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  • 1.
    Ahlgren, Sara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Andersson, Kristofer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Kit formulation for 99mTc-labeling of recombinant anti-HER2 Affibody molecules with a C-terminally engineered cysteine2010In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 37, no 5, p. 539-546Article in journal (Refereed)
    Abstract [en]

    Introduction: Molecular imaging of HER2-expression in malignant tumors provides potentially important information for patient management. Affibody molecules have shown to be suitable tracers for imaging applications using SPECT or PET. Results from an earlier evaluation of the application of site specific 99mTc-labeling on the Affibody molecule, ZHER2:2395-C, were favorable.

    Methods: As a preparation for clinical application of this tracer we have developed and evaluated a robust single-vial freeze-dried kit, allowing labeling of the Affibody molecule, ZHER2:2395-C, with 99mTc.

    Results: The composition of the kit (containing glucoheptonate, EDTA and tin(II)-chloride), as well as the protein amount and the pertechnetate volume were optimized for a high labeling yield (> 90 %) and minimal presence of reduced hydrolyzed technetium colloids (< 1 %). The specificity to HER2 receptors, the binding competence and the stability in PBS and murine serum were verified in vitro. The shelf-life was also evaluated in vitro, showing no reduction in labeling yield or binding capacity to HER2-expressing cells after over 400 days of storage of the single-vial freeze-dried kit.

    Conclusions: ZHER2:2395-C labeled with 99mTc using the lyophilized kit was stable and resulted in a favorable biodistribution in an in vivo evaluation in normal NMRI mice.

  • 2.
    Ahlgren, Sara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Radionuclide molecular imaging using affibody molecules2010In: Current Pharmaceutical Biotechnology, ISSN 1389-2010, E-ISSN 1873-4316, Vol. 11, no 6, p. 581-589Article, review/survey (Refereed)
    Abstract [en]

    The current way to increase efficacy of cancer therapy is the use of molecular recognition of aberrantly expressed gene products for selective treatment. However, only a fraction of the patients have tumors with a particular molecular target. Radionuclide imaging of molecular targets might help to stratify patient for cancer treatment. Affibody molecules are scaffold proteins, which can be selected for high affinity recognition of proteinaceous molecular targets. The capacity to re-fold under physiological conditions allows labeling of Affibody molecules in a broad range of pH and temperatures with preserved binding properties. Peptide synthesis or introduction of a unique cysteine enables site-specific labeling of Affibody molecules, resulting in uniform conjugates with well-defined pharmacological characteristics. The small size (7 kDa) of Affibody molecules provides rapid extravasation, rapid tumor penetration, and rapid clearance of unbound tracer from healthy organs and tissues. In combination with sub-nanomolar affinity, this results in high contrast in vivo imaging a few hours after injection. Excellent targeting has been demonstrated in pre-clinical studies with HER2-targeting Affibody molecules labeled with (99m)Tc and (111)In for single photon computed tomography (SPECT), and (18)F, (64)Cu, (124)I and (68)Ga for positron emission tomography (PET). Pilot clinical data confirm the high potential of Affibody molecules.

  • 3. Ahlstedt, J.
    et al.
    Orbom, A.
    Akesson, A.
    Frejd, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Strand, S.
    Tran, T.
    Simultaneous dual-radionuclide SPECT-imaging of HER2 expression using 99mTc-Affibody/111In-trastuzumab2014In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, no S2, p. S274-S274, article id OP522Article in journal (Other academic)
  • 4. Algenas, Cajsa
    et al.
    Agaton, Charlotta
    Fagerberg, Linn
    Asplund, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Bjorling, Lisa
    Bjorling, Erik
    Kampf, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Lundberg, Emma
    Nilsson, Peter
    Persson, Anja
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Wernerus, Henrik
    Uhlen, Mathias
    Takanen, Jenny Ottosson
    Hober, Sophia
    Antibody performance in western blot applications is context-dependent2014In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 9, no 3, p. 435-445Article in journal (Refereed)
    Abstract [en]

    An important concern for the use of antibodies in various applications, such as western blot (WB) or immunohistochemistry (IHC), is specificity. This calls for systematic validations using well-designed conditions. Here, we have analyzed 13000 antibodies using western blot with lysates from human cell lines, tissues, and plasma. Standardized stratification showed that 45% of the antibodies yielded supportive staining, and the rest either no staining (12%) or protein bands of wrong size (43%). A comparative study of WB and IHC showed that the performance of antibodies is application-specific, although a correlation between no WB staining and weak IHC staining could be seen. To investigate the influence of protein abundance on the apparent specificity of the antibody, new WB analyses were performed for 1369 genes that gave unsupportive WBs in the initial screening using cell lysates with overexpressed full-length proteins. Then, more than 82% of the antibodies yielded a specific band corresponding to the full-length protein. Hence, the vast majority of the antibodies (90%) used in this study specifically recognize the target protein when present at sufficiently high levels. This demonstrates the context- and application-dependence of antibody validation and emphasizes that caution is needed when annotating binding reagents as specific or cross-reactive. WB is one of the most commonly used methods for validation of antibodies. Our data implicate that solely using one platform for antibody validation might give misleading information and therefore at least one additional method should be used to verify the achieved data.

  • 5.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Honarvar, Hadis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Wållberg, Helena
    Strand, Joanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Varasteh, Zohreh
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Dunås, Finn
    Sandström, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science.
    Rosestedt, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Löfblom, John
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Ståhl, Stefan
    Selection of an optimal cysteine-containing peptide-based chelator for labeling of Affibody molecules with 188-Re2013In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, no Suppl. 2, p. S219-S220Article in journal (Other academic)
    Abstract [en]

    Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of 188Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all 188Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The 188Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of 188Re-ZHER2:V2 (3.1±0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental 188Re-ZHER2:2395 (172±32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of 188Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.

  • 6.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Honarvar, Hadis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Wållberg, Helena
    Strand, Joanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Varasteh, Zohreh
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Rosestedt, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Dunås, Finn
    Sandström, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Medical Radiation Sciences.
    Löfblom, John
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Ståhl, Stefan
    Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with 188Re2014In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 87, p. 519-528Article in journal (Refereed)
    Abstract [en]

    Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of 188Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all 188Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The 188Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of 188Re-ZHER2:V2 (3.1 ± 0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental 188Re-ZHER2:2395 (172 ± 32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of 188Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.

  • 7.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Orlova, Anna
    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.
    Radiolabeled Probes Targeting Tyrosine-Kinase Receptors For Personalized Medicine2014In: Current pharmaceutical design, ISSN 1381-6128, E-ISSN 1873-4286, Vol. 20, no 14, p. 2275-2292Article in journal (Refereed)
    Abstract [en]

    Receptor tyrosine kinases (RTK) are transmembrane receptors regulating cellular proliferation, differentiation, apoptosis, motility and recruitment of the vasculature. Aberrant expression and/or function of RTK have been detected in many malignant tumors and are considered to be a part of the transformed phenotype. The action of several classes of anti-cancer drugs is based on specific recognition of RTK. Monoclonal antibodies target extracellular binding domains, while tyrosine kinase inhibitors (TKI) bind to intracellular kinase domains to suppress RTK signaling. The issues regarding the efficient use of RTK targeting are the inter- and intra-patient heterogeneity of RTK expression and the changes of expression levels during the course of disease and in response to therapy. Radionuclide molecular imaging of RTK expression may aid in selecting patients who would benefit from RTK-targeting therapy and in identifying non-responders. Therefore, the therapy would be more personalized. Currently, radiolabeled proteins (monoclonal antibodies and their fragments, natural peptides ligands to RTK and de novo selected affinity proteins) and TKI and their analogues are under development for the visualization of RTK. In this review, we discuss the advantages and disadvantages of these approaches.

