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  • 101.
    Nordberg, Erika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    EGFR and HER2 Targeting for Radionuclide-Based Imaging and Therapy: Preclinical Studies2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The optimal way to detect and treat cancer is to target cancer cells exclusively without affecting the surrounding tissue. One promising approach is to use radiolabelled molecules to target receptors that are overexpressed in cancer cells. Since the epidermal growth factor receptor (EGFR) family is overexpressed in many types of cancer, it is an attractive target for both diagnostic and therapeutic applications.

    This thesis can be divided into two parts. In part one (paper I), studies were conducted to modulate radionuclide uptake in tumour cells. The results showed that it was possible to modulate the cellular uptake of 125I delivered by trastuzumab (targeting HER2) by adding EGF (targeting EGFR).

    In part two (papers II-V) a high affinity EGFR-targeting affibody molecule (ZEGFR:955)2 was selected and analysed both in vitro and in vivo. In papers II, III and V, the results obtained when using (ZEGFR:955)2 were compared with those obtained with the two EGFR-binding molecules, EGF and cetuximab. These studies demonstrated that the affibody molecule bound specifically to EGFR (probably to subdomain III) with high affinity (~50 nM in biosensor analysis and ~1 nM in cellular studies) and produced intracellular signalling changes similar to those with cetuximab. In paper IV, in vivo studies were made, demonstrating that [111In](ZEGFR:955)2 gave a tumour-specific 111In uptake of 3.8±1.4% of injected dose per gram tumour tissue, 4 h post-injection. The tumours could be easily visualized with a gamma camera at this time-point.

    The results of these studies indicated that the affibody molecule (ZEGFR:955)2 is a possible candidate for radionuclide-based imaging of EGFR-expressing tumours. The biological effects of (ZEGFR:955)2 might be of interest for therapy applications.

    List of papers
    1.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
    2. Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor
    Open this publication in new window or tab >>Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor
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    2007 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 20, no 4, p. 189-199Article in journal (Refereed) Published
    Abstract [en]

    Affibody molecules specific for the epidermal growth factor receptor (EGFR) have been selected by phage display technology from a combinatorial protein library based on the 58-residue, protein A-derived Z domain. EGFR is overexpressed in various malignancies and is frequently associated with poor patient prognosis, and the information provided by targeting this receptor could facilitate both patient diagnostics and treatment. Three selected Affibody variants were shown to selectively bind to the extracellular domain of EGFR (EGFR-ECD). Kinetic biosensor analysis revealed that the three monomeric Affibody molecules bound with similar affinity, ranging from 130 to 185 nM. Head-to-tail dimers of the Affibody molecules were compared for their binding to recombinant EGFR-ECD in biosensor analysis and in human epithelial cancer A431 cells. Although the dimeric Affibody variants were found to bind in a range of 25-50 nM affinities in biosensor analysis, they were found to be low nanomolar binders in the cellular assays. Competition assays using radiolabeled Affibody dimers confirmed specific EGFR-binding and demonstrated that the three Affibody molecules competed for the same epitope. Immunofluorescence microscopy demonstrated that the selected Affibody dimers were initially binding to EGFR at the cell surface of A431, and confocal microscopy analysis showed that the Affibody dimers could thereafter be internalized. The potential use of the described Affibody molecules as targeting agents for radionuclide based imaging applications in various carcinomas is discussed.

    Keywords
    Affibod, EGFR, phage display, selection, targeting
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-97198 (URN)10.1093/protein/gzm011 (DOI)000246966800005 ()17452435 (PubMedID)
    Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2017-12-14Bibliographically approved
    3. Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule
    Open this publication in new window or tab >>Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule
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    2007 (English)In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 34, no 6, p. 609-618Article in journal (Refereed) Published
    Abstract [en]

    INTRODUCTION: The cellular binding and processing of an epidermal growth factor receptor (EGFR) targeting affibody molecule, (Z(EGFR:955))(2), was studied. This new and small molecule is aimed for applications in nuclear medicine. The natural ligand epidermal growth factor (EGF) and the antibody cetuximab were studied for comparison. METHODS: All experiments were made with cultured A431 squamous carcinoma cells. Receptor specificity, binding time patterns, retention and preliminary receptor binding site localization studies were all made after (125)I labeling. Internalization was studied using Oregon Green 488, Alexa Fluor 488 and CypHer5E markers. RESULTS: [(125)I](Z(EGFR:955))(2) and [(125)I]cetuximab gave a maximum cellular uptake of (125)I within 4 to 8 h of incubation, while [(125)I]EGF gave a maximum uptake already after 2 h. The retention studies showed that the cell-associated fraction of (125)I after 48 h of incubation was approximately 20% when delivered as [(125)I](Z(EGFR:955))(2) and approximately 25% when delivered as [(125)I]cetuximab. [(125)I]EGF-mediated delivery gave a faster (125)I release, where almost all cell-associated radioactivity had disappeared within 24 h. All three substances were internalized as demonstrated with confocal microscopy. Competitive binding studies showed that both EGF and cetuximab inhibited binding of (Z(EGFR:955))(2) and indicated that the three substances competed for an overlapping binding site. CONCLUSION: The results gave information on cellular processing of radionuclides when delivered with (Z(EGFR:955))(2) in comparison to delivery with EGF and cetuximab. Competition assays suggested that [(125)I](Z(EGFR:955))(2) bind to Domain III of EGFR. The affibody molecule (Z(EGFR:955))(2) can be a candidate for EGFR imaging applications in nuclear medicine.

    Keywords
    A431, Affibody molecule, EGFR, Internalization, Radionuclide, Retention
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-97199 (URN)10.1016/j.nucmedbio.2007.05.010 (DOI)000249157300003 ()17707800 (PubMedID)
    Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2017-12-14Bibliographically approved
    4. In vivo and in vitro uptake of In-111, delivered with the affibody molecule(Z(EGFR:955))(2), in EGFR expressing tumour cells
    Open this publication in new window or tab >>In vivo and in vitro uptake of In-111, delivered with the affibody molecule(Z(EGFR:955))(2), in EGFR expressing tumour cells
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    2008 (English)In: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 19, no 4, p. 853-857Article in journal (Refereed) Published
    Abstract [en]

    The epidermal growth factor receptor, EGFR, is overexpressed in many carcinomas. Targeting this receptor with radionuclides is important for imaging and therapy applications in nuclear medicine. We investigated the in vitro and in vivo properties of a new high affinity EGFR binding affibody molecule, (Z(EGFR:955))(2), when conjugated with CHXA"-DTPA and labelled with In-111. The binding time patterns and retention studies were performed using cultured squamous carcinoma A431 cells that overexpress EGFR. In the in vivo studies, female BALB/c nu/nu mice carrying tumours from xenografted A431 cells were used. The in vitro studies showed EGFR specific binding, high uptake and good retention of In-111 when delivered as [In-111](Z(EGFR:955))(2). The retention after 72 h of incubation was 38.0 +/- 1.15% of the initial level. The biodistribution study showed a tumour specific In-111 uptake of 3.8 +/- 1.4% of injected dose per gram turnout tissue 4 h post-injection. The tumour to blood ratio was 9.1 and the tumours could easily be visualized with a gamma camera at this time-point. In-111 delivered with [In-111](Z(EGFR:955))(2) gave an EGFR specific uptake and the results indicated that the (Z(EGFR:955))(2) affibody molecule is a candidate for radionuclide-based tumour imaging. Potential therapy applications are discussed.

    Keywords
    a431, affibody molecule, EGFR, mouse, radionuclide
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-152588 (URN)000254496100003 ()18357367 (PubMedID)
    Available from: 2011-04-28 Created: 2011-04-28 Last updated: 2017-12-11Bibliographically approved
    5. Effects of an EGFR-binding affibody molecule on intracellular signaling pathways
    Open this publication in new window or tab >>Effects of an EGFR-binding affibody molecule on intracellular signaling pathways
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    2010 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 36, no 4, p. 967-972Article in journal (Refereed) Published
    Abstract [en]

    Effects on intracellular signaling were studied in cells treated with the affibody molecule (Z(EGFR:955))(2) that targets the epithelial growth factor receptor (EGFR). EGFR is over-expressed in many types of cancers and plays a fundamental role in cell signaling and it is of interest to find targeting agents capable of blocking the receptor. The clinically approved antibody cetuximab (Erbitux (R)) and the natural ligand EGF were included as reference molecules. Two EGFR-rich cell lines, A-431 and U-343, were exposed to the three targeting agents and lysed. The cell lysates were immunoprecipitated with the receptors, or directly separated by SDS-Pace. Autophosphorylation of the receptors and phosphorylation of the downstream signaling proteins Erk and Akt, were evaluated by Western blotting. Although the three different agents compete for the same binding site on EGFR, they influenced the signaling differently. The affibody molecule did not induce autophosphorylation of EGFR or my other receptor in the EGFR-family but, in spite of this, induced phosphorylation of Erk in both cell lines and Akt in the A-431 cells. Thus, the results suggest that the signaling pattern induced by (Z(EGFR:955))(2) is only partly similar to that induced by cetuximab. This makes the affibody molecule a potentially interesting alternative to cetuximab for EGFR-targeted therapy since it might give different therapy-related effects on tumor cells and different side effects on normal tissues.

    Keywords
    A-431, affibody, Akt, epithelial growth factor receptor, Erk, signaling, U-343
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-97201 (URN)10.3892/ijo_00000576 (DOI)000275794300027 ()
    Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2017-12-14Bibliographically approved
  • 102.
    Nordberg, Erika
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Ekerljung, Lina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Häggblad Sahlberg, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Lennartsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Glimelius, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
    Effects of an EGFR-binding affibody molecule on intracellular signaling pathways2010In: International Journal of Oncology, ISSN 1019-6439, Vol. 36, no 4, p. 967-972Article in journal (Refereed)
    Abstract [en]

    Effects on intracellular signaling were studied in cells treated with the affibody molecule (Z(EGFR:955))(2) that targets the epithelial growth factor receptor (EGFR). EGFR is over-expressed in many types of cancers and plays a fundamental role in cell signaling and it is of interest to find targeting agents capable of blocking the receptor. The clinically approved antibody cetuximab (Erbitux (R)) and the natural ligand EGF were included as reference molecules. Two EGFR-rich cell lines, A-431 and U-343, were exposed to the three targeting agents and lysed. The cell lysates were immunoprecipitated with the receptors, or directly separated by SDS-Pace. Autophosphorylation of the receptors and phosphorylation of the downstream signaling proteins Erk and Akt, were evaluated by Western blotting. Although the three different agents compete for the same binding site on EGFR, they influenced the signaling differently. The affibody molecule did not induce autophosphorylation of EGFR or my other receptor in the EGFR-family but, in spite of this, induced phosphorylation of Erk in both cell lines and Akt in the A-431 cells. Thus, the results suggest that the signaling pattern induced by (Z(EGFR:955))(2) is only partly similar to that induced by cetuximab. This makes the affibody molecule a potentially interesting alternative to cetuximab for EGFR-targeted therapy since it might give different therapy-related effects on tumor cells and different side effects on normal tissues.

