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  • 1.
    Al-Ramadan, Afkar
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Mortensen, Anja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Nestor, Marika V.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Analysis of radiation effects in two irradiated tumor spheroid models2018In: Oncology Letters, ISSN 1792-1074, E-ISSN 1792-1082, Vol. 15, no 3, p. 3008-3016Article in journal (Refereed)
    Abstract [en]

    Multicellular spheroids have proven suitable as three-dimensional in vivo-like models of non-vascularized micrometastases. Unlike monolayer-based models, spheroids mirror the cellular milieu and the pathophysiological gradients inside tumor nodules. However, there is limited knowledge of the radiation effects at the molecular level in spheroids of human origin. The present study is a presentation of selected cell biological processes that may easily be analyzed with methods available at routine pathology laboratories. Using gamma irradiated pancreatic neuroendocrine BON1 and colonic adenocarcinoma HCT116 spheroids as model systems, the present study assessed the radiobiological response in these models. Spheroid growth after irradiation was followed over time and molecular responses were subsequently assessed with immunohistochemistry (IHC) staining for descriptive analyses and semi-automatic grading of apoptosis, G(2)-phase and senescence in thin sections of the spheroids. Growth studies demonstrated the BON1 spheroids were slower growing and less sensitive to radiation compared with the HCT116 spheroids. IHC staining for G2-phase was primarily observed in the outer viable P-cell layers of the spheroids, with the 6 Gy irradiated HCT116 spheroids demonstrating a very clear increase in staining intensity compared with unirradiated spheroids. Apoptosis staining results indicated increased apoptosis with increasing radiation doses. No clear association between senescence and radiation exposure in the spheroids were observed. The present results demonstrate the feasibility of the use of multicellular spheroids of human origin in combination with IHC analyses to unravel radiobiological responses at a molecular level. The present findings inspire further investigations, including other relevant IHC-detectable molecular processes in time-and radiation dose-dependent settings.

  • 2.
    Bondza, Sina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Ridgeview Instruments AB, Uppsala.
    Björkelund, Hanna
    Ridgeview Instruments AB, Uppsala.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Ridgeview Instruments AB, Uppsala.
    Buijs, Jos
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Ridgeview Instruments AB, Uppsala.
    Novel Real-Time Proximity Assay for Characterizing Multiple Receptor Interactions on Living Cells2017In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 89, no 24, p. 13212-13218Article in journal (Refereed)
    Abstract [en]

    Cellular receptor activity is often controlled through complex mechanisms involving interactions with multiple molecules, which can be soluble ligands and/or other cell surface molecules. In this study, we combine a fluorescence-based technology for real-time interaction analysis with fluorescence quenching to create a novel time-resolved proximity assay to study protein-receptor interactions on living cells. This assay extracts the binding kinetics and affinity for two proteins if they bind in proximity on the cell surface. One application of real-time proximity interaction analysis is to study relative levels of receptor dimerization. The method was primarily evaluated using the HER2 binding antibodies Trastuzumab and Pertuzumab and two EGFR binding antibodies including Cetuximab. Using Cetuximab and Trastuzumab, proximity of EGFR and HER2 was investigated before and after treatment of cells with the tyrosine-kinase inhibitor Gefitinib. Treated cells displayed 50% increased proximity signal, whereas the binding characteristics of the two antibodies were not significantly affected, implying an increase in the EGFR-HER2 dimer level. These results demonstrate that real-time proximity interaction analysis enables determination of the interaction rate constants and affinity of two ligands while simultaneously quantifying their relative colocalization on living cells.

  • 3.
    Bondza, Sina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Stenberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Björkeund, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Conjugation Effects on Antibody-Drug Conjugates: Evaluation of Interaction Kinetics in Real Time on Living Cells2014In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 11, no 11, p. 4154-4163Article in journal (Refereed)
    Abstract [en]

    Antibody-drug conjugates (ADC) have shown promising effects in cancer therapy by combining the target specificity of an antibody with the toxicity of a chemotherapeutic drug. As the number of therapeutic antibodies is significantly larger than those used as ADCs, there is unused potential for more effective therapies. However, the conjugation of an additional molecule to an antibody may affect the interaction with its target, altering association rate, dissociation rate, or both. Any changes of the binding kinetics can have subsequent effects on the efficacy of the ADCs, thus the kinetics are important to monitor during ADC development and production. This paper describes a method for the analysis of conjugation effects on antibody binding to its antigen, using the instrument LigandTracer and a fluorescent monovalent anti-IgG binder denoted FIBA, which did not affect the interaction. All measurements were done in real time using living cells which naturally expressed the antigens. With this method the binding profiles of different conjugations of the therapeutic anti-EGFR antibody cetuximab and the anti-CD44v6 antibody fragment AbD15171 were evaluated and compared. Even comparatively small modifications of cetuximab altered the interaction with the epidermal growth factor receptor (EGFR). In contrast, no impact on the AbD15171-CD44v6 interaction was observed upon conjugation. This illustrates the importance to study the binding profile for each ADC combination, as it is difficult to draw any general conclusion about conjugation effects. The modification of interaction kinetics through conjugation opens up new possibilities when optimizing an antibody or an ADC, since the conjugations can be used to create a binding profile more apt for a specific clinical need.

  • 4.
    Cheng, Junping
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Ekberg, Tomas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Engström, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Jensen, Holger J.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Anniko, Matti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Radioimmunotherapy with astatine-211 using chimeric monoclonal antibody U36 in head and neck squamous cell carcinoma2007In: The Laryngoscope, ISSN 0023-852X, E-ISSN 1531-4995, Vol. 117, no 6, p. 1013-1018Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES: In advanced head and neck squamous cell carcinoma (HNSCC), there is a need for an adjuvant treatment. We aim to evaluate the biodistribution and therapeutic effect of radioimmunotherapy using the alpha emitting, astatine-211-labeled, chimeric monoclonal antibody U36 (U36) on the HNSCC cell line UT-SCC7 in vivo. STUDY DESIGN: Xenograft tumors were inoculated subcutaneously in nude mice. Astatine-211-labeled U36 was injected intravenously with or without blocking of target with nonlabeled U36. METHODS: In the biodistribution experiments, radioactivity was measured in tumors and various organs at set time points. In the therapeutic experiments, two groups (with or without blocking) received therapy, and the tumor growth was compared with that of controls. In addition, one group received nonlabeled U36 only. RESULTS: The biodistribution experiments demonstrated that astatine-211-labeled U36 could target UT-SCC7 xenografts in nude mice. With time, uptake increased in tumors and decreased in normal organs. Nonlabeled U36 did not influence tumor growth. In the two therapy groups, 18 of 20 tumors responded to therapy by decreasing or stabilizing their volumes. Significant difference was seen between the treated groups and the controls (P < .05). CONCLUSION: The study illustrates the specific binding of astatine-211-labeled U36 to HNSCC and suggests radioimmunotherapy with the alpha emitting radionuclide to be a useful treatment modality.

  • 5.
    Cheng, Junping
    et al.
    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.
    Ekberg, Tomas
    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.
    Anniko, Matti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    The use of closo-dodecaborate-containing linker improves targeting of HNSCC xenografts with radioiodinated chimeric monoclonal antibody U362010In: Molecular Medicine Reports, ISSN 1791-2997, Vol. 3, no 1, p. 155-160Article in journal (Refereed)
    Abstract [en]

    Radionuclide imaging of head and neck squamous cell carcinoma (HNSCC) using monoclonal antibodies (MAbs) has the potential to contribute to improved diagnosis and staging, thereby making more effective treatment possible. Chimeric monoclonal antibody U36 (cMAb U36), specific to CD44v6 antigen. is a candidate for the targeting of HNSCC. The aim of this study was to compare the influence of indirect iodination via closo-dodecaborate-based linker (DABI) with the influence of direct radioiodination on the biodistribution of the chimeric anti-CD44v6 antibody U36. The study was performed using nude mice bearing UT-SCC7 HNSCC xenografts using the paired-label method. The biodistribution of cMAb U36 labelled directly with I-131 and using DABI with I-125 was compared in the same animals. The influence of DABI on the tumour-to-organ ratio was evaluated. For both conjugates, radioactivity uptake in blood and organs decreased with time, except in tumours and the thyroid. DABI-labelled cMAb U36 was characterised by fast blood clearance and an elevated uptake in the liver and spleen. The use of DABI enabled a 1.5 to 2-fold improvement in the tumour-to-blood and tumour-to-organ ratios in comparison with direct radioiodination, with the exception of the liver and spleen. These results indicate that DABI is a promising linker for the coupling of radioiodine to HNSCC-targeting antibodies.