  • 8.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Perols, Anna
    Eriksson Karlström, Amelie
    Sandström, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Medical Physics.
    Boschetti, Frederic
    Orlova, Anna
    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.
    Preclinical evaluation of anti-HER2 Affibody molecules site-specifically labeled with 111In using a maleimido derivative of NODAGA2012In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 39, no 4, p. 518-529Article in journal (Refereed)
    Abstract [en]

    Introduction

    Affibody molecules have demonstrated potential for radionuclide molecular imaging. The aim of this study was to synthesize and evaluate a maleimido derivative of the 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) for site-specific labeling of anti-HER2 Affibody molecule.

    Methods

    The maleimidoethylmonoamide NODAGA (MMA-NODAGA) was synthesized and conjugated to ZHER2:2395 Affibody molecule having a C-terminal cysteine. Labeling efficiency, binding specificity to and cell internalization by HER2-expressing cells of [111In-MMA-NODAGA-Cys61]-ZHER2:2395 were studied. Biodistribution of [111In-MMA-NODAGA-Cys61]-ZHER2:2395 and [111In-MMA-DOTA-Cys61]-ZHER2:2395 was compared in mice.

    Results

    The affinity of [MMA-NODAGA-Cys61]-ZHER2:2395 binding to HER2 was 67 pM. The 111In-labeling yield was 99.6%±0.5% after 30 min at 60°C. [111In-MMA-NODAGA-Cys61]-ZHER2:2395 bound specifically to HER2-expressing cells in vitro and in vivo. Tumor uptake of [111In-MMA-NODAGA-Cys61]-ZHER2:2395 in mice bearing DU-145 xenografts (4.7%±0.8% ID/g) was lower than uptake of [111In-MMA-DOTA-Cys61]-ZHER2:2395 (7.5%±1.6% ID/g). However, tumor-to-organ ratios were higher for [111In-MMA-NODAGA-Cys61]-ZHER2:2395 due to higher clearance rate from normal tissues.

    Conclusions

    MMA-NODAGA is a promising chelator for site-specific labeling of targeting proteins containing unpaired cysteine. Appreciable influence of chelators on targeting properties of Affibody molecules was demonstrated.

  • 9.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Strand, Joanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Rosik, D.
    Selvaraju, Ram Kumar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Karlstrom, A. Eriksson
    Orlova, Anna
    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 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.
    Comparative evaluation of anti-HER2 affibody molecules labeled with 68Ga and 111In using maleimido derivatives of DOTA and NODAGA2012In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 39, no S2, p. S299-S299Article in journal (Other academic)
  • 10.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Strand, Joanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Rosik, Daniel
    Selvaraju, Ram Kumar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Eriksson Karlström, Amelie
    Orlova, Anna
    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.
    Influence of Nuclides and Chelators on Imaging Using Affibody Molecules: Comparative Evaluation of Recombinant Affibody Molecules Site-Specifically Labeled with 68Ga and 111In via Maleimido Derivatives of DOTA and NODAGA2013In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 24, no 6, p. 1102-1109Article in journal (Refereed)
    Abstract [en]

    Accurate detection of cancer-associated molecular abnormalities in tumors could make cancer treatment more personalized. Affibody molecules enable high contrast imaging of tumor-associated protein expression shortly after injection. The use of the generator-produced positron-emitting radionuclide 68Ga should increase sensitivity of HER2 imaging. The chemical nature of radionuclides and chelators influences the biodistribution of Affibody molecules, providing an opportunity to further increase the imaging contrast. The aim of the study was to compare maleimido derivatives of DOTA and NODAGA for site-specific labeling of a recombinant ZHER2:2395 HER2-binding Affibody molecule with 68Ga. DOTA and NODAGA were site-specifically conjugated to the ZHER2:2395 Affibody molecule having a C-terminal cysteine and labeled with 68Ga and 111In. All labeled conjugates retained specificity to HER2 in vitro. Most of the cell-associated activity was membrane-bound with a minor difference in internalization rate. All variants demonstrated specific targeting of xenografts and a high tumor uptake. The xenografts were clearly visualized using all conjugates. The influence of chelator on the biodistribution and targeting properties was much less pronounced for 68Ga than for 111In. The tumor uptake of 68Ga-NODAGA-ZHER2:2395 and 68Ga-DOTA-ZHER2:2395 and tumor-to-blood ratios at 2 h p.i. did not differ significantly. However, the tumor-to-liver ratio was significantly higher for 68Ga-NODAGA- ZHER2:2395 (8 ± 2 vs 5.0 ± 0.3) offering the advantage of better liver metastases visualization. In conclusion, influence of chelators on biodistribution of Affibody molecules depends on the radionuclides and reoptimization of labeling chemistry is required when a radionuclide label is changed.

  • 11.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Varasteh, Zohreh
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Eek, Annemarie
    Boerman, Otto
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET.
    In Vivo and In Vitro Studies on Renal Uptake of Radiolabeled Affibody Molecules for Imaging of HER2 Expression in Tumors2013In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 28, no 3, p. 187-195Article in journal (Refereed)
    Abstract [en]

    Affibody molecules (6-7 kDa) are a new class of small robust three-helical scaffold proteins. Radiolabeled subnanomolar anti-HER2 affibody Z(HER2:342) was developed for imaging of HER2 expression in tumors, and a clinical study has demonstrated that the In-111- and Ga-68-labeled affibody molecules can efficiently detect HER2 expressing metastases in breast cancer patients. However, a significant renal accumulation of radioactivity after systemic injection of a radiolabeled anti-HER2 affibody conjugate is observed. The aim of this study was to investigate the mechanism of renal reabsorption of anti-HER2 affibody at the molecular level. Renal accumulation of radiolabeled anti-HER2 affibody molecules was studied in a murine model and in vitro using opossum-derived proximal tubule (OK) cells. It was found that kidney reabsorption of affibody molecule was not driven by megalin/cubilin. Amino acids in the target-binding side of affibody molecule were involved in binding to OK cells. On OK cells, two types of receptors for anti-HER2 affibody molecule were found: K-D1 = 0.8 nM, B-max1 = 71,500 and K-D2 = 9.2 nM, B-max2 = 367,000. The results of the present study indicate that affibody molecule and other scaffold-based targeting proteins with a relatively low kidney uptake can be selected using in vitro studies with tubular kidney cells.