  • 103.
    Nordberg, Erika
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Friedman, Mikaela
    Göstring, Lovisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Adams, Gregory
    Brismar, Hjalmar
    Nilsson, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Ståhl, Stefan
    Glimelius, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule2007In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 34, no 6, p. 609-618Article in journal (Refereed)
    Abstract [en]

    INTRODUCTION: The cellular binding and processing of an epidermal growth factor receptor (EGFR) targeting affibody molecule, (Z(EGFR:955))(2), was studied. This new and small molecule is aimed for applications in nuclear medicine. The natural ligand epidermal growth factor (EGF) and the antibody cetuximab were studied for comparison. METHODS: All experiments were made with cultured A431 squamous carcinoma cells. Receptor specificity, binding time patterns, retention and preliminary receptor binding site localization studies were all made after (125)I labeling. Internalization was studied using Oregon Green 488, Alexa Fluor 488 and CypHer5E markers. RESULTS: [(125)I](Z(EGFR:955))(2) and [(125)I]cetuximab gave a maximum cellular uptake of (125)I within 4 to 8 h of incubation, while [(125)I]EGF gave a maximum uptake already after 2 h. The retention studies showed that the cell-associated fraction of (125)I after 48 h of incubation was approximately 20% when delivered as [(125)I](Z(EGFR:955))(2) and approximately 25% when delivered as [(125)I]cetuximab. [(125)I]EGF-mediated delivery gave a faster (125)I release, where almost all cell-associated radioactivity had disappeared within 24 h. All three substances were internalized as demonstrated with confocal microscopy. Competitive binding studies showed that both EGF and cetuximab inhibited binding of (Z(EGFR:955))(2) and indicated that the three substances competed for an overlapping binding site. CONCLUSION: The results gave information on cellular processing of radionuclides when delivered with (Z(EGFR:955))(2) in comparison to delivery with EGF and cetuximab. Competition assays suggested that [(125)I](Z(EGFR:955))(2) bind to Domain III of EGFR. The affibody molecule (Z(EGFR:955))(2) can be a candidate for EGFR imaging applications in nuclear medicine.

  • 104.
    Nordberg, Erika
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Steffen, Ann-Charlott
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Persson, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Sundberg, Åsa L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Glimelius, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Cellular uptake of radioiodine delivered by trastuzumab can be modified by the addition of epidermal growth factor.2005In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 32, no 7, p. 771-7Article in journal (Refereed)
    Abstract [en]

    PURPOSE: The purpose of this study was to analyse whether non-radiolabelled epidermal growth factor (EGF) can modify the cellular uptake of 125I when delivered as [125I]trastuzumab. 125I was used as a marker for the diagnostically and therapeutically more interesting isotopes 123I (SPECT), 124I (PET) and 131I (therapy). METHODS: The cell-associated radioactivity was measured in squamous carcinoma A431 cells following addition of [125I]trastuzumab. Different concentrations of [125I]trastuzumab and unlabelled EGF were used, and the total, membrane-bound and internalised radioactivity was measured. We also analysed how EGF and trastuzumab affected the cell growth. RESULTS: It was generally found that the cellular 125I uptake was decreased by the addition of EGF when [125I]trastuzumab was added for short incubation times. However, if the incubation times were longer, EGF increased the 125I uptake. This shift came earlier when higher [125I]trastuzumab concentrations were applied. The addition of EGF also influenced cell proliferation, and concentrations above 10 ng/ml reduced cell growth by approximately 20% after 24 h of incubation. CONCLUSION: By adding unlabelled EGF, it was possible to modify the cellular uptake of [125I]trastuzumab. This points towards new approaches for the modification of radionuclide uptake in EGFR- and HER2-positive tumours.

  • 105.
    Onell, Annica
    et al.
    Biacore AB.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Kinetic determinations of molecular interactions using Biacore: minimum data requirements for efficient experimental design2005In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 18, no 4, p. 307-17Article in journal (Refereed)
    Abstract [en]

    Reliable kinetic estimates can be obtained from significantly less data than is commonly used today, particularly in the characterization of 1:1 interactions involving low molecular weight compounds and proteins. We have designed a rational and cost-effective strategy to determine kinetic constants using Biacore's surface plasmon resonance-based biosensors and show that the number of measurements necessary for accurate kinetic determinations can be greatly reduced, increasing sample throughput and saving sample material. Simulated and measured data for a range of possible 1:1 interactants were studied to find the minimum requirements of a data set for kinetic analysis. The results showed that kinetic constants in the region 10(4) < k(a) < 10(7) M(-1) s(-1) (association) and 10(-4) < k(d) < 10(-1) s(-1) (dissociation) could easily be determined in a 1:1 interaction model. Owing to the information-dense nature of Biacore data, only two sample concentrations were necessary to reliably determine the kinetics. A standard sample concentration series consisting of 10-fold dilutions between approximately 10 microM and approximately 1 nM consistently provided at least two concentrations with sufficient information about the interaction in this region. Determinations of the constants became increasingly unreliable outside this region. If the rate constants prove to be outside the specified region or the data fits poorly to the 1:1-MTL model, more experiments are required. General recommendations for the design of a cost-effective assay to deliver reliable kinetic measurements are provided.

  • 106.
    Orlova, Anna
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Indirect Radiohalogenation of Targeting Proteins: Labelling Chemistry and Biological Characterisation2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In about half of all newly diagnosed cancer cases, conventional treatment is not adequately curative, mainly due to the failure of conventional techniques to find and kill residual cells and metastases, which might consist of only a few malignant cells, without causing unacceptable complications to healthy tissue. To solve the problem a more selective delivery of cytotoxic substances to tumour cells is needed. The approach applied here is called ‘tumour targeting’ and implies the use of biomolecules that recognise specific molecular structures on the malignant cell surface. Such molecules are then used for a selective transport of toxic agents to the cancer cells.

    The use of radionuclides as cytotoxic substances has a number of advantages: 1) radiation does not cause severe resistance; 2) there is a cross-fire effect and 3) smaller amounts of nuclides are required than other cytotoxic substances to cause the same damage. Such an approach is called radionuclide tumour therapy. Several factors are important for the success of radionuclide therapy, such as the pharmacokinetics of the radiolabelled substance and its radiocatabolites, as well as the physical and chemical properties of the radiolabel used.

    Nuclear properties of the label should be consistent with the problem to be solved: primary diagnostics; quantification of pharmacokinetics and dose planning; or therapy. From this point of view, radiohalogens are an attractive group of radiolabels. Halogens have nuclides with a variety of physical properties while the chemical and biological properties of halogens are very similar. The same labelling procedures can be used for all heavy halogens, i.e. bromine, iodine and astatine. It has been demonstrated that the biodistribution of proteins labelled with different heavy halogens is quite similar.

    The main goal of the study was to develop protein radiohalogenation methods that provide a stable halogen-protein bond, convenient labelling chemistry that preserves the binding properties of proteins, long intracellular retention of radioactivity in targeted cells and quick release of radiohalogenated catabolites from the blood circulation. Radiohalogenation of proteins using indirect methods was studied, including optimisation of labelling chemistry and biological characterisation of some labelled conjugates. Two groups for indirect radiohalogenation were used, representing two different labelling principles: activated ester of benzoic acid (1) and the derivative of closo-dodecaborate anion (2). The non-phenolic linker (1) as well as the borate-halogen moiety (2) probably prevent dehalogenation. The negative charge of the potential catabolic products of (2) might trap radiohalogens intracellularly.

    List of papers
    1. Optimization of iodination of [125I]-N-succinimidyl-para-iodobenzoate using Chloromine-T for labeling of proteins
    Open this publication in new window or tab >>Optimization of iodination of [125I]-N-succinimidyl-para-iodobenzoate using Chloromine-T for labeling of proteins
    2000 (English)In: Journal of Radioanalytical and Nuclear Chemistry, ISSN 0236-5731, E-ISSN 1588-2780, Vol. 246, no 1, p. 207-213Article in journal (Refereed) Published
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-90194 (URN)
    Available from: 2003-04-03 Created: 2003-04-03 Last updated: 2017-12-14Bibliographically approved
    2.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
    3.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
    4. Polyhedral boron clasters as linkers for attachment of radiohalogens to tumor targeting proteins. II. Radioiodination of monoclonal antibody using potassium [125I]-4-(isothiocyanatobenzylammonio)-iodo-decahydro-closo-dodecaborate (Iodo-DABI)
    Open this publication in new window or tab >>Polyhedral boron clasters as linkers for attachment of radiohalogens to tumor targeting proteins. II. Radioiodination of monoclonal antibody using potassium [125I]-4-(isothiocyanatobenzylammonio)-iodo-decahydro-closo-dodecaborate (Iodo-DABI)
    Show others...
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-90197 (URN)
    Available from: 2003-04-03 Created: 2003-04-03 Last updated: 2015-03-24Bibliographically approved
    5.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
  • 107.
    Orlova, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Bruskin, Alexander
    Sivaev, Igor
    Sjoberg, Stefan
    Lundqvist, Hans
    Tolmachev, Vladimir
    Polyhedral boron clasters as linkers for attachment of radiohalogens to tumor targeting proteins. II. Radioiodination of monoclonal antibody using potassium [125I]-4-(isothiocyanatobenzylammonio)-iodo-decahydro-closo-dodecaborate (Iodo-DABI)Manuscript (Other academic)
  • 108.
    Orlova, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Bruskin, Alexander
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Sivaev, Igor
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry.
    Sjöberg, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry.
    Lundqvist, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Radio-iodination of monoclonal antibody using potassium [125I]-(4-isothiocyanatobenzylammonio)-iodo-decahydro-closo-dodecaborate (iodo-DABI)2006In: Anticancer Research, ISSN 0250-7005, E-ISSN 1791-7530, Vol. 26, no 2A, p. 1217-1223Article, book review (Other academic)
    Abstract [en]

    BACKGROUND:

    Negatively-charged polyhedral boron clusters can be easily halogenated with highly stable boron-halogen bonds and are promising in radionuclide diagnostics and cancer therapy.

    MATERIALS AND METHODS:

    The radio-iodination conditions for the closo-dodecaborate anion and for the conjugation of its labeled isothiocyanatobenzylammonio derivative to the monoclonal antibody (mAb) were optimized.