  • 6.
    Ekberg, Thomas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences. 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.
    Engström, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Nordgren, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Wester, Kenneth
    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. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Anniko, Matti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Expression of EGFR, HER2, HER3, and HER4 in metastatic squamous cell carcinomas of the oral cavity and base of tongue2005In: International Journal of Oncology, ISSN 1019-6439, Vol. 26, no 5, p. 1177-85Article in journal (Refereed)
    Abstract [en]

    The expressions of all four receptors in the epidermal growth factor receptor family, EGFR. HER2, HER3, and HER4 were evaluated by immunohistochemistry in 19 cases of metastatic squamous cell carcinoma of the oral cavity and base of tongue. EGFR had a similar and high expression in both primary tumours and the corresponding metastases, while the expression in normal epithelium was lower in most cases. HER2 was not expressed to the same extent as EGFR. However, when HER2 was well expressed, it was in most cases expressed to the same extent and intensity in the primary tumours, metastases, and normal epithelium. The expression of HER3 and HER4 varied and was mainly cytoplasmic in all cases studied. No overexpression of HER3 and HER4 in tumours was seen as compared to normal epithelium. In order to further investigate the distribution of HER3, two HER3 expressing cell lines originating from tongue cancer were analysed in vitro, using radiolabelled anti-HER3 antibodies directed to the extracellular domains of the receptor. The results indicated that HER3 was not present in measurable amounts in the cellular membrane. There is a need for improved diagnostics and therapy for the studied type of tumours, e.g. using radiolabelled antibodies or ligands, and EGFR seemed suitable as target since the expression was high, membrane associated and similar in the primary tumours and the corresponding metastases.

  • 7.
    Elmsjö, Albert
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Haglöf, Jakob
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Engskog, Mikael K. R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Arvidsson, Torbjörn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Med Prod Agcy, Uppsala, Sweden.
    Pettersson, Curt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    The co-feature ratio, a novel method for the measurement of chromatographic and signal selectivity in LC-MS-based metabolomics.2017In: Analytica Chimica Acta, ISSN 0003-2670, E-ISSN 1873-4324, Vol. 956, p. 40-47Article in journal (Refereed)
    Abstract [en]

    Evaluation of analytical procedures, especially in regards to measuring chromatographic and signal selectivity, is highly challenging in untargeted metabolomics. The aim of this study was to suggest a new straightforward approach for a systematic examination of chromatographic and signal selectivity in LC-MS-based metabolomics. By calculating the ratio between each feature and its co-eluting features (the co-features), a measurement of the chromatographic selectivity (i.e. extent of co-elution) as well as the signal selectivity (e.g. amount of adduct formation) of each feature could be acquired, the co-feature ratio. This approach was used to examine possible differences in chromatographic and signal selectivity present in samples exposed to three different sample preparation procedures. The capability of the co-feature ratio was evaluated both in a classical targeted setting using isotope labelled standards as well as without standards in an untargeted setting. For the targeted analysis, several metabolites showed a skewed quantitative signal due to poor chromatographic selectivity and/or poor signal selectivity. Moreover, evaluation of the untargeted approach through multivariate analysis of the co-feature ratios demonstrated the possibility to screen for metabolites displaying poor chromatographic and/or signal selectivity characteristics. We conclude that the co-feature ratio can be a useful tool in the development and evaluation of analytical procedures in LC-MS-based metabolomics investigations. Increased selectivity through proper choice of analytical procedures may decrease the false positive and false negative discovery rate and thereby increase the validity of any metabolomic investigation.

  • 8.
    Fortin, Marc-André
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science.
    Salnikov, Alexei V.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Heldin, Nils-Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Rubin, Kristofer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lundqvist, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Immuno-PET of undifferentiated thyroid carcinoma with radioiodine-labelled antibody cMAb U36: application to antibody tumour uptake studies2007In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 34, no 9, p. 1376-1387Article in journal (Refereed)
    Abstract [en]

    PURPOSE: We tested the suitability of the chimeric monoclonal anti-human CD44 splice version 6 antibody (cMAb U36) for targeting and visualising human anaplastic thyroid carcinoma with PET. We also performed experiments aimed at elucidating the relation between tumour interstitial fluid pressure (TIFP) and the tumour uptake of antibodies. METHODS: The affinity and specificity of the cMAb U36 for KAT-4 cells were evaluated in vitro, as was the Na+/I- symporter (NIS) expression. Biodistribution studies were performed on KAT-4 carcinoma-bearing mice injected with 124I-cMAb U36 or free iodine. Biodistribution studies were also performed in animals treated with the specific TGF-beta1 and -beta3 inhibitor Fc:TbetaRII, which lowers TIFP. Treated and non-treated animals were scanned by microPET. RESULTS: Cultured human undifferentiated/anaplastic thyroid carcinoma KAT-4 cells expressed low levels of NIS and uptake of free iodine was insignificant. The cMAb U36 expressed an affinity (KD) of 11+/-2 nM. Tumour radioactivity uptake reached maximum values 48 h after injection of 124I-cMAb U36 (approximately 22%IA/g). KAT-4 carcinomas were readily identified in all 124I-immuno-PET images. Radioactivity tumour uptake in Fc:TbetaRII-treated animals was significantly lower at 24 and 48 h after injection, and five times higher thyroid uptake was also noted. CONCLUSION: We successfully used 124I-cMAb U36 to visualise CD44v6-expressing human anaplastic thyroid carcinoma. Given the lack of NIS expression in KAT-4, tumour visualisation is not due to free iodine uptake. Lowering the TIFP in KAT-4 carcinomas did not increase the uptake of mAbs into tumour tissue.

  • 9.
    Haylock, Anna-Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery. Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Nilvebrant, Johan
    Royal Inst Technol, Sch Biotechnol, Div Prot Technol, Stockholm, Sweden..
    Mortensen, Anja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Velikyan, Irina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Radiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Falk, Ronny
    Karolinska Inst, Dept Neurosci, Stockholm, Sweden..
    Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers2017In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 8, no 39, p. 65152-65170Article in journal (Refereed)
    Abstract [en]

    Aim: The aim of this study was to generate and characterize scFv antibodies directed to human CD44v6, as well as to radiolabel and evaluate top candidates in vitro and in vivo for their potential use in CD44v6-targeted molecular imaging in cancer patients.

    Materials and methods: Phage display selections were used to isolate CD44v6-specific scFvs. A chain shuffling strategy was employed for affinity maturation based on a set of CD44v6-specific first-generation clones. Two second-generation scFv clones were then chosen for labeling with 111In or 125I and assessed for CD44v6-specific binding on cultured tumor cells. In vivo uptake and distribution was evaluated in tumor-bearing mice using a dual tumor model. Finally, a proof-of-concept small animal PET-CT study was performed on one of the candidates labeled with 124I.

    Results: Two affinity-matured clones, CD44v6-scFv-A11 and CD44v6-scFv-H12, displayed promising binding kinetics. Seven out of eight radiolabeled conjugates demonstrated CD44v6-specific binding. In vivo studies on selected candidates demonstrated very advantageous tumor-to-organ ratios, in particular for iodinated conjugates, where 125I-labeled scFvs exhibited favorable kinetics and tumor-to-blood ratios above five already at 24 hours p. i.. The small animal PET-CT study using 124I-labeled CD44v6-scFv-H12 was in line with the biodistribution data, clearly visualizing the high CD44v6-expressing tumor.