  • 12.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Wållberg, Helena
    Honarvar, Hadis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Strand, Joanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Varasteh, Zohreh
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Sandström, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Medical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET.
    Löfblom, John
    Larsson, Erik
    Strand, Sven-Erik
    Lubberink, Mark
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Medical Physics.
    Ståhl, Stefan
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    188Re-ZHER2:V2, a promising affibody-based targeting agent against HER2-expressing tumors: preclinical assessment2014In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 55, no 11, p. 1842-1848Article in journal (Refereed)
    Abstract [en]

    Affibody molecules are small (7 kDa) nonimmunoglobulin scaffold proteins with favorable tumor-targeting properties. Studies concerning the influence of chelators on biodistribution of 99mTc-labeled Affibody molecules demonstrated that the variant with a C-terminal glycyl-glycyl-glycyl-cysteine peptide–based chelator (designated ZHER2:V2) has the best biodistribution profile in vivo and the lowest renal retention of radioactivity. The aim of this study was to evaluate 188Re-ZHER2:V2 as a potential candidate for radionuclide therapy of human epidermal growth factor receptor type 2 (HER2)–expressing tumors.

    Methods:

    ZHER2:V2 was labeled with 188Re using a gluconate-containing kit. Targeting of HER2-overexpressing SKOV-3 ovarian carcinoma xenografts in nude mice was studied for a dosimetry assessment.

    Results:

    Binding of 188Re-ZHER2:V2 to living SKOV-3 cells was demonstrated to be specific, with an affinity of 6.4 ± 0.4 pM. The biodistribution study showed a rapid blood clearance (1.4 ± 0.1 percentage injected activity per gram [%ID/g] at 1 h after injection). The tumor uptake was 14 ± 2, 12 ± 2, 5 ± 2, and 1.8 ± 0.5 %IA/g at 1, 4, 24, and 48 h after injection, respectively. The in vivo targeting of HER2-expressing xenografts was specific. Already at 4 h after injection, tumor uptake exceeded kidney uptake (2.1 ± 0.2 %IA/g). Scintillation-camera imaging showed that tumor xenografts were the only sites with prominent accumulation of radioactivity at 4 h after injection. Based on the biokinetics, a dosimetry evaluation for humans suggests that 188Re-ZHER2:V2 would provide an absorbed dose to tumor of 79 Gy without exceeding absorbed doses of 23 Gy to kidneys and 2 Gy to bone marrow. This indicates that future human radiotherapy studies may be feasible.

    Conclusion:

    188Re-ZHER2:V2 can deliver high absorbed doses to tumors without exceeding kidney and bone marrow toxicity limits.

  • 13.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Wållberg, Helena
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Rosestedt, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Hosseinimehr, Seyed Jalal
    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.
    Ståhl, Stefan
    Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of (99m)Tc-labeled recombinant Affibody molecules2012In: Amino Acids, ISSN 0939-4451, E-ISSN 1438-2199, Vol. 42, no 5, p. 1975-1985Article in journal (Refereed)
    Abstract [en]

    Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide (99m)Tc. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of (99m)Tc-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied (99m)Tc-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of (99m)Tc. The biodistribution of all (99m)Tc-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of (99m)Tc was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.

  • 14.
    Altschuh, Danièle
    et al.
    Biotechnologie et signalisation cellulaire, Université de Strasbourg, France.
    Björkelund, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Strandgård, John
    Ridgeview Instruments AB, Uppsala, Sweden.
    Chouliera, Laurence
    Biotechnologie et signalisation cellulaire, Université de Strasbourg, France.
    Malmqvist, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Deciphering complex protein interaction kinetics using Interaction Map2012In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 428, no 1, p. 74-79Article in journal (Refereed)
    Abstract [en]

    Cellular receptor systems are expected to present complex ligand interaction patterns that cannot beevaluated assuming a simple one ligand:one receptor interaction model. We have previously evaluatedheterogeneous interactions using an alternative method to regression analysis, called Interaction Map(IM). IM decomposes a time-resolved binding curve into its separate components. By replacing the reductionistic,scalar kinetic association rate constant ka and dissociation rate constant kd with a two-dimensionaldistribution of ka and kd, it is possible to display heterogeneous data as a map where each peakcorresponds to one of the components that contribute to the cumulative binding curve. Here we challengethe Interaction Map approach by artificially generating heterogeneous data from two known interactions,on either LigandTracer or Surface Plasmon Resonance devices. We prove the ability of IM toaccurately decompose these man-made heterogeneous binding curves composed of two different interactions.We conclude that the Interaction Map approach is well suited for the analysis of complex bindingdata and forecast that it has a potential to resolve previously uninterpretable data, in particular thosegenerated in cell-based assays.

  • 15. Andersen, Jan Terje
    et al.
    Pehrson, Rikard
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Daba, Muluneh Bekele
    Abrahmsén, Lars
    Ekblad, Caroline
    Extending Half-life by Indirect Targeting of the Neonatal Fc Receptor (FcRn) Using a Minimal Albumin Binding Domain2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 7, p. 5234-5241Article in journal (Refereed)
    Abstract [en]

    The therapeutic and diagnostic efficiency of engineered small proteins, peptides, and chemical drug candidates is hampered by short in vivo serum half-life. Thus, strategies to tailor their biodistribution and serum persistence are highly needed. An attractive approach is to take advantage of the exceptionally long circulation half-life of serum albumin or IgG, which is attributed to a pH-dependent interaction with the neonatal Fc receptor (FcRn) rescuing these proteins from intracellular degradation. Here, we present molecular evidence that a minimal albumin binding domain (ABD) derived from streptococcal protein G can be used for efficient half-life extension by indirect targeting of FcRn. We show that ABD, and ABD recombinantly fused to an Affibody molecule, in complex with albumin does not interfere with the strictly pH-dependent FcRn-albumin binding kinetics. The same result was obtained in the presence of IgG. An in vivo study performed in rat confirmed that the clinically relevant human epidermal growth factor 2 (HER2)-targeting Affibody molecule fused to ABD has a similar half-life and biodistribution profile as serum albumin. The proof-of-concept described may be broadly applicable to extend the in vivo half-life of short lived biological or chemical drugs ultimately resulting in enhanced therapeutic or diagnostic efficiency, a more favorable dosing regimen, and improved patient compliance.

  • 16. Andersson, K. G.
    et al.
    Varasteh, Zohreh
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Rosestedt, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Malm, M.
    Sandström, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Medical 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.
    Lofblom, J.
    Stahl, S.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    111In-labeled NOTA-conjugated Affibody molecules for visualization of HER3 expression in malignant tumors2014In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, no S2, p. S311-S311, article id OP681Article in journal (Other academic)
  • 17.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Bringing time into molecular and cellular biology2013In: Journal of Analytical Oncology, ISSN 1927-7229, Vol. 2, no 2, p. 65-68Article in journal (Refereed)
    Abstract [en]

    In conjunction with the defense of a doctoral thesis on the deciphering of complex protein interactions on living cells, six scientists shared their view on time in molecular and cellular biology. This brief review takes the form of a conference report and summarizes the contributions of the speakers and the defense. Opportunities and challenges for time resolved assays in molecular and cellular biology were vividly discussed during two days with a pan-European audience. Awareness of biological timeframes and understanding the temporal aspects were claimed critical for analytical applications in biology.