    RESULTS:

    The labeling yield was about 90% and the overall conjugation yield was 55-60%. The in vitro stability of the radio-iodinated mAb was good under physiological and non-physiological conditions. The immunoreactivity of the labeled mAb (SK-BR-3 cells) was retained in the one-pot two-step labeling.

    CONCLUSION:

    Negatively-charged polyhedral boron clusters can be used for indirect radio-iodination of mAbs.

  • 109.
    Orlova, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Bruskin, Alexander
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Sivaev, Igor
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Sjöberg, Stefan
    Lundqvist, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Radio-iodination of monoclonal antibody using potassium [125I]-(4-isothiocyanatobenzylammonio)-iodo-decahydro-closo-dodecaborate (iodo-DABI)2006In: Anticancer Research, ISSN 0250-7005, E-ISSN 1791-7530, Vol. 26, no 2A, p. 1217-23Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Negatively-charged polyhedral boron clusters can be easily halogenated with highly stable boron-halogen bonds and are promising in radionuclide diagnostics and cancer therapy. MATERIALS AND METHODS: The radio-iodination conditions for the closo-dodecaborate anion and for the conjugation of its labeled isothiocyanatobenzylammonio derivative to the monoclonal antibody (mAb) were optimized. RESULTS: The labeling yield was about 90% and the overall conjugation yield was 55-60%. The in vitro stability of the radio-iodinated mAb was good under physiological and non-physiological conditions. The immunoreactivity of the labeled mAb (SK-BR-3 cells) was retained in the one-pot two-step labeling. CONCLUSION: Negatively-charged polyhedral boron clusters can be used for indirect radio-iodination of mAbs.

  • 110.
    Orlova, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Bruskin, Alexander
    Sjöström, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Lundqvist, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Gedda, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tolmavhev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Cellular processing of 125I- and 111in-labeled epidermal growth factor (EGF) bound to cultured A431 tumor cells2000In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 27, no 8, p. 827-835Article, book review (Other academic)
    Abstract [en]

    Low molecular weight of epidermal growth factor (EGF) enables better intratumoral penetration in comparison with larger targeting proteins, but the cellular retention of EGF-associated radioactivity is poor for directly iodinated EGF. An attempt was made to improve intracellular retention by the use of metal-diethylenetriaminepentaacetic acid or nonphenolic linker (N-succinimidyl-para-iodobenzoate) as labeling agents. The use of nonphenolic linker did not improve retention of the radioactivity in A431 carcinoma cell line. The use of the radiometal label provided an appreciable prolongation of radioactivity residence inside the cell.

  • 111.
    Orlova, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Feldwisch, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Abrahmsén, Lars
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Update: Affibody molecules for molecular imaging and therapy for cancer2007In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 22, no 5, p. 573-584Article in journal (Refereed)
    Abstract [en]

    Affibody molecules are scaffold proteins, having a common frame of amino acids determining the overall fold or tertiary structure, but with each member characterized by a unique amino acid composition in an exposed binding surface determining binding specificity and affinity for a certain target. Affibody molecules represent a new class of affinity proteins based on a 58-amino acid residue protein domain, derived from one of the IgG binding domains of staphylococcal protein A. They combine small size ( approximately 6.5 kDa) with high affinity and specificity. Affibody molecules with nanomolar affinities were selected from an initial library (3 x 10(9) members) and, after affinity maturation, picomolar binders were obtained. The small size and simple structure of affibody molecules allow their production by chemical synthesis with homogeneous site-specific incorporation of moieties for further labeling using a wide range of labeling chemistries. The robustness and the refolding properties of affibody molecules make them amenable to labeling conditions that denature most proteins, including incubation at pH 11 at 60 degrees C for up to 60 minutes. Affibody molecules meet the requirements which are key for successful clinical use as imaging agents: high-affinity binding to the chosen target; short plasma half-life time; rapid renal clearance for nonbound drug substance and, high, continuously increasing tumor-to-organ ratios, resulting in high-contrast in vivo images shortly after injection of the diagnostic agent.

  • 112.
    Orlova, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Feldwisch, Joachim
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Influence of an aliphatic linker between DOTA and synthetic Affibody molecule on targeting properties2010In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 37, no 6, p. 695-695Article in journal (Other academic)
  • 113.
    Orlova, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Magnusson, Mikaela
    Affibody AB.
    Eriksson, Tove L. J.
    Affibody AB.
    Nilsson, Martin
    Affibody AB.
    Larsson, Barbro
    Affibody AB.
    Höidén-Guthenberg, Ingmarie
    Affibody AB.
    Widström, Charles
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Ståhl, Stefan
    KTH Sthlm.
    Nilsson, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tumor imaging using a picomolar affinity HER2 binding affibody molecule2006In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 66, no 8, p. 4339-48Article in journal (Refereed)
    Abstract [en]

    The detection of cell-bound proteins that are produced due to aberrant gene expression in malignant tumors can provide important diagnostic information influencing patient management. The use of small radiolabeled targeting proteins would enable high-contrast radionuclide imaging of cancers expressing such antigens if adequate binding affinity and specificity could be provided. Here, we describe a HER2-specific 6 kDa Affibody molecule (hereinafter denoted Affibody molecule) with 22 pmol/L affinity that can be used for the visualization of HER2 expression in tumors in vivo using gamma camera. A library for affinity maturation was constructed by re-randomization of relevant positions identified after the alignment of first-generation variants of nanomolar affinity (50 nmol/L). One selected Affibody molecule, Z(HER2:342) showed a >2,200-fold increase in affinity achieved through a single-library affinity maturation step. When radioiodinated, the affinity-matured Affibody molecule showed clear, high-contrast visualization of HER2-expressing xenografts in mice as early as 6 hours post-injection. The tumor uptake at 4 hours post-injection was improved 4-fold (due to increased affinity) with 9% of the injected dose per gram of tissue in the tumor. Affibody molecules represent a new class of affinity molecules that can provide small sized, high affinity cancer-specific ligands, which may be well suited for tumor imaging.

  • 114.
    Orlova, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Nilsson, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Wikman, Maria
    KTH Sthlm.
    Widström, Charles
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Section of Medical Physics.
    Ståhl, Stefan
    KTH Sthlm.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Comparative in vivo evaluation of technetium and iodine labels on an anti-HER2 affibody for single-photon imaging of HER2 expression in tumors2006In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 47, no 3, p. 512-519Article in journal (Refereed)
    Abstract [en]

    In vivo diagnosis with cancer-specific targeting agents that have optimal characteristics for imaging is an important development in treatment planning for cancer patients. Overexpression of the HER2 antigen is high in several types of carcinomas and has predictive and prognostic value, especially for breast cancer. A new type of targeting agent, the Affibody molecule, was described recently. An Affibody dimer, His6-(ZHER2:4)2 (15.4 kDa), binds to HER2 with an affinity of 3 nmol/L and might be used for the imaging of HER2 expression. The use of 99mTc might improve the availability of the labeled conjugate, and Tc(I)-carbonyl chemistry enables the site-specific labeling of the histidine tag on the Affibody molecule. The goals of the present study were to prepare 99mTc-labeled His6-(ZHER2:4)2 and to evaluate its targeting properties compared with the targeting properties of 125I-4-iodobenzoate-His6-(ZHER2:4)2 [125I-His6-(ZHER2:4)2]. METHODS: The labeling of His6-(ZHER2:4)2 with 99mTc was performed with an IsoLink kit. The specificity of 99mTc-His6-(ZHER2:4)2 binding to HER2 was evaluated in vitro with SK-OV-3 ovarian carcinoma cells. The comparative biodistributions of 99mTc-His6-(ZHER2:4)2 and 125I-His6-(ZHER2:4)2 in tumor-bearing BALB/c nu/nu mice were determined. RESULTS: The labeling yield for 99mTc-His6-(ZHER2:4)2 was approximately 60% (50 degrees C), and the radiochemical purity was greater than 97%. The conjugate was stable during storage and under histidine and cysteine challenges and demonstrated receptor-specific binding. The biodistribution study demonstrated tumor-specific uptake levels (percentage injected activity per gram of tissue [%IA/g]) of 2.6 %IA/g for 99mTc-His6-(ZHER2:4)2 and 2.3 %IA/g for 125I-His6-(ZHER2:4)2 at 4 h after injection. Both conjugates provided clear imaging of SK-OV-3 xenografts at 6 h after injection. The tumor-to-nontumor ratios were much more favorable for the radioiodinated Affibody. CONCLUSION: The use of Tc(I)-carbonyl chemistry enabled us to prepare a stable, site-specifically labeled 99mTc-His6-(ZHER2:4)2 conjugate that was able to bind to HER2-expressing cells in vitro and in vivo. The indirectly radioiodinated conjugate provided better tumor-to-liver ratios. The labeling of Affibody molecules with 99mTc should be investigated further.

  • 115.
    Orlova, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Sivaev, I.
    Sjöberg, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry.
    Lundqvist, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Radioiodination of ammonio-closo-monocarborane, 1-H3N-1-CB1 H11: Aspects of labelling chemistry in aqueous solution using Chloramine-T2004In: Radiochimica Acta, ISSN 0033-8230, E-ISSN 2193-3405, no 92, p. 311-Article in journal (Refereed)
  • 116.
    Orlova, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Sjöström, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Ericson, A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Lebeda, Ondrej
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Lundqvist, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Cellular processing of indirectly astatinated and iodinated mAb A33 in SW1222 cultured cells2001In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 44, no suppl 1, p. S715-S717Article, book review (Other academic)
    Abstract [en]

    In principle, alpha-emitting radionuclides, such as 211At, are more efficient than beta-emitters to inactive single disseminated cancer cells. However, cellular processing of astatinated proteins has not yet been studied in detail. In this study an anti-colorectal cancer monoclonal antibody (mAb) A33 was indirectly labeled with 211At and for comparison with 125I. Binding and retention of radioactivity was studied in the colorectal cancer cell-line SW1222. A similar pattern of binding and retention of the two radiohalogens was seen. The main difference found, that the retention time of astatinated mAb in SW1222 was almost two times longer, might be of advantage in radionuclide therapy.

  • 117.
    Orlova, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Sjöström, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Lebeda, Ondrej
    Nuclear Physics Institute, Rez, Czech Republic.
    Lundqvist, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Carlsson, Jorgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Targeting against epidermal growth factor receptors: Cellular processing of astatinated EGF after binding to cultured carcinoma cells2004In: Anticancer Research, ISSN 0250-7005, E-ISSN 1791-7530, Vol. 24, no 6, p. 4035-4042Article in journal (Refereed)
    Abstract [en]

    BACKGROUND:

    The alpha-emitting nuclide 211At is of great interest for radionuclide therapy when coupled to a tumor-targeting biomolecule, e.g. epidermal growth factor (EGF) the receptors of which are overexpressed in many malignancies. However, almost no information concerning the cellular processing of astatinated targeting agents is available.