    Conclusion: The single chain fragments, CD44v6-scFv-A11 and CD44v6-scFv-H12 specifically bind to CD44v6, and the radiolabeled counterparts provide high tumor-to-blood ratios and fast clearance from organs and blood. We conclude that radioiodinated CD44v6-scFv-A11 and CD44v6-scFv-H12 possess features highly suitable for stringent molecular imaging.

  • 10.
    Haylock, Anna-Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Spiegelberg, Diana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Mortensen, Anja C.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Selvaraju, Ram K.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Nilvebrant, Johan
    Royal Inst Technol, Sch Biotechnol, Div Prot Technol, Stockholm, Sweden.
    Eriksson, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Nestor, Marika V
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Evaluation of a novel type of imaging probe based on a recombinant bivalent mini-antibody construct for detection of CD44v6-expressing squamous cell carcinoma2015In: International journal of oncology, ISSN 1791-2423, Vol. 48, no 2, p. 461-470Article in journal (Refereed)
    Abstract [en]

    We have developed the CD44v6-targeting human bivalent antibody fragment AbD19384, an engineered recombinant human bivalent Fab antibody formed via dimerization of dHLX (synthetic double helix loop helix motif) domains, for potential use in antibody-based molecular imaging of squamous cell carcinoma in the head and neck region. This is a unique construct that has, to the best of our knowledge, never been assessed for molecular imaging in vivo before. The objective of the present study was to evaluate for the first time the in vitro and in vivo binding properties of radio-iodinated AbD19384, and to assess its utility as a targeting agent for molecular imaging of CD44v6-expressing tumors. Antigen specificity and binding properties were assessed in vitro. In vivo specificity and biodistribution of 125I-AbD19384 were next evaluated in tumor-bearing mice using a dual-tumor setup. Finally, AbD19384 was labeled with 124I, and its imaging properties were assessed by small animal PET/CT in tumor bearing mice, and compared with 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG). In vitro studies demonstrated CD44v6-specific binding with slow off-rate for AbD19384. A favorable biodistribution profile was seen in vivo, with tumor-specific uptake. Small animal PET/CT images of 124I-AbD19384 supported the results through clearly visible high CD44v6-expressing tumors and faintly visible low expressing tumors, with superior imaging properties compared to 18F-FDG. Tumor-to-blood ratios increased with time for the conjugate (assessed up to 72 h p.i.), although 48 h p.i. proved best for imaging. Biodistribution and small-animal PET studies demonstrated that the recombinant Fab-dHLX construct AbD19384 is a promising tracer for imaging of CD44v6 antigen expression in vivo, with the future aim to be used for individualized diagnosis and early detection of squamous cell carcinomas in the head and neck region. Furthermore, this proof-of-concept research established the feasibility of using recombinant Fab-dHLX constructs for in vivo imaging of tumor biomarkers.

  • 11.
    Haylock, Anna-Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Spiegelberg, Diana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Nilvebrant, Johan
    Royal Inst Technol, AlbaNova Univ Ctr, Sch Biotechnol, Div Prot Technol, SE-10691 Stockholm, Sweden.
    Sandström, Karl
    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 Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    In vivo characterization of the novel CD44v6-targeting Fab fragment AbD15179 for molecular imaging of squamous cell carcinoma: a dual-isotope study2014In: EJNMMI Research, ISSN 2191-219X, E-ISSN 2191-219X, Vol. 4, article id 11Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Patients with squamous cell carcinoma in the head and neck region (HNSCC) offer a diagnostic challenge due to difficulties to detect small tumours and metastases. Imaging methods available are not sufficient, and radio-immunodiagnostics could increase specificity and sensitivity of diagnostics. The objective of this study was to evaluate, for the first time, the in vivo properties of the radiolabelled CD44v6-targeting fragment AbD15179 and to assess its utility as a targeting agent for radio-immunodiagnostics of CD44v6-expressing tumours.

    METHODS: The fully human CD44v6-targeting Fab fragment AbD15179 was labelled with 111In or 125I, as models for radionuclides suitable for imaging with SPECT or PET. Species specificity, antigen specificity and internalization properties were first assessed in vitro. In vivo specificity and biodistribution were then evaluated in tumour-bearing mice using a dual-tumour and dual-isotope setup.

    RESULTS: Both species-specific and antigen-specific binding of the conjugates were demonstrated in vitro, with no detectable internalization. The in vivo studies demonstrated specific tumour binding and favourable tumour targeting properties for both conjugates, albeit with higher tumour uptake, slower tumour dissociation, higher tumour-to-blood ratio and higher CD44v6 sensitivity for the 111In-labelled fragment. In contrast, the 125I-Fab demonstrated more favourable tumour-to-organ ratios for liver, spleen and kidneys.

    CONCLUSIONS: We conclude that AbD15179 efficiently targets CD44v6-expressing squamous cell carcinoma xenografts, and particularly, the 111In-Fab displayed high and specific tumour uptake. CD44v6 emerges as a suitable target for radio-immunodiagnostics, and a fully human antibody fragment such as AbD15179 can enable further clinical imaging studies.

  • 12.
    Häggblad Sahlberg, Sara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Mortensen, Anja C.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Haglöf, Jakob
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Engskog, Mikael K. R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Arvidsson, Torbjörn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Pettersson, Curt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Glimelius, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Stenerlöw, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Different functions of AKT1 and AKT2 in molecular pathways, cell migration and metabolism in colon cancer cells2017In: International Journal of Oncology, ISSN 1019-6439, Vol. 50, no 1, p. 5-14Article in journal (Refereed)
    Abstract [en]

    AKT is a central protein in many cellular pathways such as cell survival, proliferation, glucose uptake, metabolism, angiogenesis, as well as radiation and drug response. The three isoforms of AKT (AKT1, AKT2 and AKT3) are proposed to have different physiological functions, properties and expression patterns in a cell type-dependent manner. As of yet, not much is known about the influence of the different AKT isoforms in the genome and their effects in the metabolism of colorectal cancer cells. In the present study, DLD-1 isogenic AKT1, AKT2 and AKT'/2 knockout colon cancer cell lines were used as a model system in conjunction with the parental cell line in order to further elucidate the differences between the AKT isoforms and how they are involved in various cellular pathways. This was done using genome wide expression analyses, metabolic profiling and cell migration assays. In conclusion, downregulation of genes in the cell adhesion, extracellular matrix and Notch-pathways and upregulation of apoptosis and metastasis inhibitory genes in the p53-pathway, confirm that the knockout of both AKT1 and AKT2 will attenuate metastasis and tumor cell growth. This was verified with a reduction in migration rate in the AKT1 KO and AKT2 KO and most explicitly in the AKT1/2 KO. Furthermore, the knockout of AKT1, AKT2 or both, resulted in a reduction in lactate and alanine, suggesting that the metabolism of carbohydrates and glutathione was impaired. This was further verified in gene expression analyses, showing downregulation of genes involved in glucose metabolism. Additionally, both AKT1 KO and AKT2 KO demonstrated an impaired fatty acid metabolism. However, genes were upregulated in the Wnt and cell proliferation pathways, which could oppose this effect. AKT inhibition should therefore be combined with other effectors to attain the best effect.