  • 18. Barta, P.
    et al.
    Trejtnar, F.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Preclinical Evaluation of Ligand-Target Binding Properties Using Competitive Interaction Analysis in Real-Time Radioimmunoassay on Living Cells2013In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, no Suppl. 2, p. S323-S324Article in journal (Other academic)
  • 19. Barta, P.
    et al.
    Volkova, M.
    Buijs, Jos
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Trejtnar, F.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Application of Automatic Radioimmunoassay to Facilitate In Vitro Tracer Development2014In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, no S2, p. S441-S441, article id P291Article in journal (Other academic)
  • 20.
    Barta, Pavel
    et al.
    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.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Circumventing the requirement of binding saturation for receptor quantification using interaction kinetic extrapolation2011In: Nuclear medicine communications, ISSN 0143-3636, E-ISSN 1473-5628, Vol. 32, no 9, p. 863-867Article in journal (Refereed)
    Abstract [en]

    Quantification of the number of receptors per cell (NRPC) is important when assessing whether a tumor surface biomarker is suitable for medical imaging. One common method for NPRC quantification is to use a binding saturation assay, which is time consuming and requires large amounts of reagents. The aim of this study was to evaluate an alternative method based on kinetic extrapolation (KEX) and compare it with the classical manual saturation technique with regard to accuracy as well as time and reagent consumption. Epidermal growth factor receptor (EGFR) and HER2 receptor surface expression were quantified on five tumor cell lines using three (125)I-labeled and (131)I-labeled ligands (cetuximab and EGF for EGFR, trastuzumab for HER2 receptor) for both techniques. The KEX method involved interaction measurements in the LigandTracer, followed by KEX through computerized real-time interaction analysis to correct for nonsaturation on cells. Variability and NRPC estimates of the EGFR and HER2 receptor levels using the KEX method were comparable with the results from the classical saturation technique. However, the ligand consumption for the KEX method was 26-46% of the classical saturation technique. Furthermore, the KEX method reduced the workload radically. From the observations described in this study, we believe that the KEX method enables fast, credible, and easy NRPC quantification with a reduction in reagent consumption.

  • 21. Barta, Pavel
    et al.
    Malmberg, Jennie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Melicharova, Ludmila
    Strandgård, John
    Orlova, Anna
    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.
    Laznicek, Milan
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Protein interactions with HER-family receptors can have different characteristics depending on the hosting cell line2012In: International Journal of Oncology, ISSN 1019-6439, Vol. 40, no 5, p. 1677-1682Article in journal (Refereed)
    Abstract [en]

    Cell lines are common model systems in the development of therapeutic proteins and in the research on cellular functions and dysfunctions. In this field, the protein interaction assay is a frequently used tool for assessing the adequacy of a protein for diagnostic and therapeutic purposes. In this study, we investigated the extent to which the interaction characteristics depend on the choice of cell line for HER-family receptors. The interaction characteristics of two therapeutic antibodies (trastuzumab and cetuximab) and one Affibody molecule (ZHER2:342), interacting with the intended receptor were characterized with high precision using an automated real-time interaction method, in different cell lines (HaCaT, A431, HEP-G2, SKOV3, PC3, DU-145). Clear differences in binding affinity and kinetics, up to one order of magnitude, were found for the interaction of the same protein binding to the same receptor on different cells for all three proteins. For HER-family receptors, it is therefore important to refer to the measured affinity for a protein-receptor interaction together with the hosting cell line. The ability to accurately measure affinity and kinetics of a protein-receptor interaction on cell lines of different origins may increase the understanding of underlying receptor biology, and impact the selection of candidates in the development of therapeutic or diagnostic agents.

  • 22. Barta, Pavel
    et al.
    Volkova, Marie
    Dascalu, Adrian
    Spiegelberg, Diana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Trejtnar, Frantisek
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Determination of receptor protein binding site specificity and relative binding strength using a time-resolved competition assay2014In: Journal of pharmacological and toxicological methods, ISSN 1056-8719, E-ISSN 1873-488X, Vol. 70, no 2, p. 145-151Article in journal (Refereed)
    Abstract [en]

    Introduction: Competitive binding assays can be used to decipher not only the binding kinetics of studied ligands but also the binding site preference. Such assays are an essential step in the characterization of radioligands. However, the currently used competition assays require high concentrations of usually expensive ligands and still provide only binding site preference. By employing the time-resolved competition assay presented in this paper, binding characteristics including binding site preference can be obtained using less ligand. Methods: To demonstrate the appropriateness of the time-resolved competition assay, we developed an assay in which the ligand binding was interrupted with a competitor. Experiments were performed on human carcinoma cell lines expressing epidermal growth factor receptor (EGFR). The targeting of the receptor was performed with radio-iodinated epidermal growth factor (EGF). The employed competitors involved either natural ligand transforming growth factor alpha (TGF-alpha) or anti-EGFR antibodies cetuximab and panitumumab targeting the same EGFR domain. Results: Radio-iodinated EGF bound to EGFR was displaced with either low concentrations of cetuximab or high concentrations of panitumumab. In the case of TGF-alpha, we observed no competitive displacement of bound EGF at either high or low concentrations. When comparing the time-resolved competition assay with a manual competition assay, the resulting data of measured inhibition constants were in agreement. Discussion: The results summarised in this study confirm the appropriateness of the time-resolved competition assay for assessing ligand binding properties. The assay has the potential to complement or replace conventional competition assays for determining binding site preference in the future.  

  • 23.
    Beshara, Soheir
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Sörensen, Jens
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Physiology.
    Lubberink, Mark
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Långström, Bengt
    PET Centre, University Hospital, Uppsala, Sweden.
    Antoni, Gunnar
    PET Centre, University Hospital, Uppsala, Sweden.
    Danielsson, Bo G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Internal Medicine.
    Lundqvist, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Pharmacokinetics and red cell utilization of 52Fe/59Fe-labelled iron polymaltose in anaemic patients using positron emission tomography2003In: British Journal of Haematology, ISSN 0007-1048, E-ISSN 1365-2141, Vol. 120, no 5, p. 853-859Article in journal (Other academic)
    Abstract [en]

    Parenteral iron-polysaccharide complexes are increasingly applied. The pharmacokinetics of iron sucrose have been assessed by our group using positron emission tomography (PET). A single intravenous injection of 100 mg iron as iron (III) hydroxide-polymaltose complex, labelled with a tracer in the form of 52Fe/59Fe, was similarly assessed in six patients using PET for about 8 h. Red cell utilization was followed for 4 weeks. Iron polymaltose was similarly distributed to the liver, spleen and bone marrow. However, a larger proportion of this complex was rapidly distributed to the bone marrow. The shorter equilibration phase for the liver, about 25 min, indicates the minimal role of the liver for direct distribution. Splenic uptake also reflected the reticuloendothelial handling of this complex. Red cell utilization ranged from 61% to 99%. Despite the relatively higher uptake by the bone marrow, there was no saturation of marrow transport systems at this dose level. In conclusion, high red cell utilization of iron polymaltose occurred in anaemic patients. The major portion of the injected dose was rapidly distributed to the bone marrow. In addition, the reticuloendothelial uptake of this complex may reflect the safety of polysaccharide complexes. Non-saturation of transport systems to the bone marrow indicated the presence of a large interstitial transport pool, which might possibly be transferrin.