    MATERIALS AND METHODS:

    We indirectly astatinated EGF ([211At]-benzoate-EGF) and studied its cellular processing in A-431 carcinoma cells in comparison with data concerning [125I]-benzoate-EGF.

    RESULTS:

    The biological half-life of astatine (3.5 h) was longer than the half-life of the iodine label (1.5 h). The increase of the half-life was due to longer retention of the internalised astatine radioactivity. The maximum accumulation for the astatine label occurred later (4-6h) than that for the iodine label (2-4h), indicating a slower excretion of astatine that was confirmed in experiment with 211At/1251-benzoate-EGF.

    CONCLUSION:

    The long retention of astatine might be advantageous for radionuclide therapy.

  • 118.
    Orlova, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Sundberg, Å.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Bruskin, Alexander
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Gedda, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    [111In] benzyl-DTPA-EGF, a new potential radiopeptide for targeting of glioma2002In: European Journal of Nuclear Medicine, ISSN 0340-6997, E-ISSN 1432-105X, Vol. 29, p. S367-Article, book review (Other academic)
  • 119.
    Orlova, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tran, Thuy A
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Ekblad, Torun
    Karlström, Amelie Eriksson
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Re-186-maSGS-Z(HER2:342), a potential Affibody conjugate for systemic therapy of HER2-expressing tumours2010In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 37, no 2, p. 260-269Article in journal (Refereed)
    Abstract [en]

    PURPOSE: Affibody molecules are a novel class of tumour-targeting proteins, which combine small size (7 kDa) and picomolar affinities. The Affibody molecule Z(HER2:342) has been suggested for imaging of HER2 expression in order to select patients for trastuzumab therapy. When optimizing chelators for (99m)Tc-labelling, we have found that synthetic Z(HER2:342) conjugated with mercaptoacetyl-glycyl-glycyl-glycyl (maGGG) and mercaptoacetyl-glycyl-seryl-glycyl (maGSG) chelators provides relatively low renal uptake of radioactivity and could be suitable for therapy. METHODS: maGGG-Z(HER2:342) and maGSG-Z(HER2:342) were labelled with (186)Re and their biodistribution was studied in normal mice. Dosimetric evaluation and tumour targeting to HER2-overexpressed xenografts (SKOV-3) by (186)Re-maGSG-Z(HER2:342) were studied. RESULTS: Gluconate-mediated labelling of maGGG-Z(HER2:342) and maGSG-Z(HER2:342) with (186)Re provided a yield of more than 95% within 60 min. The conjugates were stable and demonstrated specific binding to HER2-expressing SKOV-3 cells. Biodistribution in normal mice demonstrated rapid blood clearance, low accumulation of radioactivity in the kidney and other organs, accumulating free perrhenate. Both (186)Re-maGGG-Z(HER2:342) and (186)Re-maGSG-Z(HER2:342) demonstrated lower renal uptake than their (99m)Tc-labelled counterparts. (186)Re-maGSG-Z(HER2:342) provided the lowest uptake in healthy tissues. Biodistribution of (186)Re-maGSG-Z(HER2:342) in nude mice bearing SKOV-3 xenografts showed specific targeting of tumours. Tumour uptake 24 h after injection (5.84+/-0.54%ID/g) exceeded the concentration in blood by more than 500-fold, and uptake in kidneys by about 8-fold. Preliminary dosimetric evaluation showed that dose-to-tumour should exceed dose-to-kidney by approximately 5-fold. CONCLUSION: Optimization of chelators improves biodistribution properties of rhenium-labelled small scaffold proteins and enables selection of promising radiotherapeutic agents based on the Affibody molecule.

  • 120.
    Orlova, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tran, Thuy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Widström, Charles
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Section of Medical Physics.
    Engfeldt, Torun
    Eriksson Karlström, Amelie
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Pre-clinical evaluation of [111In]-benzyl-DOTA-Z(HER2:342), a potential agent for imaging of HER2 expression in malignant tumors2007In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 20, no 3, p. 397-404Article in journal (Refereed)
    Abstract [en]

    Imaging of expression of human epidermal growth factor receptor type 2 (HER2) in breast carcinomas may help to select patients eligible for trastuzumab therapy. The Affibody molecule Z(HER2:342) is a small (7-kDa) non-immunoglobulin affinity protein, which binds to HER2 with a picomolar affinity. Previously, a benzyl-DTPA conjugate of Z(HER2:342) was labeled with 111In and demonstrated good targeting in murine xenografts. We considered that the use of the macrocyclic chelator DOTA could increase the label stability and enhance a choice of nuclides, which could be used as a label for Z(HER2:342). The goal of this study was the preparation and pre-clinical evaluation of the indium-111- labeled DOTA-derivative of Z(HER2:342). Isothiocyanate-benzyl-DOTA was coupled to recombinant Z(HER2:342), and the conjugate was efficiently labeled with 111In at 60 degrees C. The specificity of 111In-benzyl-DOTA-Z(HER2:342) binding to HER2 was confirmed in vitro using HER2-expressing breast carcinoma BT474 and ovarian carcinoma SKOV-3 cell lines. Biodistribution of 111In-benzyl-DOTA-Z(HER2:342) was performed in nude mice bearing LS174T xenografts and compared directly with the biodistribution of 111In-benzyl-DTPA-Z(HER2:342). In vivo, 111In-benzyl-DOTA-Z(HER2:342) demonstrated quick clearance from blood and non-specific organs except the kidneys. Four hours post injection (pi), the tumor uptake of 111In-benzyl-DOTA-Z(HER2:342) (4.4+/-1.0% IA/g) was specific and the tumor-to-blood ratio was 23. The use of benzyl-DTPA provided higher tumor-to-blood and tumor-to-liver ratios. gamma-camera imaging showed clear visualization of HER2-expressing xenografts using 111In-benzyl-DOTA-Z(HER2:342). 111In-benzyl-DOTA-Z(HER2:342) has a potential for imaging of HER2 expression in malignant tumors.

  • 121.
    Orlova, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Wållberg, Helena
    Stone-Elander, Sharon
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    On the selection of a tracer for PET imaging of HER2-expressing tumors: direct comparison of 124I-labelled Affibody molecule and trastuzumab in a murine xenograft model2009In: European Journal of Nuclear Medicine, ISSN 0340-6997, E-ISSN 1432-105X, Vol. 50, no 3, p. 417-425Article in journal (Refereed)
    Abstract [en]

    Human epidermal growth factor receptor type 2 (HER2) is a tyrosine kinase, which is often overexpressed in many carcinomas. Imaging HER2 expression in malignant tumors can provide important prognostic and predictive diagnostic information. The use of anti-HER2 tracers labeled with positron-emitting radionuclides may increase the sensitivity of HER2 imaging. The goal of this study was to compare directly 2 approaches for developing anti-HER2 PET tracers: a (124)I-labeled monoclonal antibody and a small (7-kDa) scaffold protein, the Affibody molecule. METHODS: The anti-HER2 Affibody Z(HER2:342) and humanized monoclonal antibody trastuzumab were labeled with (124/125)I using p-iodobenzoate (PIB) as a linker. Cellular processing of both tracers by HER2-expressing cells was investigated. The biodistributions of (124)I-PIB-Z(HER2:342) and (125)I-PIB-trastuzumab were compared in BALB/C nu/nu mice bearing HER2-expressing NCI-N87 xenografts using paired labels. Small-animal PET of (124)I-PIB-Z(HER2:342) and (124)I-PIB-trastuzumab in tumor-bearing mice was performed at 6, 24, and 72 h after injection. RESULTS: Both radioiodinated Z(HER2:342) and trastuzumab bound specifically to HER2-expressing cells in vitro and specifically targeted HER2-expressing xenografts in vivo. Radioiodinated trastuzumab was more rapidly internalized and degraded, which resulted in better retention of radioactivity delivered by Z(HER2:342). Total uptake of trastuzumab in tumors was higher than that of (124)I-PIB-Z(HER2:342). However, tumor-to-organ ratios were appreciably higher for (124)I-PIB-Z(HER2:342) due to the more rapid clearance of radioactivity from blood and normal organs. The ex vivo results were confirmed by small-animal PET. CONCLUSION: The use of the small scaffold targeting Affibody provides better contrast in HER2 imaging than does the monoclonal antibody.

  • 122. Orre, Lukas M.
    et al.
    Stenerlöw, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Dhar, Sumeer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Lewensohn, Rolf
    Lehtiö, Janne
    p53 is involved in clearance of ionizing radiation-induced RAD51 foci in a human colon cancer cell line2006In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 342, no 4, p. 1211-7Article in journal (Refereed)
    Abstract [en]

    We have investigated p53-related differences in cellular response to DNA damaging agents, focusing on p53s effects on RAD51 protein level and sub-cellular localization post exposure to ionizing radiation. In a human colon cancer cell line, HCT116 and its isogenic p53-/- subcell line we show here p53-independent RAD51 foci formation but interestingly the resolution of RAD51 foci showed clear p53 dependence. In p53 wt cells, but not in p53-/- cells, RAD51 protein level decreased 48 h post irradiation and fluorescence immunostaining showed resolution of RAD51 foci and relocalization of RAD51 to nucleoli at time points corresponding to the decrease in RAD51 protein level. Both cell lines rejoined DNA double strand breaks efficiently with similar kinetics and p53 status did not influence sensitivity to DNA damaging agents. We suggest that p53 has a role in RAD51 clearance post DSB repair and that nucleoli might be sites of RAD51 protein degradation.

  • 123.
    Persson, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Antibody Mediated Radionuclide Targeting of HER-2 for Cancer Diagnostics and Therapy: Preclinical Studies2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Targeted radionuclide therapy (TRT) holds great promise for the treatment of cancer. In TRT, radioactive nuclides are delivered specifically to tumours by molecules that recognise and bind to structures overexpressed by, or specific to, cancer cells. Human epidermal growth factor receptor like protein 2 (HER-2) is an oncogene product overexpressed in e.g. urological, breast, or ovarian cancers that have been correlated to poor prognosis and resistance to hormonal therapy. There is also evidence that tumour cells retain their HER-2 overexpression in metastases.

    Trastuzumab and pertuzumab are two humanised monoclonal antibodies targeting different parts of HER-2. This thesis describes the radiolabelling of these antibodies for use in TRT and diagnostics. The thesis also investigates possible methods for modifying uptake and retention of radioactivity delivered with antibodies binding to HER-2. Modification of the cellular retention of 125I by using polyhedral boron anion based linker molecules (DABI and NBI) is investigated, and it is shown that linking 125I to trastuzumab using DABI increases cellular accumulation of radioactivity by 33%. It is also shown that trastuzumab can be efficiently coupled to the positron emitter 76Br by using NBI. Furthermore, it is shown that cellular uptake of 125I can be modified by stimulating EGFR (HER-1) with EGF.