  • 13.
    Häggblad Sahlberg, Sara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Spiegelberg, Diana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Glimelius, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    Stenerlöw, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Nestor, Marika
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Evaluation of cancer stem cell markers CD133, CD44, CD24: association with AKT isoforms and radiation resistance in colon cancer cells.2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 4, p. e94621-Article in journal (Refereed)
    Abstract [en]

    The cell surface proteins CD133, CD24 and CD44 are putative markers for cancer stem cell populations in colon cancer, associated with aggressive cancer types and poor prognosis. It is important to understand how these markers may predict treatment outcomes, determined by factors such as radioresistance. The scope of this study was to assess the connection between EGFR, CD133, CD24, and CD44 (including isoforms) expression levels and radiation sensitivity, and furthermore analyze the influence of AKT isoforms on the expression patterns of these markers, to better understand the underlying molecular mechanisms in the cell. Three colon cancer cell-lines were used, HT-29, DLD-1, and HCT116, together with DLD-1 isogenic AKT knock-out cell-lines. All three cell-lines (HT-29, HCT116 and DLD-1) expressed varying amounts of CD133, CD24 and CD44 and the top ten percent of CD133 and CD44 expressing cells (CD133(high)/CD44(high)) were more resistant to gamma radiation than the ten percent with lowest expression (CD133(low)/CD44(low)). The AKT expression was lower in the fraction of cells with low CD133/CD44. Depletion of AKT1 or AKT2 using knock out cells showed for the first time that CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. There were several genes in the cell adhesion pathway which had significantly higher expression in the AKT2 KO cell-line compared to the AKT1 KO cell-line; however important genes in the epithelial to mesenchymal transition pathway (CDH1, VIM, TWIST1, SNAI1, SNAI2, ZEB1, ZEB2, FN1, FOXC2 and CDH2) did not differ. Our results demonstrate that CD133(high)/CD44(high) expressing colon cancer cells are associated with AKT and increased radiation resistance, and that different AKT isoforms have varying effects on the expression of cancer stem cell markers, which is an important consideration when targeting AKT in a clinical setting.

  • 14.
    Kareem, Heewa
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Sandström, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Elia, Ronny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Gedda, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    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 Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Blocking EGFR in the liver improves the tumor-to-liver uptake ratio of radiolabeled EGF2010In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 31, no 2, p. 79-87Article in journal (Refereed)
    Abstract [en]

    Overexpression of epidermal growth factor receptor (EGFR) in several types of malignant tumors correlates with disease progression. EGFR could, therefore, be an excellent candidate for targeted radionuclide diagnostics. However, the high natural expression of EGFR in the liver may be problematic. The aim of this study was to improve the tumor-to-liver ratio of radiolabeled epidermal growth factor (EGF) by blocking its uptake by the liver with a nonradiolabeled EGFR-targeting molecule in tumorbearing mice. Intraperitoneally injected nonradiolabeled EGF was first evaluated as a blocking agent, preadministered at various time intervals before intravenous injection of 125I-labeled EGF. The anti-EGFR Affibody molecule (ZEGFR:955)2 was then assessed as a blocking agent of 111In-labeled EGF in a dual isotope study (50, 100, and 200μg, preadministered 30 or 60 min before 111In-EGF). The 30-min preadministration of nonradiolabeled EGF significantly decreased 125I-EGF uptake in the liver, whereas uptake in the tumor remained unchanged. Furthermore, preadministration of only 50μg (ZEGFR:955)2 as a blocking agent 30 min before the 111In-EGF decreased the uptake of 111In-EGF by the liver and increased its uptake by the tumor, thereby increasing the tumor-to-liver ratio sixfold. We conclude that the Affibody molecule (ZEGFR:955)2 shows promise as a blocking agent that could enhance the outcome of radionuclide-based EGFRexpressing tumor diagnostics and imaging.

  • 15.
    Kennedy, Patrick J.
    et al.
    Univ Porto, Inst Invest & Inovacao Saude, Porto, Portugal;Univ Porto, INEB Inst Engn Biomed, Porto, Portugal;Univ Porto, IPATIMUP Inst Patol & Imunol Mol, Porto, Portugal;Univ Porto, ICBAS, Porto, Portugal.
    Perreira, Ines
    Univ Porto, Inst Invest & Inovacao Saude, Porto, Portugal;Univ Porto, INEB Inst Engn Biomed, Porto, Portugal.
    Ferreira, Daniel
    Univ Porto, Inst Invest & Inovacao Saude, Porto, Portugal;Univ Porto, IPATIMUP Inst Patol & Imunol Mol, Porto, Portugal.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Oliveira, Carla
    Univ Porto, Inst Invest & Inovacao Saude, Porto, Portugal;Univ Porto, IPATIMUP Inst Patol & Imunol Mol, Porto, Portugal;Univ Porto, Fac Med, Porto, Portugal.
    Granja, Pedro L.
    Univ Porto, Inst Invest & Inovacao Saude, Porto, Portugal;Univ Porto, INEB Inst Engn Biomed, Porto, Portugal;Univ Porto, ICBAS, Porto, Portugal;Univ Porto, Fac Engn, Porto, Portugal.
    Sarmento, Bruno
    Univ Porto, Inst Invest & Inovacao Saude, Porto, Portugal;Univ Porto, INEB Inst Engn Biomed, Porto, Portugal;Inst Invest & Formacao Avancada Ciencias & Tecnol, CESPU, Gandra, Portugal;Inst Univ Ciencias Saude, Gandra, Portugal.
    Impact of surfactants on the target recognition of Fab-conjugated PLGA nanoparticles2018In: European journal of pharmaceutics and biopharmaceutics, ISSN 0939-6411, E-ISSN 1873-3441, Vol. 127, p. 366-370Article in journal (Refereed)
    Abstract [en]

    Targeted drug delivery with nanoparticles (NPs) requires proper surface ligand presentation and availability. Surfactants are often used as stabilizers in the production of targeted NPs. Here, we evaluated the impact of surfactants on ligand functionalization and downstream molecular recognition. Our model system consisted of fluorescent poly(lactic-co-glycolic acid) (PLGA) NPs that were nanoprecipitated in one of a small panel of commonly-used surfactants followed by equivalent washes and conjugation of an engineered Fab antibody fragment. Size, polydispersity index and zeta potential were determined by dynamic light scattering and laser Doppler anemometry, and Fab presence on the NPs was assessed by enzyme-linked immunosorbent assay. Most importantly, Fab-decorated NP binding to the cell surface receptor was monitored by fluorescence-activated cell sorting. 2% polyvinyl alcohol, 1% sodium cholate, 0.5% Pluronic F127 (F127) and 2% Tween-80 were initially tested. Of the four surfactants tested, PLGA NPs in 0.5% F127 and 2% Tween-80 had the highest cell binding. These two surfactants were then retested in two different concentrations, 0.5% and 2%. The Fab-decorated PLGA NPs in 2% F127 had the highest cell binding. This study highlights the impact of common surfactants and their concentrations on the downstream targeting of ligand-decorated NPs. Similar principles should be applied in the development of future targeted nanosystems where surfactants are employed.

  • 16.
    Kullberg, Erika Bohl
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Nestor, Marika
    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.
    Tumor-cell targeted EGF liposomes loaded with boronated acridine: Uptake and processing2003In: Pharmaceutical research, ISSN 0724-8741, E-ISSN 1573-904X, Vol. 20, no 2, p. 229-236Article in journal (Refereed)
    Abstract [en]

    PURPOSE:

    The aim of this work was to investigate the cellular binding and processing of polyethylene glycol-stabilized epidermal growth factor (EGF) liposomes. The liposomes were actively loaded with water-soluble boronated acridine (WSA), primarily developed for boron neutron capture therapy.

    METHODS:

    The uptake, internalization, and retention of EGF-liposome conjugates were studied in two cultured monolayer cell-lines, A-431 and U-343, with regard to the nuclide-label on the targeting agent, the carrier, and the load. The subcellular localization of WSA was studied using confocal microscopy.

    RESULTS:

    We found that the liposome complex was internalized after specific binding to the EGF receptor. After internalization in the tumor cells, WSA was distributed mainly in the cytoplasm and was shown to have long cellular retention, with 80% of the boron remaining after 48 h.

    CONCLUSIONS:

    The long retention of the compound and the cellular boron concentration reached makes these targeted liposomes interesting for further development toward boron neutron capture therapy.