  • 24.
    Billström, Gry Hulsart
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    Piskounova, Sonya
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Gedda, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Bowden, Tim
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Larsson, Sune
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    Improved bone formation by altering surface area of hyaluronan-based hydrogel carrier for bone morphogenetic protein-22012In: Bone, ISSN 8756-3282, E-ISSN 1873-2763, Vol. 50, p. S114-S114Article in journal (Other academic)
  • 25.
    Björkelund, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Novel Methods for Analysis of Heterogeneous Protein-Cell Interactions: Resolving How the Epidermal Growth Factor Binds to Its Receptor2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Cells are complex biological units with advanced signalling systems, a dynamic capacity to adapt to its environment, and the ability to divide and grow. In fact, they are of such high level of complexity that it has deemed extremely difficult or even impossible to completely understand cells as complete units. The search for comprehending the cell has instead been divided into small, relatively isolated research fields, in which simplified models are used to explain cell biology. The result produced through these reductionistic investigations is integral for our current description of biology. However, there comes a time when it is possible to go beyond such simplifications and investigate cell biology at a higher level of complexity. That time is now.

    This thesis describes the development of mathematical tools to investigate intricate biological systems, with focus on heterogeneous protein interactions. By the use of simulations, real-time measurements and kinetic fits, standard assays for specificity measurements and receptor quantification were scrutinized in order to find optimal experimental settings and reduce labour time as well as reagent cost. A novel analysis platform, called Interaction Map, was characterized and applied on several types of interactions. Interaction Map decomposes a time-resolved binding curve and presents information on the kinetics and magnitude of each interaction that contributed to the curve. This provides a greater understanding of parallel interactions involved in the same biological system, such as a cell. The heterogeneity of the epidermal growth factor receptor (EGFR) system was investigated with Interaction Map applied on data from the instrument LigandTracer, together with complementing manual assays. By further introducing disturbances to the system, such as tyrosine kinase inhibitors and variation in temperature, information was obtained about dimerization, internalization and degradation rates.

    In the long term, analysis of binding kinetics and combinations of parallel interactions can improve the understanding of complex biomolecular mechanisms in cells and may explain some of the differences observed between cell lines, medical treatments and groups of patients.

    List of papers
    1. Avoiding false negative results in specificity analysis of protein-protein interactions
    Open this publication in new window or tab >>Avoiding false negative results in specificity analysis of protein-protein interactions
    2011 (English)In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 24, no 1, p. 81-89Article in journal (Refereed) Published
    Abstract [en]

    The competition measurement using simultaneous incubation of labeled and unlabeled Ligand is a common method to assess the specificity of a biomolecular interaction. In this paper we show that invalid assumptions about the interactions may lead to improper experimental setups which in turn can result in inaccurate conclusions about the specificity. To improve understanding of competition measurements, simulations in MATLAB as well as real-time interaction analysis using LigandTracer have been performed. We show that use of a concentration of unlabeled Ligand of at least 10 × K(D) is necessary for assay accuracy. Increasing the incubation time to assure equilibrium, adding a pre-incubation phase, and a general understanding of the reversibility of an interaction may also improve the reliability of the measurement and the conclusions drawn about specificity. These findings may lower the risk of false negative results as well as reducing the amount of reagent needed.

    Keywords
    specificity, affinity, kinetics, competition assay
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-147025 (URN)10.1002/jmr.1026 (DOI)000289781900008 ()21194119 (PubMedID)
    Available from: 2011-02-23 Created: 2011-02-23 Last updated: 2018-12-04
    2. Circumventing the requirement of binding saturation for receptor quantification using interaction kinetic extrapolation
    Open this publication in new window or tab >>Circumventing the requirement of binding saturation for receptor quantification using interaction kinetic extrapolation
    2011 (English)In: Nuclear medicine communications, ISSN 0143-3636, E-ISSN 1473-5628, Vol. 32, no 9, p. 863-867Article in journal (Refereed) Published
    Abstract [en]

    Quantification of the number of receptors per cell (NRPC) is important when assessing whether a tumor surface biomarker is suitable for medical imaging. One common method for NPRC quantification is to use a binding saturation assay, which is time consuming and requires large amounts of reagents. The aim of this study was to evaluate an alternative method based on kinetic extrapolation (KEX) and compare it with the classical manual saturation technique with regard to accuracy as well as time and reagent consumption. Epidermal growth factor receptor (EGFR) and HER2 receptor surface expression were quantified on five tumor cell lines using three (125)I-labeled and (131)I-labeled ligands (cetuximab and EGF for EGFR, trastuzumab for HER2 receptor) for both techniques. The KEX method involved interaction measurements in the LigandTracer, followed by KEX through computerized real-time interaction analysis to correct for nonsaturation on cells. Variability and NRPC estimates of the EGFR and HER2 receptor levels using the KEX method were comparable with the results from the classical saturation technique. However, the ligand consumption for the KEX method was 26-46% of the classical saturation technique. Furthermore, the KEX method reduced the workload radically. From the observations described in this study, we believe that the KEX method enables fast, credible, and easy NRPC quantification with a reduction in reagent consumption.

    Keywords
    kinetics, ligand-receptor interaction, receptor quantification
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-158879 (URN)10.1097/MNM.0b013e3283483e1c (DOI)000294345300015 ()
    Available from: 2011-09-20 Created: 2011-09-19 Last updated: 2017-12-08Bibliographically approved
    3. Deciphering complex protein interaction kinetics using Interaction Map
    Open this publication in new window or tab >>Deciphering complex protein interaction kinetics using Interaction Map
    Show others...
    2012 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 428, no 1, p. 74-79Article in journal (Refereed) Published
    Abstract [en]

    Cellular receptor systems are expected to present complex ligand interaction patterns that cannot beevaluated assuming a simple one ligand:one receptor interaction model. We have previously evaluatedheterogeneous interactions using an alternative method to regression analysis, called Interaction Map(IM). IM decomposes a time-resolved binding curve into its separate components. By replacing the reductionistic,scalar kinetic association rate constant ka and dissociation rate constant kd with a two-dimensionaldistribution of ka and kd, it is possible to display heterogeneous data as a map where each peakcorresponds to one of the components that contribute to the cumulative binding curve. Here we challengethe Interaction Map approach by artificially generating heterogeneous data from two known interactions,on either LigandTracer or Surface Plasmon Resonance devices. We prove the ability of IM toaccurately decompose these man-made heterogeneous binding curves composed of two different interactions.We conclude that the Interaction Map approach is well suited for the analysis of complex bindingdata and forecast that it has a potential to resolve previously uninterpretable data, in particular thosegenerated in cell-based assays.