    When labelled with the alpha emitter 211At, trastuzumab could specifically kill cells in vitro. This cell killing effect could be prevented by saturating the receptors of the target cells with non-radiolabelled trastuzumab.

    Pertuzumab was radiolabelled with the low energy beta emitter 177Lu without losing affinity or immunocompetence. [177Lu]pertuzumab was specific to HER-2 in vitro and in vivo. This targeting conjugate was shown to increase median time to tumour progression in mice bearing xenografts of the radioresistant SKOV-3 cell line.

    In conclusion, antibodies against HER-2, especially pertuzumab radiolabelled with 177Lu, show promise as TRT agents.

    List of papers
    1.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
    2. In vitro evaluation of two polyhedral boron anion derivatives as linkers for attachment of radioiodine to the anti-HER2 monoclonal antibody trastuzumab
    Open this publication in new window or tab >>In vitro evaluation of two polyhedral boron anion derivatives as linkers for attachment of radioiodine to the anti-HER2 monoclonal antibody trastuzumab
    Show others...
    2007 (English)In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 22, no 5, p. 585-596Article in journal (Refereed) Published
    Abstract [en]

    Improving intracellular retention is important for the use of radiohalogens in radionuclide therapy usinginternalizing antibodies. Two putative linkers for residualization of radioiodine labels, 7-(4-isothiocyanato-phenyl)undecahydro-7,8-dicarba-nido-undecaborate(1Ϫ) ion (NBI) and (4-isothiocyanato-benzylammo-nio)undecahydro-closo-dodecaborate(1Ϫ) (DABI), were analyzed. The anti-HER-2 antibody, trastuzumab,was labeled with iodine-125 using NBI and DABI linkers, and, for comparison, with the para-[125I]iodoben-zoate (PIB), and Chloramine-T (CAT) methods. The different labels were tested for residualizing prop-erties using the HER-2 overexpressing SKBR-3 cells. The cellular radioactivity retention showed thatDABI provided a 55% better retention than CAT and was 42% better than PIB after 20 hours. NBI didnot improve retention. Accumulation tests up to 21 hours showed that the HER-2-specific accumulationof radioactivity delivered with DABI was, on average, 33% higher than with the use of PIB. These DABI-dependent improvements could, with high probability, be attributed to the good residualizing propertiesof DABI. The affinity of DABI-labeled trastuzumab to SKBR-3 cells was not better than the affinity of thePIB labeled (3.2 Ϯ 1.9 nM and 0.77 Ϯ 0.39 nM, respectively). In conclusion, the use of the DABI linkerimproved intracellular retention in vitro in comparison with the other labeling methods.

    Keywords
    antibody, radiolabeling, polyhedral boron anion, HER-2
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-94330 (URN)10.1089/cbr.2006.338 (DOI)000250821800002 ()17979561 (PubMedID)
    Available from: 2006-04-20 Created: 2006-04-20 Last updated: 2018-12-04
    3.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
    4.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
    5.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
    6. [Lu-177]pertuzumab: Experimental therapy of HER-2-expressing xenografts
    Open this publication in new window or tab >>[Lu-177]pertuzumab: Experimental therapy of HER-2-expressing xenografts
    Show others...
    2007 (English)In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 67, no 1, p. 326-331Article in journal (Refereed) Published
    Abstract [en]

    Pertuzumab (Omnitarg) is a novel antibody against HER-2, domain II. HER-2 is a tyrosine kinase receptor that is overexpressed in several carcinomas, especially breast cancer. Pertuzumab, labeled with the low-energy beta emitter Lu-177, might be a candidate for targeted radiotherapy of disseminated HER-2-positive micrometastases. The radiolabeled antibody [Lu-177]pertuzumab showed favorable targeting properties in BALB/c (nu/nu) mice with HER-2-overexpressing xenografts. The absorbed dose in tumors was more than five times higher than the absorbed dose in blood and more than seven times the absorbed dose in any other normal organ. Experimental therapy showed that [Lu-177]pertuzumab delayed tumor progression compared with controls (no treatment, P < 0.0001; nonlabeled pertuzumab antibody, P < 0.0001; and Lu-177-labeled irrelevant antibody, P < 0.01). No adverse side effects of the treatment could be detected. Thus, the experimental results support the planning of clinical studies applying [Lu-177]pertuzumab for therapy.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-94334 (URN)10.1158/0008-5472.CAN-06-2363 (DOI)000243320000041 ()17210714 (PubMedID)
    Available from: 2006-04-20 Created: 2006-04-20 Last updated: 2018-12-04
  • 124.
    Persson, Mikael
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Gedda, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Lundqvist, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Nordgren, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Malmström, Per-Uno
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Urology.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    [Lu-177]pertuzumab: Experimental therapy of HER-2-expressing xenografts2007In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 67, no 1, p. 326-331Article in journal (Refereed)
    Abstract [en]

    Pertuzumab (Omnitarg) is a novel antibody against HER-2, domain II. HER-2 is a tyrosine kinase receptor that is overexpressed in several carcinomas, especially breast cancer. Pertuzumab, labeled with the low-energy beta emitter Lu-177, might be a candidate for targeted radiotherapy of disseminated HER-2-positive micrometastases. The radiolabeled antibody [Lu-177]pertuzumab showed favorable targeting properties in BALB/c (nu/nu) mice with HER-2-overexpressing xenografts. The absorbed dose in tumors was more than five times higher than the absorbed dose in blood and more than seven times the absorbed dose in any other normal organ. Experimental therapy showed that [Lu-177]pertuzumab delayed tumor progression compared with controls (no treatment, P < 0.0001; nonlabeled pertuzumab antibody, P < 0.0001; and Lu-177-labeled irrelevant antibody, P < 0.01). No adverse side effects of the treatment could be detected. Thus, the experimental results support the planning of clinical studies applying [Lu-177]pertuzumab for therapy.

  • 125.
    Persson, Mikael I.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Gedda, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Jensen, H. J.
    Danmark.
    Lundqvist, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Malmström, Per-Uno
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Astatinated trastuzumab, a putative agent for radionuclide immunotherapy of ErbB2-expressing tumours2006In: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 15, no 3, p. 673-80Article in journal (Refereed)
    Abstract [en]

    The anti-ErbB2 antibody trastuzumab is used for the treatment of patients with advanced breast cancer, resulting in a response rate of 40-60%. Coupling with a cytotoxic nuclide, e.g. alpha-emitting 211At, may further increase tumour response. The tumour-targeting properties of trastuzumab, astatinated using N-succinimidyl-para-(tri-n-methylstannyl)-benzoate, were evaluated and compared with those of radioiodinated trastuzumab in this study. We found that astatinated trastuzumab retains high specificity towards ErbB2. While the immunoreactive fraction of radioiodinated trastuzumab was higher than that of astatinated trastuzumab (76+/-9% versus 54+/-28%), both radioconjugates showed high affinity (KD 0.75+/-0.16 nM versus 1.8+/-0.3 nM). A growth inhibition study indicated a dose-dependent cell deactivation, in which approximately 74 cell-associated astatine decays per cell gave a survival fraction of 4.5+/-0.8x10(-4). Results of a comparative animal study on normal mice gave no indication that astatination would have any adverse effects on the biodistribution of the antibody. In conclusion, the results of the study suggest that astatinated trastuzumab is a promising candidate for treating ErbB2-expressing tumours.

  • 126.
    Persson, Mikael
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Sivaev, Igor
    Winberg, Karl-Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry.
    Gedda, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Malmström, Per-Uno
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Urology.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    In vitro evaluation of two polyhedral boron anion derivatives as linkers for attachment of radioiodine to the anti-HER2 monoclonal antibody trastuzumab2007In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 22, no 5, p. 585-596Article in journal (Refereed)
    Abstract [en]

    Improving intracellular retention is important for the use of radiohalogens in radionuclide therapy usinginternalizing antibodies. Two putative linkers for residualization of radioiodine labels, 7-(4-isothiocyanato-phenyl)undecahydro-7,8-dicarba-nido-undecaborate(1Ϫ) ion (NBI) and (4-isothiocyanato-benzylammo-nio)undecahydro-closo-dodecaborate(1Ϫ) (DABI), were analyzed. The anti-HER-2 antibody, trastuzumab,was labeled with iodine-125 using NBI and DABI linkers, and, for comparison, with the para-[125I]iodoben-zoate (PIB), and Chloramine-T (CAT) methods. The different labels were tested for residualizing prop-erties using the HER-2 overexpressing SKBR-3 cells. The cellular radioactivity retention showed thatDABI provided a 55% better retention than CAT and was 42% better than PIB after 20 hours. NBI didnot improve retention. Accumulation tests up to 21 hours showed that the HER-2-specific accumulationof radioactivity delivered with DABI was, on average, 33% higher than with the use of PIB. These DABI-dependent improvements could, with high probability, be attributed to the good residualizing propertiesof DABI. The affinity of DABI-labeled trastuzumab to SKBR-3 cells was not better than the affinity of thePIB labeled (3.2 Ϯ 1.9 nM and 0.77 Ϯ 0.39 nM, respectively). In conclusion, the use of the DABI linkerimproved intracellular retention in vitro in comparison with the other labeling methods.

  • 127.
    Persson, Mikael
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Gedda, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Sandström, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    [(177)Lu]pertuzumab: experimental studies on targeting of HER-2 positive tumour cells2005In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 32, no 12, p. 1457-62Article in journal (Refereed)
    Abstract [en]

    PURPOSE: The new antibody pertuzumab (Omnitarg) targets the dimerisation subdomain of HER-2. The purpose of this study was to analyse whether pertuzumab retains HER-2 targeting capacity after labelling with the therapeutically interesting beta emitter (177)Lu and to make initial characterizations in vitro and in vivo. METHODS: Pertuzumab was conjugated with isothiocyanate-benzyl-CHX-A''-DTPA and chelated to (177)Lu. Immunoreactivity, affinity, cellular retention and internalisation were analysed using SKOV-3 cells. The affinity of non-radioactive pertuzumab was measured using a surface plasmon resonance biosensor. In vivo targeting and specific binding were assessed in Balb/c (nu/nu) mice carrying SKOV-3 xenografts. The biodistribution of (177)Lu was determined 1, 3 and 7 days after [(177)Lu]pertuzumab administration. Gamma camera images were taken after 3 days. RESULTS: The immunoreactivity of [(177)Lu]pertuzumab was 85.8+/-1.3%. The affinity of non-radioactive pertuzumab was 1.8+/-1.1 nM, and that of [(177)Lu]pertuzumab, 4.1+/-0.7 nM. The cellular retention after 5 h pre-incubation was 90+/-2% at 20 h. The targeting was HER-2 specific both in vitro and in vivo, since excess amounts of non-labelled antibody inhibited the uptake of labelled antibody (p<0.0001 and p<0.01, respectively). The biodistribution and gamma camera images of (177)Lu showed extensive tumour uptake. Normal tissues had a surprisingly low uptake. CONCLUSION: Pertuzumab was efficiently labelled with (177)Lu and showed good intracellular retention and HER-2 specific binding both in vitro and in vivo. The gamma camera images and the biodistribution study gave excellent tumour targeting results. Thus, [(177)Lu]pertuzumab is of interest for further studies aimed at radionuclide therapy.