  • 17.
    Kundu, S K
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Targeted therapy in head and neck cancer2012In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 33, no 3, p. 707-721Article in journal (Refereed)
    Abstract [en]

    Head and neck squamous cell carcinoma (HNSCC) of multi-factorial etiopathogenesis is rising worldwide. Treatment-associated toxicity problems and treatment failure in advanced disease stages with conventional therapies have necessitated a focus on alternative strategies. Molecular targeted therapy, with the potential for increased selectivity and fewer adverse effects, hold promise in the treatment of HNSCC. In an attempt to improve outcomes in HNSCC, targeted therapeutic strategies have been developed. These strategies are focusing on the molecular biology of HNSCC in an attempt to target selected pathways involved in carcinogenesis. Inhibiting tumor growth and metastasis by focusing on specific protein or signal transduction pathways or by targeting the tumor microenvironment or vasculature are some of the new approaches. Targeted agents for HNSCC expected to improve the effectiveness of current therapy include EGFR inhibitors (Cetuximab, Panitumumab, Zalutumumab), EGFR tyrosine kinase inhibitors (Gefitinib, Erloitinib), VEGFR inhibitors (Bevacizumab, Vandetanib), and various inhibitors of, e.g., Src-family kinase, PARP, proteasome, mTOR, COX, and heat shock protein. Moreover, targeted molecular therapy can also act as a complement to other existing cancer therapies. Several studies have demonstrated that the combination of targeting techniques with conventional current treatment protocols may improve the treatment outcome and disease control, without exacerbating the treatment related toxicities. Some of the targeted approaches have been proved as promising therapeutic potentials and are already in use, whereas remainder exhibits mixed result and necessitates further studies. Identification of predictive biomarkers of resistance or sensitivity to these therapies remains a fundamental challenge in the optimal selection of patients most likely to benefit from targeted treatment.

  • 18.
    Mortensen, Anja C.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Morin, Eric
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Brown, Christopher J
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Lane, David P
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Enhancing therapeutic effects of radio-immunotherapy using the novel stapled MDM2/X-p53 antagonist PM2Manuscript (preprint) (Other academic)
  • 19.
    Mortensen, Anja C.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Spegelberg, Diana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Haylock, Anna-Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Selvaraju, Ram Kumar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    In vivo characterization of a novel engineered CD44v6-targeting bivalent antibody fragment AbD19384 for molecular imaging of squamous cell carcinoma2014In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, no S2, p. S447-S447, article id P309Article in journal (Other academic)
  • 20.
    Mortensen, Anja C.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Spiegelberg, Diana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Scott, A. M.
    Olivia Newton John Canc Res Inst, Ludwig Inst Canc Res, Melbourne, Vic, Australia.;La Trobe Univ, Melbourne, Vic, Australia..
    Lane, D. P.
    ASTAR, P53Lab, Singapore, Singapore..
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Enhancing the effect of radiotherapy in cultured tumour cells using p53 therapy2015In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 42, no S1, p. S429-S430Article in journal (Other academic)
  • 21.
    Mortensen, Anja
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Spiegelberg, Diana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Haylock, Anna-Karin
    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 Immunology, Genetics and Pathology, Medical Radiation Science.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Preclinical evaluation of a novel engineered recombinant human anti-CD44v6 antibody for potential use in radio-immunotherapy2018In: International Journal of Oncology, ISSN 1019-6439, Vol. 52, no 6, p. 1875-1885Article in journal (Refereed)
    Abstract [en]

    CD44v6 is overexpressed in a variety of cancers, rendering it a promising target for radio-immunotherapy (RIT). In this study, we have characterized a novel engineered recombinant monoclonal anti-CD44v6 antibody, AbN44v6, and assessed its potential for use in RIT using either Lu-177 or I-131 as therapeutic radionuclides. In vitro affinity and specificity assays characterized the binding of the antibody labeled with Lu-177, I-125 or I-131. The therapeutic effects of Lu-177-AbN44v6 and I-131-AbN44v6 were investigated using two in vitro 3D tumor models with different CD44v6 expression. Finally, the normal tissue biodistribution and dosimetry for Lu-177-AbN44v6 and I-125-AbN44v6/I-131-AbN44v6 were assessed in vivo using a mouse model. All AbN44v6 radioconjugates demonstrated CD44v6-specific binding in vitro. In the in vitro 3D tumor models, dose-dependent therapeutic effects were observed with both Lu-177-AbN44v6 and I-131-AbN44v6, with a greater significant therapeutic effect observed on the cells with a higher CD44v6 expression. Biodistribution experiments demonstrated a greater uptake of Lu-177-AbN44v6 in the liver, spleen and bone, compared to I-125-AbN44v6, whereas I-125-AbN44v6 demonstrated a longer circulation time. In dosimetric calculations, the critical organs for Lu-177-AbN44v6 were the liver and spleen, whereas the kidneys and red marrow were considered the critical organs for I-131-AbN44v6. The effective dose was in the order of 0.1 mSv/MBq for both labels. In conclusion, AbN44v6 bound specifically and with high affinity to CD44v6. Furthermore, in vitro RIT demonstrated growth inhibition in a CD44v6-specific activity-dependent manner for both radioconjugates, demonstrating that both Lu-177-AbN44v6 and I-131-AbN44v6 may be promising RIT candidates. Furthermore, biodistribution and dosimetric analysis supported the applicability of both conjugates for RIT. The CD44v6-specific therapeutic effects observed with radiolabeled AbN44v6 in the 3D tumor models in vitro, combined with the beneficial dosimetry in vivo, render AbN44v6 a potential candidate for RIT.

  • 22.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Effect of cetuximab treatment in squamous cell carcinomas2010In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 31, no 2, p. 141-147Article in journal (Refereed)
    Abstract [en]

    The purpose of this study was to assess the effects of the monoclonal antibody cetuximab in a panel of cultured squamous cell carcinoma cell lines. This antibody, targeting the epidermal growth factor receptor (EGFR), is emerging as a promising agent for treatment of several cancers. As this antibody comes into clinical use, the identification of predictive markers of therapeutic benefit remains a pressing issue. Cells were first characterized according to EGFR expression, cell doubling time, and BRAF and K-ras mutations. The effects of cetuximab on cell-cycle distribution, proliferation, as well as cell growth rate were then evaluated. Cetuximab decreased cell proliferation in three out of four cell lines in a time-dependent manner, and all cell lines were found to exhibit wild type K-ras and BRAF genes. A possible correlation between EGFR expression and cetuximab effect on growth inhibition rate was observed, whereas reduction of cell doubling time seemed to be more dependent on initial growth rate. In addition, other factors may further influence the long-term treatment response of cetuximab. Moreover, the time-dependent manner of cetuximab response demonstrates the importance of long-term measurements for this substance.

  • 23.
    Nestor, Marika
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Andersson, Karl
    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.
    Characterization of In-111 and Lu-177-labeled antibodies binding to CD44v6 using a novel automated radioimmunoassay2008In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 21, no 3, p. 179-183Article in journal (Refereed)
    Abstract [en]

    Targeted cancer therapies rely on bifunctional molecules, typically a protein that specifically recognizes tumor cells and a toxic component which is linked to the protein. Therefore, development of such therapies includes detailed characterizations of protein-cell interactions in order to find a good targeting agent. Knowledge of factors such as antibody-antigen specificity, as well as cellular uptake, retention and affinity of the antibody are necessary in order to be successful. In this paper, we have used a novel instrument, LigandTracer (R) Yellow, to characterize the interactions of In-111 and Lu-177-labeled monoclonal antibodies (MAbs) with CD44v6. Uptake studies with varying specific radioactivity of the chimeric MAb U36 and with an irrelevant antibody for the CD44v6 receptor verified the reliability of the method, as well as the specificity of the antibody-receptor binding. Uptake, retention, and affinity were very similar for the In-111 and Lu-177-labeled conjugate, and were in line with earlier studies using manual methods. The fact that no adverse effects from labeling were seen, together with the high retention, could make these conjugates promising candidates for imaging and therapy of certain cancer types in the future. The novel LigandTracer technology reduced the workload and reagent spending while providing data with superior time resolution. The obtained results were in agreement with previously reported findings. In addition the real-time detection and higher time resolution made more detailed studies of the interactions possible.