    Keywords
    Real-time analysis, Kinetics, Heterogeneity, LigandTracer, SPR
    National Category
    Medical and Health Sciences
    Research subject
    Molecular Biotechnology
    Identifiers
    urn:nbn:se:uu:diva-183869 (URN)10.1016/j.bbrc.2012.10.008 (DOI)000311523200013 ()
    Available from: 2012-11-05 Created: 2012-11-05 Last updated: 2017-12-07Bibliographically approved
    4. Comparing the epidermal growth factor interaction with four different cell lines: intriguing effects imply strong dependency of cellular context
    Open this publication in new window or tab >>Comparing the epidermal growth factor interaction with four different cell lines: intriguing effects imply strong dependency of cellular context
    2011 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 6, no 1, p. e16536-Article in journal (Refereed) Published
    Abstract [en]

    The interaction of the epidermal growth factor (EGF) with its receptor (EGFR) is known to be complex, and the common over-expression of EGF receptor family members in a multitude of tumors makes it important to decipher this interaction and the following signaling pathways. We have investigated the affinity and kinetics of (125)I-EGF binding to EGFR in four human tumor cell lines, each using four culturing conditions, in real time by use of LigandTracer®.Highly repeatable and precise measurements show that the overall apparent affinity of the (125)I-EGF - EGFR interaction is greatly dependent on cell line at normal culturing conditions, ranging from K(D)≈200 pM on SKBR3 cells to K(D)≈8 nM on A431 cells. The (125)I-EGF - EGFR binding curves (irrespective of cell line) have strong signs of multiple simultaneous interactions. Furthermore, for the cell lines A431 and SKOV3, gefitinib treatment increases the (125)I-EGF - EGFR affinity, in particular when the cells are starved. The (125)I-EGF - EGFR interaction on cell line U343 is sensitive to starvation while as on SKBR3 it is insensitive to gefitinib and starvation.The intriguing pattern of the binding characteristics proves that the cellular context is important when deciphering how EGF interacts with EGFR. From a general perspective, care is advisable when generalizing ligand-receptor interaction results across multiple cell-lines.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-147027 (URN)10.1371/journal.pone.0016536 (DOI)000286834300081 ()21304974 (PubMedID)
    Available from: 2011-02-23 Created: 2011-02-23 Last updated: 2018-12-04
    5. Gefitinib Induces Epidermal Growth Factor Receptor Dimers Which Alters the Interaction Characteristics with (125)I-EGF
    Open this publication in new window or tab >>Gefitinib Induces Epidermal Growth Factor Receptor Dimers Which Alters the Interaction Characteristics with (125)I-EGF
    Show others...
    2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 9, p. e24739-Article in journal (Refereed) Published
    Abstract [en]

    The tyrosine kinase inhibitor gefitinib inhibits growth in some tumor types by targeting the epidermal growth factor receptor (EGFR). Previous studies show that the affinity of the EGF-EGFR interaction varies between hosting cell line, and that gefitinib increases the affinity for some cell lines. In this paper, we investigate possible mechanisms behind these observations. Real-time interaction analysis in LigandTracer (R) Grey revealed that the HER2 dimerization preventing antibody pertuzumab clearly modified the binding of (125)I-EGF to EGFR on HER2 overexpressing SKOV3 cells in the presence of gefitinib. Pertuzumab did not affect the binding on A431 cells, which express low levels of HER2. Cross-linking measurements showed that gefitinib increased the amount of EGFR dimers 3.0-3.8 times in A431 cells in the absence of EGF. In EGF stimulated SKOV3 cells the amount of EGFR dimers increased 1.8-2.2 times by gefitinib, but this effect was cancelled by pertuzumab. Gefitinib treatment did not alter the number of EGFR or HER2 expressed in tumor cell lines A431, U343, SKOV3 and SKBR3. Real-time binding traces were further analyzed in a novel tool, Interaction Map, which deciphered the different components of the measured interaction and supports EGF binding to multiple binding sites. EGFR and HER2 expression affect the levels of EGFR monomers, homodimers and heterodimers and EGF binds to the various monomeric/dimeric forms of EGFR with unique binding properties. Taken together, we conclude that dimerization explains the varying affinity of EGF - EGFR in different cells, and we propose that gefitinib induces EGFR dimmers, which alters the interaction characteristics with (125)I-EGF.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-159470 (URN)10.1371/journal.pone.0024739 (DOI)000294803200045 ()
    Available from: 2011-10-04 Created: 2011-10-03 Last updated: 2018-12-04
    6. Resolving the EGF-EGFR interaction characteristics through a multiple-temperature, multiple-inhibitor, real-time interaction analysis approach
    Open this publication in new window or tab >>Resolving the EGF-EGFR interaction characteristics through a multiple-temperature, multiple-inhibitor, real-time interaction analysis approach
    2013 (English)In: Molecular and Clinical Oncology, ISSN 2049-9469, Vol. 1, no 2, p. 343-352Article in journal (Refereed) Published
    Abstract [en]

    Overexpression and aberrant activity of the epidermal growth factor (EGF) have been observed in various cancer types, rendering it an important target in oncology research. The interaction between EGF and its receptor (EGFR), as well as subsequent internalization, is complex and may be affected by various factors including tyrosine kinase inhibitors (TKIs). By combining real‑time binding curves produced in LigandTracer® with internalization assays conducted at different temperatures and with different TKIs, the processes of ligand binding, internalization and excretion was visualized. SKOV3 cells had a slower excretion rate compared to A431 and U343 cells, and the tested TKIs (gefitinib, lapatinib, AG1478 and erlotinib) reduced the degree of internalization. The kinetic analysis of the binding curves further demonstrated TKI‑dependent balances of EGFR monomer and dimer populations, where lapatinib promoted the monomeric form, while the other TKIs induced dimers. The dimer levels were found to be associated with the apparent affinity of the EGF‑EGFR interaction, with EGF binding stronger to EGFR dimers compared to monomers. This study analyzed how real‑time molecular interaction analysis may be utilized in combination with perturbations in order to understand the kinetics of a ligand‑receptor interaction, as well as some of its associated intracellular processes. Our multiple‑temperature and ‑inhibitor assay setup renders it possible to follow the EGFR monomer, dimer and internalized populations in a detailed manner, allowing for a new perspective of the EGFR biology.

    Keywords
    epidermal growth factor, tyrosine kinase inhibitors, internalization, kinetics, dimerization, heterogeneity
    National Category
    Medical and Health Sciences
    Research subject
    Medicine; Molecular Biotechnology; Medical Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-183868 (URN)10.3892/mco.2012.37 (DOI)
    Available from: 2012-11-05 Created: 2012-11-05 Last updated: 2018-12-04
  • 26.
    Björkelund, Hanna
    et al.
    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.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Avoiding false negative results in specificity analysis of protein-protein interactions2011In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 24, no 1, p. 81-89Article in journal (Refereed)
    Abstract [en]

    The competition measurement using simultaneous incubation of labeled and unlabeled Ligand is a common method to assess the specificity of a biomolecular interaction. In this paper we show that invalid assumptions about the interactions may lead to improper experimental setups which in turn can result in inaccurate conclusions about the specificity. To improve understanding of competition measurements, simulations in MATLAB as well as real-time interaction analysis using LigandTracer have been performed. We show that use of a concentration of unlabeled Ligand of at least 10 × K(D) is necessary for assay accuracy. Increasing the incubation time to assure equilibrium, adding a pre-incubation phase, and a general understanding of the reversibility of an interaction may also improve the reliability of the measurement and the conclusions drawn about specificity. These findings may lower the risk of false negative results as well as reducing the amount of reagent needed.