  • 128. Polischouk, A.G.
    et al.
    Stenerlöw, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Edgren, M.R.
    Lewensohn, R.
    Difference in the induction, but not in the repair, of X-ray- and nitrogen ion-induced DNA single-strand breaks as measured using human cell extracts2003In: International Journal of Radiation Biology, ISSN 0955-3002, E-ISSN 1362-3095, Vol. 79, no 12, p. 965-71Article in journal (Refereed)
    Abstract [en]

    PURPOSE: To compare the repair efficiency of X-ray (low linear energy transfer [LET]) and nitrogen ion (high LET)-induced single-strand breaks (SSB) in a human cell-free end-joining system. MATERIALS AND METHODS: SSB were introduced into a bacterial plasmid, pBR322, by X-rays (4 MeV photons) and nitrogen ions with an LET=125 keV micro m(-1). Repair efficiency was studied under incubation with the protein extracts from human squamous carcinoma cells, UT-SCC-5. RESULTS: A several fold higher dose of nitrogen ion radiation compared with X-ray radiation was needed to induce a similar loss of supercoiled plasmid DNA. There was no difference in the repair efficiency of SSB induced by these two types of radiation. CONCLUSION: The data indicate that X-rays at 25 Gy and nitroging ions at 100 Gy radiation doses, under condition of low scavenging capacity (10 mM Tris), induce SSB of similar complexity or, alternatively, differences in SSB complexity do not alter the repair rate.

  • 129. Polischouk, Anya G.
    et al.
    Holgersson, Åsa
    Zong, Dali
    Stenerlöw, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Karlsson, Hanna L.
    Möller, Lennart
    Viktorsson, Kristina
    Lewensohn, Rolf
    The antipsychotic drug trifluoperazine inhibits DNA repair and sensitizes non-small cell lung carcinoma cells to DNA double-strand break-induced cell death2007In: Molecular Cancer Therapeutics, ISSN 1535-7163, E-ISSN 1538-8514, Vol. 6, no 8, p. 2303-2309Article in journal (Refereed)
    Abstract [en]

    Trifluoperazine (TFP), a member of the phenothiazine class of antipsychotic drugs, has been shown to augment the cytotoxicity of the DNA-damaging agent bleomycin. In the present study, we investigated the effect of trifluoperazine on (a) survival of bleomycin-treated human non-small cell lung carcinoma U1810 cells, (b) induction and repair of bleomycin-induced DNA strand breaks, and (c) nonhomologous end-joining (NHEJ), the major DNA double-strand break (DSB) repair pathway in mammalian cells. By using a clonogenic survival assay, we show here that concomitant administration of trifluoperazine at a subtoxic concentration enhances the cytotoxicity of bleomycin. Moreover, trifluoperazine also increases the longevity of bleomycin-induced DNA strand breaks in U1810 cells, as shown by both comet assay and fraction of activity released (FAR)-assay. This action seems to be related to suppression of cellular DNA DSB repair activities because NHEJ-mediated rejoining of DSBs occurs with significantly lower efficiency in the presence of trifluoperazine. We propose that TFP might be capable of inhibiting one or more elements of the DNA DSB repair machinery, thereby increasing the cytotoxicity of bleomycin in lung cancer cells.

  • 130.
    Pålsgård, Eva
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Lindh, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Juntti-Berggren, Lisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Berggren, Per-Olof
    Roomans, Godfried M
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Effects of K+-induced depolarization and purinergic receptor activation on elemental content in insulin-producing RINm5F-cells1995In: Cell Biology International, ISSN 1065-6995, E-ISSN 1095-8355, Vol. 19, no 1, p. 25-34Article in journal (Refereed)
    Abstract [en]

    X-ray microanalysis was used to detect elemental changes in the insulin-producing tumor cell-lineRINm5F. To improve discrimination between mobile ions and ions bound to macromolecules a new approach was employed, consisting of multivariate statistical analysis of correlations between the concentrations of Na, Mg, P, S, CI, K, and Ca. RINm5F cells, cultured an Formvar-coated titanium grids, were stimulated with high K+ or ATP, that are both known to stimulate insulin release. The buffers used contained Ca2+ or one of the Ca2+-analogues Sr2+ and Ba2+, to represent Ca2+ uptake inresponse to stimulation. After stimulation the cells were shock-frozen and freeze-dried overnight. Incubation for 10-20 seconds in a Ca2+-containing buffer did not significantly affect elementalcomposition, whereas cellular Mg, P and K decreased in a Sr2+-containing buffer. Depolarization with high K+ concentration caused an increase in the cellular Na content, both in Ca2+- and Sr2+-containing buffers, but not in the buffer where Ca2+ had been replaced by Ba2+. X-ray microanalysis is useful for detection of elemental changes subsequent to stimulation of cultured cells. Moreover, multivariate statistical analysis strengthens the idea that stimulation of RINm5F cells causes redistribution of ions possibly due to changes in the state of binding of some elements to cellular proteins.

  • 131.
    Pålsgård, Eva
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Lindh, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Juntti-Berggren, Lisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Berggren, Per-Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Roomans, Gotfried
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Grime, G W
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Proton-induced and electron-induced X-ray microanalysis of insulin-secreting cells1994In: Scanning Microscopy Suppl., Vol. 8, p. 325-333Article in journal (Refereed)
    Abstract [en]

    Elemental redistribution induced by insulin secretion, was investigated by electron and proton probe X-ray microanalysis. In particular, ion fluxes following immediately upon stimulation were studied. As the sensitivity of the electron probe was insufficient, the proton microprobe was employed. In order to see whether the cell is asymmetric with respect to Ca2+ influx, the cells were stimulated in the presence of Sr2+ (as a Ca2+ analog). Insulin-secreting cells (RINm5F cells and isolated mouse beta-cells) were cultured on grids and shock-frozen at 2-30 seconds after stimulation. In a large number of cells, the major elements and and large fluxes were analyzed by the electron microprobe. In the proton microprobe, selected cells were analyzed and elemental maps were compared with electron micrographs of the same cells. The proton microprobe, but not the electron microprobe, could detect an influx of Sr in response to K+-stimulation for 2 seconds, in RINm5F cells. No polarization of Sr2+ uptake in RINm5F-cells could be detected, and the beta-cells did not respond to high K+ by uptake of Sr. Momentary stimulation of beta-cells also resulted in a significant increase in Na, detected by the electron probe. Spreading of the beta-cells on the substrate appears to influence the subcellular elemental distribution. Thus, the proton probe has potential to detect small changes in elements such as those occurring after short-time stimulation.

  • 132.
    Pålsgård, Eva
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Roomans, Godfried
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Human Anatomy.
    Lindh, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Ion dynamics in cells-preparation for studies of intracellular processes1995In: Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms, ISSN 0168-583X, E-ISSN 1872-9584, Vol. 104, no 1-4, p. 324-327Article in journal (Refereed)
    Abstract [en]

    A proton beam of about 1 mu m allows the study of inner structures of cells. These studies demand sophisticated preparation methods, not to destroy the morphology or the elemental distribution. Analysing a well-preserved cell may lead to important knowledge about basic regulatory processes at the cellular level. Freezing followed by removal of water by drying or by substitution with an organic solvent will be exemplified. Insulin-producing cells were studied to reach a further understanding of the signal transduction between stimulation to secrete insulin and the secretion.

  • 133.
    Qvarnström, O. F
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Simonsson, M
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Eriksson, V
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Turesson, I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Carlsson, J
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    gamma H2AX and cleaved PARP-1 as apoptotic markers in irradiated breast cancer BT474 cellular spheroids2009In: International Journal of Oncology, ISSN 1019-6439, Vol. 35, no 1, p. 41-47Article in journal (Refereed)
    Abstract [en]

    Chemo- and radiotherapy induce apoptosis in tumours and surrounding tissues. In a search for robust and reliable apoptosis markers, we have evaluated immunostaining patterns of gamma H2AX and cleaved PARP-1 in paraffin-embedded cellular spheroids. Breast cancer BT474 cells were grown as cell spheroids to diameters of 700-800 pm. The spheroids contained an outer cell layer with proliferative cells, a deeper region with quiescent cells and a central area with necrosis. They were irradiated with 5 Gy and the frequency of apoptotic cells was determined at several time points (0-144 h) and distances (0-150 mu m) from the spheroids surface. gamma H2AX and cleaved PARP-1 were quantified independently. Apoptotic frequencies for the two markers agreed both temporally and spatially in the proliferative regions of the spheroids. The gamma H2AX signal was stronger and had lower background compared to cleaved PARP-1. The central necrotic region was intensely stained with cleaved PARP-1, whereas no gamma H2AX could be detected. The apoptotic frequency increased with distance from surface for all time points. However, apoptotic frequencies, above unirradiated control levels, could only be detected for the last time point, 144 h after irradiation. We have shown that the spheroid model is a practical system for evaluation of staining patterns and specificities of apoptosis markers. Also, the radial gradient provides the opportunity to study apoptosis under a range of physiological conditions within the same system. We have further shown that gamma H2AX and cleaved PARP-1 are applicable markers for apoptosis in the proliferative regions of the spheroids. However, the more intense and clear staining patterns of gamma H2AX suggests that this marker is preferable for quantification of apoptosis in spheroids and similar paraffin-embedded materials.