  • 24.
    Nestor, Marika
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Ekberg, Tomas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Dring, John
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    van Dongen, Guus
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Anniko, Matti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Quantification of CD44v6 and EGFR expression in head and neck squamous cell carcinomas using a single-dose radioimmunoassay2007In: Tumor Biology, ISSN 1010-4283, Vol. 28, no 5, p. 253-263Article in journal (Refereed)
    Abstract [en]

    Background: In the growing field of tumor targeting, there is an urgent need to profile suitable molecular targets. In this study, CD44v6 and EGFR expression was quantified in samples of patients with head and neck squamous cell carcinoma (HNSCC) using a single-dose (SD) radioimmunoassay. Methods: The SD radioimmunoassay using 125I-chimeric monoclonal antibody (cMAb) U36 and 125I-cMAb cetuximab was first validated and then applied to quantify the expression of their target antigen molecules, CD44v6 and EGFR, in patient samples. Results were compared to immunohistochemical staining. Results: The SD assay provided sensitive quantitative values of the molecular targets studied, generally agreeing with the immunohistochemistry (IHC) results. The results indicated that expression of CD44v6 (0.2-20 nmol/μg membrane) was generally higher than that of EGFR (0.6-2.3 nmol/μg membrane) in the tumor samples analyzed, which corresponded to an average of 700,000 and 90,000 antigen molecules per cell, respectively. Conclusions: The SD radioimmunoassay is simple, reliable, and can be performed on a small amount (50 mg) of tissue. This assay could be a useful tool in the growing field of personalized cancer therapy, and can be used as a complement to IHC. In the tumors studied, CD44v6 was generally expressed at a higher level than EGFR, which might suggest that it could be more readily targeted by MAbs.

  • 25.
    Nestor, Marika
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Persson, Mikael
    Cheng, Junping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Tolmachev, Vladimir
    van Dongen, Guus
    Anniko, Matti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Kairemo, Kalevi
    Biodistribution of the chimeric monoclonal antibody U36 radioiodinated with a closo-dodecaborate-containing linker: Comparison with other radioiodination methods2003In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 14, no 4, p. 805-10Article in journal (Refereed)
    Abstract [en]

    We have evaluated the applicability of the [(4-isothiocyanatobenzylammonio)undecahydro-closo-dodecaborate (1-)] (DABI) linker molecule for antibody radiohalogenation and compared it to radiohalogenation using the linker N-succinimidyl 4-iodobenzoate (PIB) and to direct radiohalogenation using Chloramine T. These studies were performed to assess the potential of DABI conjugates and to optimize the biological properties of halogen-labeled cMAb U36. The three conjugates were evaluated in vitro for their specificity and affinity and in vivo for their biodistribution patterns in normal mice at 1.5, 6, 24, and 96 h pi. Labeling efficiencies of direct CAT labeling, indirect PIB labeling, and indirect DABI labeling were 90-95%, 60%, and 68%, respectively. This resulted in a PIB:cMAb U36 molar ratio of 1.8-2.5 and a DABI:cMAb U36 molar ratio of 4.1. The in vitro data demonstrated specific binding for all conjugates and similar affinities with values around 1 x 10(8) M(-)(1). However, the in vivo data revealed accumulation of the radioiodine uptake in thyroid for the directly labeled conjugate, with a value 10 times higher than the indirectly labeled conjugates 96 h pi. Both the (125)I-PIB-cMAb U36 and (125)I-DABI-cMAb U36 conjugates yielded a low thyroid uptake with no accumulation, indicating different catabolites for these conjugates. This may favor the use of the indirectly labeled conjugates for future studies. Apart from the specific results obtained, these findings also demonstrate how the right linker molecule will provide additional opportunities to further improve the properties of an antibody-radionuclide conjugate.

  • 26.
    Nestor, Marika
    et al.
    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, Biomedical Radiation Sciences.
    van Dongen, Guus A.
    Holland.
    Jensen, Holger 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.
    Anniko, Matti
    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.
    In vitro evaluation of the astatinated chimeric monoclonal antibody U36, a potential candidate for treatment of head and neck squamous cell carcinoma2005In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 32, no 11, p. 1296-304Article in journal (Refereed)
    Abstract [en]

    PURPOSE: The purpose of this study was to analyse the properties of the astatinated chimeric MAb (cMAb) U36 as a conjugate to selectively target and eradicate head and neck squamous cell carcinoma (HNSCC). METHODS: cMAb U36 was labelled with 211At via the linker N-succinimidyl 4-(trimethylstannyl)benzoate (SPMB). The quality of the conjugate was extensively evaluated for binding and internalisation capacity, and compared with 125I-SPMB-cMAb U36. The cellular toxicity of the astatinated conjugate was assessed in two types of in vitro growth assay and compared with 131I-labelled cMAb U36 (directly labelled). RESULTS: Comparisons between 211At-cMAb U36 and 125I-cMAb U36 demonstrated an optimal functional capacity of the labelled products. Immunoreactivity and affinity assays showed high immunoreactive fractions (>93%), and an affinity in good agreement between the astatinated and iodinated antibodies. For both conjugates, specific binding to HNSCC cells could be demonstrated, as well as some internalisation. Retention of the astatinated conjugate was just slightly lower than for the iodinated conjugate and still reasonable for therapeutic use (31+/-2% vs 42.6+/-1.0% at 22 h), demonstrating no adverse effects from astatination of the antibody. Studies on cellular toxicity demonstrated a dose-dependent and antigen-specific cellular toxicity for 211At-cMAb U36, with about 10% cell survival at 50 decays per cell. The 131I-labelled conjugate was not as efficient, with a surviving cell fraction of about 50% at 55 decays per cell. CONCLUSION: These results indicate that 211At-cMAb U36 might be a promising future candidate for eradicating HNSCC micrometastases in vivo.

  • 27.
    Nestor, Marika
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Sundström, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Anniko, Matti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Effect of cetuximab in combination with alpha-radioimmunotherapy in cultured squamous cell carcinomas2011In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 38, no 1, p. 103-112Article in journal (Refereed)
    Abstract [en]

    Aim

    The monoclonal antibody cetuximab, targeting the epidermal growth factor receptor (EGFR), is a promising molecular targeting agent to be used in combination with radiation for anticancer therapy. In this study, effects of cetuximab in combination with alpha-emitting radioimmunotherapy (RIT) in a panel of cultured human squamous cell carcinomas (SCCs) were assessed.

    Methods

    SCC cell lines were characterized and treated with cetuximab in combination with anti-CD44v6 RIT using the astatinated chimeric monoclonal antibody U36 (211At-cMAb U36). Effects on 211At-cMAb U36 uptake, internalization and cell proliferation were then assessed in SCC cells.

    Results

    Cetuximab in combination with 211At-cMAb U36 mediated increased growth inhibition compared to RIT or cetuximab alone in two cell lines. However, cetuximab also mediated radioprotective effects compared to RIT alone in two cell lines. The radioprotective effects occurred in the cell lines in which cetuximab clearly inhibited cell growth during radiation exposure. Cetuximab treatment also influenced 211At-cMAb-U36 uptake and internalization, suggesting interactions between CD44v6 and EGFR.

    Conclusions

    Results from this study demonstrate the vast importance of further clarifying the mechanisms of cetuximab and radiation response, and the relationship between EGFR and suitable RIT targets. This is important not only in order to avoid potential radioprotective effects, but also in order to find and utilize potential synergistic effects from these combinations.

  • 28. Nilvebrant, J.
    et al.
    Kuku, G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science.
    Björkelund, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Selection and characterisation of human CD44v6-binding antibody fragments for specific targeting of head and neck squamous cell carcinoma2012In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 39, no S2, p. S418-S418Article in journal (Other academic)
  • 29. Nilvebrant, Johan
    et al.
    Kuku, Gamze
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Björkelund, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Selection and in vitro characterization of human CD44v6-binding antibody fragments2012In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 59, no 5, p. 367-380Article in journal (Refereed)
    Abstract [en]

    The cluster of differentiation (CD) 44v6 antigen has been suggested to be involved in tumor formation, invasion, and metastasis formation, and has been observed in a majority of primary and metastatic squamous cell carcinomas of the head and neck. Probes specifically binding to this region may be utilized as tools for the challenging tasks of early detection and targeted treatments of small residual disease. In this project, an epitope-guided phage display selection of human fragment antigen-binding (Fab) fragments with affinity to the v6 sequence was performed. A selected set of Fab fragments was shown to specifically recognize increasingly complex forms of the target sequence, both in the form of a short synthetic or recombinant peptide and in the context of a purified extracellular domain of CD44. The binding was independent of known v6-sequence variation and posttranslational modifications that are common in the CD44 protein family. Furthermore, real-time interaction measurements on antibody fragments labeled with 125I showed specific and high-affinity binding to the antigen present on cultured head and neck squamous cell carcinoma cells. There was no cross-reactivity toward cells that lack the target protein. As hypothesized, characterization of the interaction between Fab fragments and the targets using the mathematical tool Interaction Map revealed more heterogeneous interactions on cells than with pure proteins analyzed by surface plasmon resonance. One main candidate Fab fragment with optimal affinity for all forms of the target sequence was identified. The flexible recombinant source of the Fab fragments might aid the development of tailored molecules adapted for therapeutic or diagnostic applications in the future.

