 system. The therapeutic effect of ammonium chloride treatment combined with (211)At-EGF was also studied. The effect obtained after combined treatment was found to be much better than after (211)At-EGF treatment alone. CONCLUSIONS: The encouraging results from the present study indicate that the use of lysosomotropic weak bases is a promising approach for increasing the therapeutic effect of radionuclide targeting with radiohalogens.
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| 7. |
- Sundberg, Åsa Liljegren, et al.
(författare)
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Treatment of cultured glioma cells with EGFR-TKI gefitinib (‘Iressa’, ZD1839) increases the uptake of astatinated EGF despite absence of gefitinib-mediated growth inhibition
- 2003
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Ingår i: European Journal of Nuclear Medicine. - : Springer Science and Business Media LLC. - 0340-6997 .- 1432-105X .- 1619-7070 .- 1619-7089. ; 30:5, s. 727-9
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Tidskriftsartikel (refereegranskat)abstract
- The EGFR-TKI (epidermal growth factor receptor tyrosine kinase inhibitor) gefitinib ("Iressa", ZD1839), a reversible growth inhibitor of EGFR-expressing tumour cells, has been shown to enhance the antitumour effect of ionising radiation, and also to increase the uptake of radioiodinated EGF. Thus, combination of gefitinib treatment and radionuclide targeting is an interesting option for therapy of brain tumours that are difficult to treat with conventional methods. The aim of this study was to evaluate how pre-treatment with gefitinib affects binding of astatinated EGF ((211)At-EGF) to cultured glioma U343 cells, which express high levels of EGFR. The growth of U343 cells in the presence of gefitinib was investigated, and it was found that gefitinib does not significantly inhibit the growth of these cells. Nevertheless, the uptake of (211)At-EGF in U343 cells was markedly increased (up to 3.5 times) in cells pre-treated with gefitinib (1 microM). This indicates that a combination of gefitinib treatment and radionuclide targeting to EGFR might be a useful therapeutic modality, even for patients who do not respond to treatment with gefitinib alone.
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| 8. |
- Sundberg, Åsa Liljegren, 1974-
(författare)
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Tumour Targeting Using Radiolabelled EGF Conjugates : Preclinical Studies
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Tumour targeted radiotherapy is an appealing approach for treatment of disseminated tumour cells. A targeting agent that specifically binds to a structure on tumour cells is then used to transport therapeutically relevant radionuclides. The epidermal growth factor receptor, EGFR, is overexpressed on tumour cells in several malignancies, e.g. highly malignant gliomas. In this thesis, three types of radiolabelled EGF-conjugates, aimed for targeting to EGFR-expressing tumour cells, were developed and studied: EGF-dextran labelled with 125I, EGF labelled with 211At, and two EGF-chelates, DTPA-EGF and Bz-DTPA-EGF, labelled with the radioactive metals 111In and 177Lu. The targeting properties of radioiodinated EGF-dextran were first studied in cultured glioma cells. Radioiodine coupled to the dextran part of EGF-dextran was retained in cells appreciably longer than radioiodine coupled to EGF. This can give about 100 times increased radiation dose to tumour cells.Targeting with 211At-EGF was investigated in combination with the tyrosine kinase inhibitor gefitinib (Iressa™, ZD1839). The uptake of 211At-EGF in EGFR-expressing tumour cells increased with increasing gefitinib concentrations. This was the case for both gefitinib-resistant and gefitinib-sensitive cell lines. The effect of the combined treatment on cell survival, however, differed between the cell lines in an unexpected way. In gefitinib resistant cells, combined treatment decreased cell survival approximately 3.5 times relative to 211At-EGF treatment alone. In gefitinib sensitive cells, however, combined treatment increased the cell survival (i.e. a protective effect).The EGF-chelates studied ([111In]DTPA-EGF, [111In]Bz-DTPA-EGF and [177Lu]Bz-DTPA-EGF) all bound specifically with high affinity (Kd≈2 nM) to EGFR on cultured glioma cells. They were internalised after binding, and the cellular retention of radionuclides was high (60% remained after 45 h). A biodistribution study in mice showed that liver and kidneys accumulated a majority of the radioactivity. The EGF-chelates bound EGFR specifically also in vivo. A tumour-to-blood ratio of 25 was achieved in a preliminary study.
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| 9. |
- Velikyan, Irina, et al.
(författare)
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Preparation and evaluation of (68)Ga-DOTA-hEGF for visualisation of EGFR expression in malignant tumours
- 2005
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Ingår i: Journal of Nuclear Medicine. - 0161-5505 .- 1535-5667. ; 46:11, s. 1881-1888
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Tidskriftsartikel (refereegranskat)abstract
- Detection of epidermal growth factor receptor (EGFR) overexpression in many carcinomas provides important diagnostic information, which can influence patient management. The use of PET may enable such detection in vivo by a noninvasive procedure with high sensitivity. The aim of this study was to develop a method for preparation of a positron-emitting tracer based on a natural ligand to EGFR, the recombinant human epidermal growth factor (hEGF), and to perform a preclinical evaluation of the tracer.METHODS: DOTA-hEGF (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) was prepared by coupling of a N-sulfosuccinimide ester of DOTA to hEGF. The conjugate was labeled with a generator-produced positron-emitting nuclide, (68)Ga (half-life = 68 min), using microwave heating. Binding specificity, affinity, internalization, and retention of (68)Ga-DOTA-hEGF was studied in 2 EGFR-expressing cell lines, U343 glioma cells and A431 cervical carcinoma cells. Biodistribution and microPET visualization studies were performed in BALB/c nu/nu mice bearing A431 carcinoma xenografts.RESULTS: A 1-min-long microwave-assisted labeling provided radioactivity incorporation of 77% +/- 4%. Both cell lines demonstrated receptor-specific uptake of the conjugate, rapid internalization of the tracer, and good retention of radioactivity. Binding to both cell lines occurred with high affinity, approximately 2 nmol/L. The biodistribution study demonstrated accumulation of radioactivity in xenografts and in EGFR-expressing organs. The microPET imaging study enabled visualization of tumors and demonstrated quick--within 5 min--localization of radioactivity in tumors.CONCLUSION: (68)Ga-DOTA-hEGF has potential for imaging EGFR overexpression in tumors.
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