  • 27.
    Björkelund, Hanna
    et al.
    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.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Comparing the epidermal growth factor interaction with four different cell lines: intriguing effects imply strong dependency of cellular context2011In: PLoS ONE, ISSN 1932-6203, Vol. 6, no 1, p. e16536-Article in journal (Refereed)
    Abstract [en]

    The interaction of the epidermal growth factor (EGF) with its receptor (EGFR) is known to be complex, and the common over-expression of EGF receptor family members in a multitude of tumors makes it important to decipher this interaction and the following signaling pathways. We have investigated the affinity and kinetics of (125)I-EGF binding to EGFR in four human tumor cell lines, each using four culturing conditions, in real time by use of LigandTracer®.Highly repeatable and precise measurements show that the overall apparent affinity of the (125)I-EGF - EGFR interaction is greatly dependent on cell line at normal culturing conditions, ranging from K(D)≈200 pM on SKBR3 cells to K(D)≈8 nM on A431 cells. The (125)I-EGF - EGFR binding curves (irrespective of cell line) have strong signs of multiple simultaneous interactions. Furthermore, for the cell lines A431 and SKOV3, gefitinib treatment increases the (125)I-EGF - EGFR affinity, in particular when the cells are starved. The (125)I-EGF - EGFR interaction on cell line U343 is sensitive to starvation while as on SKBR3 it is insensitive to gefitinib and starvation.The intriguing pattern of the binding characteristics proves that the cellular context is important when deciphering how EGF interacts with EGFR. From a general perspective, care is advisable when generalizing ligand-receptor interaction results across multiple cell-lines.

  • 28.
    Björkelund, Hanna
    et al.
    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.
    Barta, Pavel
    Malmqvist, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Gefitinib Induces Epidermal Growth Factor Receptor Dimers Which Alters the Interaction Characteristics with (125)I-EGF2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 9, p. e24739-Article in journal (Refereed)
    Abstract [en]

    The tyrosine kinase inhibitor gefitinib inhibits growth in some tumor types by targeting the epidermal growth factor receptor (EGFR). Previous studies show that the affinity of the EGF-EGFR interaction varies between hosting cell line, and that gefitinib increases the affinity for some cell lines. In this paper, we investigate possible mechanisms behind these observations. Real-time interaction analysis in LigandTracer (R) Grey revealed that the HER2 dimerization preventing antibody pertuzumab clearly modified the binding of (125)I-EGF to EGFR on HER2 overexpressing SKOV3 cells in the presence of gefitinib. Pertuzumab did not affect the binding on A431 cells, which express low levels of HER2. Cross-linking measurements showed that gefitinib increased the amount of EGFR dimers 3.0-3.8 times in A431 cells in the absence of EGF. In EGF stimulated SKOV3 cells the amount of EGFR dimers increased 1.8-2.2 times by gefitinib, but this effect was cancelled by pertuzumab. Gefitinib treatment did not alter the number of EGFR or HER2 expressed in tumor cell lines A431, U343, SKOV3 and SKBR3. Real-time binding traces were further analyzed in a novel tool, Interaction Map, which deciphered the different components of the measured interaction and supports EGF binding to multiple binding sites. EGFR and HER2 expression affect the levels of EGFR monomers, homodimers and heterodimers and EGF binds to the various monomeric/dimeric forms of EGFR with unique binding properties. Taken together, we conclude that dimerization explains the varying affinity of EGF - EGFR in different cells, and we propose that gefitinib induces EGFR dimmers, which alters the interaction characteristics with (125)I-EGF.

  • 29.
    Björkelund, Hanna
    et al.
    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.
    Malmqvist, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Resolving the EGF-EGFR interaction characteristics through a multiple-temperature, multiple-inhibitor, real-time interaction analysis approach2013In: Molecular and Clinical Oncology, ISSN 2049-9469, Vol. 1, no 2, p. 343-352Article in journal (Refereed)
    Abstract [en]

    Overexpression and aberrant activity of the epidermal growth factor (EGF) have been observed in various cancer types, rendering it an important target in oncology research. The interaction between EGF and its receptor (EGFR), as well as subsequent internalization, is complex and may be affected by various factors including tyrosine kinase inhibitors (TKIs). By combining real‑time binding curves produced in LigandTracer® with internalization assays conducted at different temperatures and with different TKIs, the processes of ligand binding, internalization and excretion was visualized. SKOV3 cells had a slower excretion rate compared to A431 and U343 cells, and the tested TKIs (gefitinib, lapatinib, AG1478 and erlotinib) reduced the degree of internalization. The kinetic analysis of the binding curves further demonstrated TKI‑dependent balances of EGFR monomer and dimer populations, where lapatinib promoted the monomeric form, while the other TKIs induced dimers. The dimer levels were found to be associated with the apparent affinity of the EGF‑EGFR interaction, with EGF binding stronger to EGFR dimers compared to monomers. This study analyzed how real‑time molecular interaction analysis may be utilized in combination with perturbations in order to understand the kinetics of a ligand‑receptor interaction, as well as some of its associated intracellular processes. Our multiple‑temperature and ‑inhibitor assay setup renders it possible to follow the EGFR monomer, dimer and internalized populations in a detailed manner, allowing for a new perspective of the EGFR biology.

  • 30.
    Blom, Elisabeth
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Velikyan, Irina
    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 Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET.
    Estrada, Sergio
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Hall, Håkan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Muhammad, Taj
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science.
    Ding, Chenmin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science.
    Nair, Manoj
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science.
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
    Ga-68-Labeling of RGD peptides and biodistribution2012In: International Journal of Clinical and Experimental Medicine, ISSN 1940-5901, E-ISSN 1940-5901, Vol. 5, no 2, p. 165-172Article in journal (Refereed)
    Abstract [en]

    Several peptides comprising Arg-Gly-Asp (RGD) domain and macrocyclic chelator were labeled with Ga-68 for the imaging of angiogenesis. The analogues varied in peptide constitution, linker and chelator type. The labeling efficiency did not vary with the peptide constitution and linker type, but depended on the chelator type. Four of the compounds containing 2,2', 2 '', 2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl) tetraacetic acid (DOTA) chelator were labeled at 90 +/- 5 degrees C using conventional or microwave heating reaching 90% of Ga-68 incorporation after 5 and 2 min respectively, when the concentration of the precursor was 2.5 mu M. The compound having 2,2', 2 ''-(1,4,7-triazonane1,4,7-triyl)triacetic acid (NOTA) as the chelator could be labeled at room temperature within 5 min using 2.5 mu M peptide precursor. Two of the compounds contained a poly (ethylene glycol) (PEG) linker to the chelator. The biodistribution of the analogues was studied in male rats.