  • 30.
    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.
    Haylock, Anna-Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Spiegelberg, Diana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Qvarnström, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    A novel CD44v6 targeting antibody fragment with improved tumor-to-blood ratio2012In: International Journal of Oncology, ISSN 1019-6439, Vol. 40, no 5, p. 1525-1532Article in journal (Refereed)
    Abstract [en]

    The chimeric monoclonal antibody U36 (cMAb U36) recognizes the CD44v6 antigen. Its potential as a radioimmunotargeting agent, as well as its safety, has been shown in previous studies in head and neck cancer patients. However, intact MAbs have long circulation time in the blood and tumor targeting may also be hampered due to the slow and incomplete diffusion into solid tumors. In comparison, smaller monovalent Fab' and divalent F(ab')2 fragments are expected to exhibit shorter circulating half-lives, better tumor penetration and are thus more likely to yield better imaging results. In this study, novel F(ab')2 and Fab' fragments from cMAb U36 were radiolabeled with 125I and the characteristics of the conjugates in vitro were examined. The biodistribution of the conjugates were then evaluated in nude mice bearing CD44v6-expressing xenograft tumors. Furthermore, the penetration depth and distribution in tumor tissue was assessed by autoradiography in selected tumor samples. The in vitro experiments showed that the conjugates were stable and had intact affinity to CD44v6. The biodistribution study demonstrated superior tumor-to-blood ratio for the novel cMAb U36 fragment 125I-F(ab')2 compared with both the intact MAb and the monovalent fragment form. Autoradiography also revealed better tumor penetration for 125I-F(ab')2. This study demonstrates that the use of antibody fragments may improve radioimmunotargeting and possibly improve the management of head and neck malignancies.

  • 31.
    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.
    Haylock, Anna-Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Velikyan, Irina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Spiegelberg, Diana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Kareem, Heewa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Lundqvist, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Improved Tumor-to-Organ Ratios of a Novel 67Ga-Human Epidermal Growth Factor Radionuclide Conjugate with Preadministered Antiepidermal Growth Factor Receptor Affibody Molecules2011In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 26, no 5, p. 593-601Article in journal (Refereed)
    Abstract [en]

    The over-expression of the epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinoma (HNSCC) is associated with poor prognosis. Targeted nuclear imaging of the EGFR expression could improve the diagnostics in patients with HNSCC. However, the high expression of EGFR in normal organs may conceal the tumor uptake and therefore limit the use.

    In this study, we have assessed the biodistribution of a novel hEGF radionuclide conjugate after pre-injection with anti-EGFR Affibody molecules. hEGF was conjugated with p-SCN-Bn-NOTA and labeled with 67Ga. The biodistribution of [67Ga]Ga-NOTA-Bn-NCS-hEGF in nude mice with EGFR-expressing xenografts was evaluated either alone or 45 minutes after pre-injection with one of the anti-EGFR Affibody molecules ZEGFR:1907, (ZEGFR:1907)2 or (ZEGFR:955)2.

    The novel radioimmunoconjugate, [67Ga]Ga-NOTA-Bn-NCS-hEGF demonstrated high stability in vitro and specific binding to hEGF in vitro and in vivo. Pre-injection with anti-EGFR Affibody molecules improved the tumor-to-organ ratio in the liver, salivary glands and colon. Overall, the dimeric high affinity Affibody molecule (ZEGFR:1907)2 exhibited the best results.

    These findings show that pre-blocking with an anti-EGFR Affibody molecule is a promising tool that could improve the outcome of radionuclide-based imaging of EGFR-expressing tumors.

  • 32.
    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.

  • 33.
    Spiegelberg, Diana
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Dascalu, Adrian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Mortensen, Anja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Abramenkovs, Andris
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Kuku, Gamze
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Stenerlöw, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    The novel HSP90 inhibitor AT13387 potentiates radiation effects in squamous cell carcinoma and adenocarcinoma cells2015In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 6, no 34, p. 35652-35666Article in journal (Refereed)
    Abstract [en]

    Overexpression of heat shock protein 90 (HSP90) is associated with increased tumor cell survival and radioresistance. In this study we explored the efficacy of the novel HSP90 inhibitor AT13387 and examined its radiosensitizing effects in combination with gamma-radiation in 2D and 3D structures as well as mice-xenografts. AT13387 induced effective cytotoxic activity and radiosensitized cancer cells in monolayer and tumor spheroid models, where low drug doses triggered significant synergistic effects on cell survival together with radiation. Furthermore, AT13387 treatment resulted in G2/M-phase arrest and significantly reduced the migration capacity. The expression of selected client proteins involved in DNA repair, cell-signaling and cell growth was downregulated in vitro, though the expression of most investigated proteins recurred after 8-24 h. These results were confirmed in vivo where AT13387 treated tumors displayed effective downregulation of HSP90 and its oncogenic client proteins. In conclusion, our results demonstrate that AT13387 is a potent new cancer drug and effective radiosensitizer in vitro with an excellent in vivo efficacy. AT13387 treatment has the potential to improve external beam therapy and radionuclide therapy outcomes and restore treatment efficacy in cancers that are resistant to initial therapeutic regimes.

  • 34.
    Spiegelberg, Diana
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Haylock, Anna-Karin
    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 Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    In vivo characterization of CD44v6-targeting Fab fragments for molecular imaging of squamous cell carcinoma: a dual isotope study2013In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, no Suppl. 2, p. S185-S185Article in journal (Other academic)
  • 35.
    Spiegelberg, Diana
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Kuku, Gamze
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Selvaraju, Ram
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Characterization of CD44 variant expression in head and neck squamous cell carcinomas2014In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 35, no 3, p. 2053-2062Article in journal (Refereed)
    Abstract [en]

    CD44 is a complex family of molecules, associated with aggressive malignancies and cancer stem cells. However, the role of CD44 variants in tumor progression and treatment resistance is not clear. In this study, the expression of CD44 and its variants was assessed in head and neck squamous cell carcinomas (HNSCC). Furthermore, subpopulations of cells expressing high amounts of CD44 variants were identified and characterized, for e.g., cell cycle phase and radioresistance. Results revealed high and homogenous CD44 and CD44v7 expression in four cell lines and CD44v4 and CD44v6 in three cell lines. CD44v3 was highly expressed in two cell lines, whereas CD44v5, CD44v7/8, CD44v10, CD133, and CD24 demonstrated no or moderate expression. Moreover, a subpopulation of very high CD44v4 expression was identified, which is independent of cell phase, demonstrating increased proliferation and radioresistance. In cell starvation experiments designed to enrich for cancer stem cells, a large population with dramatically increased expression of CD44, CD44v3, CD44v6, and CD44v7 was formed. Expression was independent of cell phase, and cells demonstrated increased radioresistance and migration rate. Our results demonstrate that the heterogeneity of tumor cells has important clinical implications for the treatment of HNSCC and that some of the CD44 variants may be associated with increased radioresistance. Highly expressed CD44 variants could make interesting candidates for selective cancer targeting.