  • 134.
    Sandström, Karl
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Ekberg, Tomas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Engström, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Anniko, Matti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Lundqvist, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Targeting CD44v6 expressed in head and neck squamous cell carcinoma: preclinical characterization of an 111In-labeled monoclonal antibody2008In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 29, no 3, p. 137-144Article in journal (Refereed)
    Abstract [en]

    In patients with head and neck squamous cell carcinoma (HNSCC) radioimmunodiagnosis could offer a more specific and sensitive tumor diagnostic method.Our aim was to evaluate the labeling and biodistribution of the novel radioimmunoconjugate (111)In-cMAb U36. In this study cMAb U36, targeting CD44v6, and huA33, as a negative control, were labeled with indium-111, using the chelator CHXA''-DTPA. Immunoreactivity assays and binding studies were performed in vitro. Biodistribution and tumor imaging were conducted after intravenous injection of the radioimmunoconjugate to nude mice bearing HNSCC xenografts expressing CD44v6. The immunoreactive fraction was very high and the binding was CD44v6-specific. In vivo results demonstrated a promising biodistribution, with tumors clearly accumulating radioactivity with time. At 168 h postinjection (p.i.) the tumor uptake was 54.7 +/- 16.6% injected dose/g. The cMAb U36 had significantly (p < 0.05) higher uptake in tumors 72 h p.i. compared to huA33. We produced a novel radioimmunoconjugate targeting CD44v6 for possible use in the detection of HNSCC. The conjugate demonstrates no adverse effects from labeling and a favorable biodistribution.

  • 135.
    Segerström, Lova
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Stenerlow, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Eriksson, Veronika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Effects of radiation on growth of two human tumour cell lines surviving a previous high dose, low dose-rate, radionuclide exposure2008In: International Journal of Oncology, ISSN 1019-6439, Vol. 33, no 2, p. 341-9Article in journal (Refereed)
    Abstract [en]

    Effects of radiation on growth of two human tumour cell lines that survived a previous high dose, low dose-rate radionuclide exposure simulating intensive radionuclide therapy, were analyzed. The purpose was to investigate whether the survivors gained therapy induced changes in growth and radiation response. The U118MG, ParRes (parental resistant), and U373MG, ParSen (parental sensitive), glioma cells were used because they are known to be low dose-rate radiation resistant and sensitive, respectively. These cells were initially exposed to high dose, low dose-rate radiation for 24 h and surviving U118MG and U373MG cells formed new cultures called SurRes (surviving resistant) and SurSen (surviving sensitive), respectively. All four cell types were then exposed to graded acute radiation doses, 0-8 Gy, and analyzed for radiation induced growth disturbances. They were also analyzed regarding DNA-content and cell cycle distributions. The SurRes cells regained in most cases the same growth rate, had the same growth delays and showed generally a similar response as the original ParRes cells to the 0-8 Gy exposures. In contrast, the SurSen cells had in all cases slower growth rate and longer growth delays than the original ParSen cells after the 0-8 Gy exposures. There were no signs of radiation-induced radioresistance. The slow growing SurSen cells contained about 80% more DNA and had more cells in G1 and fewer in G2 than the ParSen cells. The conclusion is that tumour cells surviving high dose, low dose-rate, radionuclide therapy, afterwards can react differently to a new radiation exposure.

  • 136. Sjöberg, S
    et al.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Radiohalogenated polyhedral boron clusters for use in targeted oncological nuclide therapy2002In: Abstracts of XIth International Meeting on Boron Chemistry (IMEBORON XI), 28 July-2 August 2002, Moscow, Russia, 2002, p. 40-Conference paper (Other academic)
  • 137.
    Steffen, Ann-Charlott
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Radiolabeled HER-2 Binding Affibody Molecules for Tumor Targeting: Preclinical Studies2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Conventional cancer treatment based on radiotherapy or chemotherapy affects all dividing cells. By directing the therapy specifically to the tumor cells, normal cells can be spared. Tumor targeting molecules carrying a cytotoxic moiety is then an attractive approach.

    In this thesis, an affibody molecule with high affinity for the protein HER-2, that is strongly associated with aggressive forms of breast cancer, was selected. After radiolabeling with 125I, the affibody molecule, in monovalent and bivalent form, was tested in vitro in HER-2 overexpressing tumor cells and in transplanted tumors in mice.

    It was shown that the HER-2 targeting affibody molecule bound its target in a specific manner, both in vitro and in vivo. The small size of the affibody molecule resulted in fast clearance through the kidneys. An impressive tumor-to-blood ratio of 10 eight hours post injection was achieved and the tumors could easily be visualized in a gamma camera.

    The biologic effects of the bivalent affibody molecule and a monovalent affinity maturated version was measured and compared with the effects of the monoclonal antibody trastuzumab. It was found that although all molecules target the same protein, the effects differed greatly.

    The affibody molecule was also labeled with the alpha-emitting radionuclide 211At. Specific decrease in survival was seen in HER-2 overexpressing cells receiving the 211At labeled affibody molecule. The sensitivity to the treatment differed between cell lines, probably as a result of differences between the cell lines in internalization and nuclear size. The 211At labeled affibody molecules were also tested in vivo, where stability of the 211At label was a problem. To circumvent this problem, more stable conjugation chemistry was tested, as well as strategies to prevent uptake of released 211At by normal organs.

    This thesis describes the selection and optimization of affibody molecules for medical use for the first time.

    List of papers
    1. Selection and characterization of HER2/neu-binding affibody ligands
    Open this publication in new window or tab >>Selection and characterization of HER2/neu-binding affibody ligands
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    2004 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 17, no 5, p. 455-462Article in journal (Refereed) Published
    Abstract [en]

    Affibody (affibody) ligands that are specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) have been selected by phage display technology from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. The predominant variants from the phage selection were produced in Escherichia coli, purified by affinity chromatography, and characterized by biosensor analyses. Two affibody variants were shown to selectively bind to the extracellular domain of HER2/neu (HER2-ECD), but not to control proteins. One of the variants, denoted His6-ZHER2/neu:4, was demonstrated to bind with nanomolar affinity (approximately 50 nM) to the HER2-ECD molecule at a different site than the monoclonal antibody trastuzumab. Furthermore, radiolabeled His6-ZHER2/neu:4 affibody showed specific binding to native HER2/neu, overexpressed on the SKBR-3 tumor cell line. Such affibody ligands might be considered in tumor targeting applications for radionuclide diagnostics and therapy of adenocarcinomas such as breast and ovarian cancers.

    Keywords
    Amino Acid Sequence, Antibodies; Monoclonal/pharmacology, Antineoplastic Agents/pharmacology, Biosensing Techniques, Humans, Iodine Radioisotopes, Ligands, Molecular Sequence Data, Protein Structure; Secondary, Receptor; erbB-2/antagonists & inhibitors/genetics/*metabolism, Research Support; Non-U.S. Gov't, Time Factors
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-94183 (URN)10.1093/protein/gzh053 (DOI)15208403 (PubMedID)
    Available from: 2006-03-30 Created: 2006-03-30 Last updated: 2017-12-14Bibliographically approved
    2.
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    3.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
    4.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
    5. Differences in sensitivity for 211At-(ZHER2:4)2 treatment between three HER-2 overexpressing cell lines
    Open this publication in new window or tab >>Differences in sensitivity for 211At-(ZHER2:4)2 treatment between three HER-2 overexpressing cell lines
    2006 (English)In: Radiation Research, ISSN 0033-7587, E-ISSN 1938-5404Article in journal (Refereed) Submitted
    Identifiers
    urn:nbn:se:uu:diva-94187 (URN)
    Available from: 2006-03-30 Created: 2006-03-30 Last updated: 2017-12-14Bibliographically approved
    6. Biodistribution and dose calculations for 211At labeled HER-2 binding affibody molecules
    Open this publication in new window or tab >>Biodistribution and dose calculations for 211At labeled HER-2 binding affibody molecules
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    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-94188 (URN)
    Available from: 2006-03-30 Created: 2006-03-30 Last updated: 2010-01-13Bibliographically approved
  • 138.
    Steffen, Ann-Charlott
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Almqvist, Ylva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Radiology.
    Chyan, Ming-Kuan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
    Lundqvist, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Wilbur, D. Scott
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Biodistribution of 211At labeled HER-2 binding affibody molecules in mice2007In: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 17, no 5, p. 1141-1147Article in journal (Refereed)
    Abstract [en]

    The size of affibody molecules makes them suitable as targeting agents for targeted radiotherapy with the alpha-emitter 211At, since their biokinetic properties match the short physical half-live of 211At. In this study, the potential for this approach was investigated in vivo. Two different HER-2 binding affibody molecules were radiolabeled with 211At using both the linker PAB (N-succinimidyl-para-astatobenzoate) and a decaborate-based linker, and the biodistribution in tumor-bearing nude mice was investigated. The influence of L-lysine and Na-thiocyanate on the 211At uptake in normal tissues was also studied. Based on the biokinetic information obtained, the absorbed dose was calculated for different organs. Compared with a previous biodistribution with 125I, the 211At biodistribution using the PAB linker showed higher uptake in lungs, stomach, thyroid and salivary glands, indicating release of free 211At. When the decaborate-based linker was used, the uptake in those organs was decreased, but instead, high uptake in kidneys and liver was found. The uptake, when using the PAB linker, could be significantly reduced in some organs by the use of L-lysine and/or Na-thiocyanate. In conclusion, affibody molecules have suitable blood-kinetics for targeted radionuclide therapy with 211At. However, the labeling chemistry affects the distribution in normal organs to a high degree and needs to be improved to allow clinical use.

  • 139.
    Steffen, Ann-Charlott
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Almqvist, Ylva
    Chyan, Ming-Kuan
    Lundqvist, Hans
    Tolmachev, Vladimir
    Wilbur, Scott D.
    Carlsson, Jörgen
    Biodistribution and dose calculations for 211At labeled HER-2 binding affibody moleculesManuscript (Other academic)
  • 140.
    Steffen, Ann-Charlott
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Göstring, Lovisa
    Tolmachev, Vladimir
    Carlsson, Jörgen
    Differences in sensitivity for 211At-(ZHER2:4)2 treatment between three HER-2 overexpressing cell lines2006In: Radiation Research, ISSN 0033-7587, E-ISSN 1938-5404Article in journal (Refereed)
  • 141.
    Steffen, Ann-Charlott
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Göstring, Lovisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Palm, Stig
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Stenerlöw, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Differences in radiosensitivity between three HER2 overexpressing cell lines2008In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 35, no 6, p. 1179-91Article in journal (Refereed)
    Abstract [en]

    PURPOSE: HER2 is a potential target for radionuclide therapy, especially when HER2 overexpressing breast cancer cells are resistant to Herceptin(R) treatment. Therefore, it is of interest to analyse whether HER2 overexpressing tumour cells have different inherent radiosensitivity. METHODS: The radiosensitivity of three often used HER2 overexpressing cell lines, SKOV-3, SKBR-3 and BT-474, was analysed. The cells were exposed to conventional photon irradiation, low linear energy transfer (LET), to characterise their inherent radiosensitivity. The analysis was made with clonogenic survival and growth extrapolation assays. The cells were also exposed to alpha particles, high LET, from (211)At decays using the HER2-binding affibody molecule (211)At-(Z(HER2:4))(2) as targeting agent. Assays for studies of internalisation of the affibody molecule were applied. RESULTS: SKOV-3 cells were most radioresistant, SKBR-3 cells were intermediate and BT-474 cells were most sensitive as measured with the clonogenic and growth extrapolation assays after photon irradiation. The HER2 dependent cellular uptake of (211)At was qualitatively similar for all three cell lines. However, the sensitivity to the alpha particles from (211)At differed; SKOV-3 was most resistant, SKBR-3 intermediate and BT-474 most sensitive. These differences were unexpected because it is assumed that all types of cells should have similar sensitivity to high-LET radiation. The sensitivity to alpha particle exposure correlated with internalisation of the affibody molecule and with size of the cell nucleus. CONCLUSION: There can be differences in radiosensitivity, which, if they also exist between patient breast cancer cells, are important to consider for both conventional radiotherapy and for HER2-targeted radionuclide therapy.