  • 31.
    Blom, Elisabeth
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Velikyan, Irina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Monazzam, Azita
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrine Tumor Biology.
    Razifar, Pasha
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Centre for Image Analysis. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Nair, Manoj
    Razifar, Payam
    Vanderheyden, Jean-Luc
    Krivoshein, Arcadius V.
    Backer, Marina
    Backer, Joseph
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Synthesis and characterization of scVEGF-PEG-[68Ga]NOTA and scVEGF-PEG-[68Ga]DOTA PET tracers2011In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 54, no 11, p. 685-692Article in journal (Refereed)
    Abstract [en]

    Vascular endothelial growth factor (VEGF) signaling via vascular endothelial growth factor receptor 2 (VEGFR-2) on tumor endothelial cells is a critical driver of tumor angiogenesis. Novel anti-angiogenic drugs target VEGF/VEGFR-2 signaling and induce changes in VEGFR-2 prevalence. To monitor VEGFR-2 prevalence in the course of treatment, we are evaluating (68)Ga positron emission tomography imaging agents based on macrocyclic chelators, site-specifically conjugated via polyethylene glycol (PEG) linkers to engineered VEGFR-2 ligand, single-chain (sc) VEGF. The (68)Ga-labeling was performed at room temperature with NOTA (2,2', 2 ''-(1,4,7-triazonane-1,4,7-triyl) triacetic acid) conjugates or at 90 degrees C by using either conventional or microwave heating with NOTA and DOTA (2,2', 2 '', 2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl) tetraacetic acid) conjugates. The fastest (similar to 2min) and the highest incorporation (>90%) of (68)Ga into conjugate that resulted in the highest specific radioactivity (similar to 400MBq/nmol) was obtained with microwave heating of the conjugates. The bioactivity of the NOTA-and DOTA-containing tracers was validated in 3-D tissue culture model of 293/KDR cells engineered to express high levels of VEGFR-2. The NOTA-containing tracer also displayed a rapid accumulation (similar to 20s after intravenous injection) to steady-state level in xenograft tumor models. A combination of high specific radioactivity and maintenance of functional activity suggests that scVEGF-PEG-[(68)Ga] NOTA and scVEGF-PEG-[(68)Ga] DOTA might be promising tracers for monitoring VEGFR-2 prevalence and should be further explored.

  • 32.
    Bohl Kullberg, E
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Edwards, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Physical Chemistry.
    Capala, J
    Sjöberg, S
    Gedda, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Introductory experiments on ligand liposomes as delivery agents for boronneutron capture therapy2003In: International Journal of Oncology, ISSN 1019-6439, Vol. 23, no 2, p. 461-467Article in journal (Refereed)
    Abstract [en]

    Liposomes are, when coupled to receptor ligands, candidates for receptor mediated delivery of boron for tumour therapy since they have capacity to deliver large amounts of boron per receptor interaction. With EGF-liposomes we present a pegylated ligand liposome delivery vehicle, containing water soluble boronated phenanthridine, WSP1, or water soluble boronated acridine, WSA1, for EGFR targeting. In the case of WSA1 a ligand dependent uptake was obtained and the boron uptake was as good as if free WSA1 was given. No ligand dependent boron uptake was seen for WSP1 containing liposomes. Thus, WSA1 is a candidate for further studies. Approximately 10(5) boron atoms were in each liposome. A critical assessment indicates that after optimization up to 10(6) boron atoms can be loaded. Since it is known that, for therapeutic effect, approximately 10(8)-10(9) boron atoms are needed in a single tumour cell it is realized that 10(2)-10(3) receptor interactions are needed to meet the demand. Tests applying cultured glioma cells indicate, without optimization of the delivery conditions, a boron uptake in the ppm range, which is necessary for successful BNCT. Thus, it seems possible to kill micro-invasive tumour cells with targeted liposomes if the delivery conditions are optimal.

  • 33.
    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.

  • 34.
    Borges, João Batista
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Physiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Anaesthesiology and Intensive Care.
    Velikyan, Irina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Sörensen, Jens
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Physiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET.
    Ulin, Johan
    Maripuu, Enn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Medical Physics.
    Sandström, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    Widström, Charles
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Medical Physics.
    Hedenstierna, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Physiology.
    Ventilation Distribution Studies Comparing Technegas and "Gallgas" Using (GaCl3)-Ga-68 as the Label2011In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 52, no 2, p. 206-209Article in journal (Refereed)
    Abstract [en]

    Ventilation distribution can be assessed by SPECT with Technegas. This study was undertaken in piglets with different degrees of ventilation inhomogeneity to compare PET using Ga-68-labeled pseudogas or "Gallgas" with Technegas. Methods: Twelve piglets were studied in 3 groups: control, lobar obstruction, and diffuse airway obstruction. Two more piglets were assessed for lung volume (functional residual capacity). Results: In controls, SPECT and PET images showed an even distribution of radioactivity. With lobar obstruction, the absence of ventilation of the obstructed lobe was visible with both techniques. In diffuse airway obstruction, SPECT images showed an even distribution of radioactivity, and PET images showed more varied radioactivity over the lung. Conclusion: PET provides detailed ventilation distribution images and a better appreciation of ventilation heterogeneity. Gallgas with PET is a promising new diagnostic tool for the assessment of ventilation distribution.

  • 35. Borzecka, Kinga
    et al.
    Plociennikowska, Agnieszka
    Björkelund, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Sobota, Andrzej
    Kwiatkowska, Katarzyna
    CD14 Mediates Binding of High Doses of LPS but Is Dispensable for TNF-alpha Production2013In: Mediators of Inflammation, ISSN 0962-9351, E-ISSN 1466-1861, p. 824919-Article in journal (Refereed)
    Abstract [en]

    Activation of macrophages with lipopolysaccharide (LPS) involves a sequential engagement of serum LPS-binding protein (LBP), plasma membrane CD14, and TLR4/MD-2 signaling complex. We analyzed participation of CD14 in TNF-alpha production stimulated with 1-1000 ng/mL of smooth or rough LPS (sLPS or rLPS) and in sLPS binding to RAW264 and J744 cells. CD14 was indispensable for TNF-alpha generation induced by a low concentration, 1 ng/mL, of sLPS and rLPS. At higher doses of both LPS forms (100-1000 ng/mL), TNF-alpha release required CD14 to much lower extent. Among the two forms of LPS, rLPS-induced TNF-alpha production was less CD14-dependent and could proceed in the absence of serumas an LBP source. On the other hand, the involvement of CD14 was crucial for the binding of 1000 ng/mL of sLPS judging from an inhibitory effect of the anti-CD14 antibody. The binding of sLPS was also strongly inhibited by dextran sulfate, a competitive ligand of scavenger receptors (SR). In the presence of dextran sulfate, sLPS-induced production of TNF-alpha was upregulated about 1.6-fold. The data indicate that CD14 together with SR participates in the binding of high doses of sLPS. However, CD14 contribution to TNF-alpha production induced by high concentrations of sLPS and rLPS can be limited.

  • 36.
    Bulenga, T. N.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Selvaraju, Ram Kumar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Estrada, Sergio
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Asplund, Veronika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Lubberink, Mark
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET.
    Velikyan, Irina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Eriksson, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Dosimetry of 68Ga and 177Lu labeled Exendin4-impact on feasibility of repeated PET imaging and radiotherapy2014In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, no S2, p. S293-S293, article id OP607Article in journal (Other academic)
  • 37.
    Buratovic, Sonja
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Environmental toxicology.