  • 36.
    Spiegelberg, Diana
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Mortensen, Anja C.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Haylock, Anna-Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Selvaraju, Ram Kumar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Stenerlöw, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Evaluation of biomarkers for imaging and radio-immunotherapy in combination with HSP90 inhibition in squamous cell carcinomas2014In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, no S2, p. S638-S638, article id P972Article in journal (Other academic)
  • 37.
    Spiegelberg, Diana
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Mortensen, Anja C
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Selvaraju, Ram K
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET-MRI Platform.
    Eriksson, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    Stenerlöw, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Molecular imaging of EGFR and CD44v6 for prediction and response monitoring of HSP90 inhibition in an in vivo squamous cell carcinoma model.2016In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, no 5, p. 974-982Article in journal (Refereed)
    Abstract [en]

    PURPOSE: Heat shock protein 90 (HSP90) is essential for the activation and stabilization of numerous oncogenic client proteins. AT13387 is a novel HSP90 inhibitor promoting degradation of oncogenic proteins upon binding, and may also act as a radiosensitizer. For optimal treatment there is, however, the need for identification of biomarkers for patient stratification and therapeutic response monitoring, and to find suitable targets for combination treatments. The aim of this study was to assess the response of surface antigens commonly expressed in squamous cell carcinoma to AT13387 treatment, and to find suitable biomarkers for molecular imaging and radioimmunotherapy in combination with HSP90 inhibition.

    METHODS: Cancer cell proliferation and radioimmunoassays were used to evaluate the effect of AT13387 on target antigen expression in vitro. Inhibitor effects were then assessed in vivo in mice-xenografts. Animals were treated with AT13387 (5 × 50 mg/kg), and were imaged with PET using either (18)F-FDG or (124)I-labelled tracers for EGFR and CD44v6, and this was followed by ex-vivo biodistribution analysis and immunohistochemical staining.

    RESULTS: AT13387 exposure resulted in high cytotoxicity and possible radiosensitization with IC50 values below 4 nM. Both in vitro and in vivo AT13387 effectively downregulated HSP90 client proteins. PET imaging with (124)I-cetuximab showed a significant decrease of EGFR in AT13387-treated animals compared with untreated animals. In contrast, the squamous cell carcinoma-associated biomarker CD44v6, visualized with (124)I-AbD19384 as well as (18)F-FDG uptake, were not significantly altered by AT13387 treatment.

    CONCLUSION: We conclude that AT13387 downregulates HSP90 client proteins, and that molecular imaging of these proteins may be a suitable approach for assessing treatment response. Furthermore, radioimmunotherapy targeting CD44v6 in combination with AT13387 may potentiate the radioimmunotherapy outcome due to radiosensitizing effects of the drug, and could potentially lead to a lower dose to normal tissues.

  • 38.
    Spiegelberg, Diana
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Mortensen, Anja C.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Selvaraju, Ram Kumar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Eriksson, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Stenerlöw, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Scott, A.
    Olivia Newton John Canc Res Inst, Ludwig Inst Canc Res, Melbourne, Vic, Australia.;La Trobe Univ, Melbourne, Vic, Australia..
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Molecular imaging of EGFR and EGFRvIII for prediction and response monitoring of HSP90 inhibition in an in vivo squamous cell carcinoma model2015In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 42, no S1, p. S263-S264Article in journal (Other academic)
  • 39.
    Spiegelberg, Diana
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Mortensen, Anja C.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Selvaraju, Ram Kumar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Stenberg, J.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Scott, A.
    Olivia Newton John Canc Res Inst, Ludwig Inst Canc Res, Melbourne, Vic, Australia.;La Trobe Univ, Melbourne, Vic, Australia..
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Improving EGFR imaging by use of an anti-EGFRvIII monoclonal antibody: characterizations in an in vivo squamous cell carcinoma model2015In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 42, no S1, p. S467-S467Article in journal (Other academic)
  • 40.
    Spiegelberg, Diana
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Characterization of CD44 Variant Expression in Cultured Head & Neck Squamous Cell Carcinomas for Possible Use in Radio-Immunotargeting2012In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 39, no S2, p. S354-S354Article in journal (Other academic)
  • 41.
    Spiegelberg, Diana
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Stenberg, J.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Haylock, Anna-Karin
    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. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    A real-time in vitro assay as a potential predictor of in vivo tumour imaging properties2015In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 42, no S1, p. S306-S307Article in journal (Other academic)
  • 42.
    Spiegelberg, Diana
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Stenberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Ridgeview Instruments AB, Vange, Sweden..
    Haylock, Anna-Karin
    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.
    A real-time in vitro assay as a potential predictor of in vivo tumor imaging properties2016In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 43, no 1, p. 12-18Article in journal (Refereed)
    Abstract [en]

    Introduction: Selective tumor targeting strategies based on cell surface molecules enable new personalized diagnosis and treatments, potentially lowering adverse effects and increasing efficacy. Radio-immunotargeting generally relies on a molecule binding to a cancer-specific target. It is therefore important to understand the properties of molecular interactions in their working environment and how to translate these properties measured in vitro into the in vivo molecular imaging situation. Methods: Time resolved interaction analysis in vitro was compared with a corresponding in vivo xenograft mouse model. The antibody fragment AbD15179 was labeled with I-125 or In-111, and analyzed on cell lines with differing CD44v6 expression in vitro, and in a dual tumor xenograft model derived from the same cell lines. In vitro LigandTracer measurements were analyzed with TraceDrawer and Interaction Map. Conjugate sensitivity, kinetics, and signal-to-background ratios were assessed for both tumor cells in vitro and xenograft tumors in vivo. Results: In vitro results revealed a general biphasic appearance of a high- and a low-affinity interaction event. The In-111-labeled fragment displayed the largest proportion of the high-affinity interaction with increased sensitivity and retention compared to I-125-Fab. In vivo results were in agreement with in vitro data, with increased retention, higher sensitivity and better contrast for the In-111-labeled fragment compared to I-125. Conclusions: Time resolved binding characteristics measured in vitro largely matched the in vivo performance for the conjugates, which is promising for future studies. In vitro time-resolved LigandTracer assays are efficient, rapid, and in this study shown to be able to predict in vivo outcomes. Advances in Knowledge and Implications for Patient Care: Further studies are needed to confirm these findings, but the method is promising considering the ethical need to reduce the use of laboratory animals, as well as reducing costs for the development of tumor targeting compounds in the future.

  • 43.
    Stenberg, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Bondza, Sina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Spiegelberg, Diana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Björkelund, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Impact of Conjugation of Antibodies and Antibody Fragments on Their Interaction With Target Antigens on Living Cells2014In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, no S2, p. S448-S448, article id P311Article in journal (Other academic)
  • 44.
    Stenberg, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Spiegelberg, Diana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Karlsson, Hampus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Choice of labeling and cell line influences interactions between the Fab fragment AbD15179 and its target antigen CD44v62014In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 41, no 2, p. 140-147Article in journal (Refereed)
    Abstract [en]

    Medical imaging by use of immunotargeting generally relies on a labeled molecule binding to a specific target on the cell surface. It is important to utilize both cell-based and time-resolved binding assays in order to understand the properties of such molecular interactions in a relevant setting. In this report we describe the detailed characterization of the interaction properties for AbD15179, a promising CD44v6-targeting antibody fragment for radio-immunotargeting. Influence of labeling and cell-line model on the protein interaction kinetics was assessed using three different labeling approaches in (In-111, I-125 and FITC) on three different squamous carcinoma cell lines. Interactions were measured using time-resolved assays on living cells, and further analyzed with Interaction Map (R). Results demonstrated a general biphasic appearance of a high- and a low-affinity binding event in all cases. The relative contribution from these two interactions differed between conjugates. For I-125-Fab, the population of low-affinity binders could be significantly increased by extending the chloramine T exposure during labeling, whereas the In-111-labeling predominantly resulted in a high-affinity interaction. Interactions were also shown to be cell line dependent, with e.g. SCC-25 cells generally mediating a faster dissociation of conjugates compared to the other cell lines. In conclusion, we report both cell line dependent and labeling associated variations in interaction kinetics for AbD15179 binding to CD44v6. This has implications for cell-based kinetic assays and applications based on labeled conjugates in general, as well as in a clinical setting, where each individual tumor may create different kinetic profiles for the same conjugate.

1 - 44 of 44
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