  • 142.
    Steffen, Ann-Charlott
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Wikman, Maria
    Nilsson, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Ståhl, Stefan
    Adams, Gregory P.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Affibody-mediated tumour targeting of HER-2 expressing xenografts in mice2006In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 33, no 6, p. 631-638Article in journal (Refereed)
    Abstract [en]

    PURPOSE: Targeted delivery of radionuclides for diagnostic and therapeutic applications has until recently largely been limited to receptor ligands, antibodies and antibody-derived molecules. Here, we present a new type of molecule, a 15-kDa bivalent affibody called (Z(HER2:4))(2), with potential for such applications. The (Z(HER2:4))(2) affibody showed high apparent affinity (K (D)=3 nM) towards the oncogene product HER-2 (also called p185/neu or c-erbB-2), which is often overexpressed in breast and ovarian cancers. The purpose of this study was to investigate the in vivo properties of the new targeting agent. METHODS: The biodistribution and tumour uptake of the radioiodinated (Z(HER2:4))(2) affibody was studied in nude mice carrying tumours from xenografted HER-2 overexpressing SKOV-3 cells. RESULTS: The radioiodinated (Z(HER2:4))(2) affibody was primarily excreted through the kidneys, and significant amounts of radioactivity were specifically targeted to the tumours. The blood-borne radioactivity was, at all times, mainly in the macromolecular fraction. A tumour-to-blood ratio of about 10:1 was obtained 8 h post injection, and the tumours could be easily visualised with a gamma camera at this time point. CONCLUSION: The results indicate that the (Z(HER2:4))(2) affibody is an interesting candidate for applications in nuclear medicine, such as radionuclide-based tumour imaging and therapy.

  • 143.
    Steffen, Ann-Charlott
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Wikman, Maria
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Adams, Gregory P.
    Nilsson, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Ståhl, Stefan
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    In vitro characterization of a bivalent anti-HER-2 affibody with potential for radionuclide-based diagnostics2005In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 20, no 3, p. 239-248Article in journal (Refereed)
    Abstract [en]

    The 185 kDa transmembrane glycoprotein human epidermal growth factor receptor 2 (HER-2) (p185/neu, c-ErbB-2) is overexpressed in breast and ovarian cancers. Overexpression in breast cancer correlates with poor patient prognosis, and visualization of HER-2 expression might provide valuable diagnostic information influencing patient management. We have previously described the generation of a new type of affinity ligand, a 58-amino-acid affibody (Z(HER2:4)) with specific binding to HER-2. In order to benefit from avidity effects, we have created a bivalent form of the affibody ligand, (Z(HER2:4))2. The monovalent and bivalent ligands were compared in various assays. The new bivalent affibody has a molecular weight of 15.6 kDa and an apparent affinity (K(D)) against HER-2 of 3 nM. After radioiodination, using the linker molecule N-succinimidyl p-(trimethylstannyl) benzoate (SPMB), in vitro binding assays showed specific binding to HER-2 overexpressing cells. Internalization of 125I was shown after delivery with both the monovalent and the bivalent affibody. The cellular retention of 125I was longer after delivery with the bivalent affibody when compared to delivery with the monovalent affibody. With approximately the same affinity as the monoclonal antibody trastuzumab (Herceptin) but only one tenth of the size, this new bivalent molecule is a promising candidate for radionuclide-based detection of HER-2 expression in tumors. 125I was used in this study as a surrogate marker for the diagnostically relevant radioisotopes 123I for single photon emission computed tomography (SPECT)/gamma-camera imaging and 124I for positron emission tomography (PET).

  • 144.
    Stenerlöw, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Radiation-induced bystander effects2006In: Acta Oncologica, ISSN 0284-186X, E-ISSN 1651-226X, Vol. 45, no 4, p. 373-4Article in journal (Refereed)
    Abstract [en]

    This Article does not have an abstract.

  • 145.
    Stenerlöw, Bo
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Ekerljung, Lina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Lennartsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Radiation Induced DNA-Damage/Repair and Associated Signaling Pathways2008In: Targeted Radionuclide Tumor Therapy: Biological Aspects / [ed] Torgny Stigbrand, Jörgen Carlsson, Gregory P. Adams, New York: Springer , 2008, p. 249-266Chapter in book (Other academic)
  • 146. Stenerlöw, Bo
    et al.
    Karlsson, Karin H
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Cooper, Brian
    Rydberg, Björn
    Measurement of Prompt DNA Double-Strand Breaks in Mammalian Cells without Including Heat-Labile Sites: Results for Cells Deficient in Nonhomologous End Joining2003In: Radiation Research, ISSN 0033-7587, Vol. 159, p. 502-510Article in journal (Refereed)
  • 147. Ståhl, Sara
    et al.
    Fung, Eva
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Adams, Christopher
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Lengqvist, Johan
    Mörk, Birgitta
    Stenerlöw, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Lewensohn, Rolf
    Lehtiö, Janne
    Zubarev, Roman
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Viktorsson, Kristina
    Proteomics and pathway analysis identifies JNK signaling as critical for high linear energy transfer radiation-induced apoptosis in non-small lung cancer cells2009In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 8, no 5, p. 1117-1129Article in journal (Refereed)
    Abstract [en]

    During the past decade, we have witnessed an explosive increase in generation of large proteomics data sets, not least in cancer research. There is a growing need to extract and correctly interpret information from such data sets to generate biologically relevant hypotheses. A pathway search engine (PSE) has recently been developed as a novel tool intended to meet these requirements. Ionizing radiation (IR) is an anticancer treatment modality that triggers multiple signal transduction networks. In this work, we show that high linear energy transfer (LET) IR induces apoptosis in a non-small cell lung cancer cell line, U-1810, whereas low LET IR does not. PSE was applied to study changes in pathway status between high and low LET IR to find pathway candidates of importance for high LET-induced apoptosis. Such pathways are potential clinical targets, and they were further validated in vitro. We used an unsupervised shotgun proteomics approach where high resolution mass spectrometry coupled to nanoflow liquid chromatography determined the identity and relative abundance of expressed proteins. Based on the proteomics data, PSE suggested the JNK pathway (p = 6.10(-6)) as a key event in response to high LET IR. In addition, the Fas pathway was found to be activated (p = 3.10(-5)) and the p38 pathway was found to be deactivated (p = 0.001) compared with untreated cells. Antibody-based analyses confirmed that high LET IR caused an increase in phosphorylation of JNK. Moreover pharmacological inhibition of JNK blocked high LET-induced apoptotic signaling. In contrast, neither an activation of p38 nor a role for p38 in high LET IR-induced apoptotic signaling was found. We conclude that, in contrast to conventional low LET IR, high LET IR can trigger activation of the JNK pathway, which in turn is critical for induction of apoptosis in these cells. Thus PSE predictions were largely confirmed, and PSE was proven to be a useful hypothesis-generating tool.

  • 148.
    Sundberg, Åsa Liljegren
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tumour Targeting Using Radiolabelled EGF Conjugates: Preclinical Studies2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    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.

    List of papers
    1.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
    2. EGF and dextran-conjugated EGF induces differential phosphorylation of the EGF receptor
    Open this publication in new window or tab >>EGF and dextran-conjugated EGF induces differential phosphorylation of the EGF receptor
    Show others...
    2002 (English)In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 10, no 5, p. 655-659Article in journal (Refereed) Published
    Abstract [en]

    Dextran-conjugated EGF (EGF-dextran) has a potential use for targeted radionuclide therapy of tumors that overexpress the epidermal growth factor receptor (EGFR). There are plans to treat both bladder carcinomas and malignant gliomas with local injections of radiolabeled EGF-dextran since these tumors often express high levels of EGFR. In this report we show that EGF and EGF-dextran differentially activate the EGFR. In the human glioma cell line U-343, activation of the serine/threonine kinases Erk and Akt is identical upon stimulation with EGF or EGF-dextran. However, the effect on phospholipase Cgamma1 (PLCgamma1) phosphorylation differs. In cells stimulated with EGF-dextran, the PLCgamma1 phosphorylation is lower than in cells stimulated with EGF. This observation could be explained by the fact that the PLCgamma1 association sites in the EGFR, tyrosine residues 992 and 1173, were phosphorylated to a lower degree when the receptor was stimulated with EGF-dextran as compared to with EGF.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-91593 (URN)12373311 (PubMedID)
    Available from: 2004-04-15 Created: 2004-04-15 Last updated: 2017-12-14Bibliographically approved
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    5. [111In]Bz-DTPA-hEGF: Preparation and in vitro characterization of a potential anti-glioblastoma targeting agent
    Open this publication in new window or tab >>[111In]Bz-DTPA-hEGF: Preparation and in vitro characterization of a potential anti-glioblastoma targeting agent
    Show others...
    2003 (English)In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 18, no 4, p. 643-54Article in journal (Refereed) Published
    Abstract [en]

    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.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-91596 (URN)10.1089/108497803322287736 (DOI)14503960 (PubMedID)
    Available from: 2004-04-15 Created: 2004-04-15 Last updated: 2018-12-04
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  • 149.
    Sundberg, Åsa Liljegren
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Gedda, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Bruskin, Alexander
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Blomquist, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    [177Lu]Bz-DTPA-EGF: Preclinical characterization of a potential radionuclide targeting agent against glioma2004In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 19, no 2, p. 195-204Article in journal (Refereed)
    Abstract [en]

    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.

  • 150.
    Sundberg, Åsa Liljegren
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Orlova, Anna
    Bruskin, Alexander
    Gedda, Lars
    Carlsson, Jörgen
    Blomquist, Erik
    Lundqvist, Hans
    Tolmachev, Vladimir
    [111In]Bz-DTPA-hEGF: Preparation and in vitro characterization of a potential anti-glioblastoma targeting agent2003In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 18, no 4, p. 643-54Article in journal (Refereed)
    Abstract [en]

    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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