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Björkelund, Hanna
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Publications (10 of 13) Show all publications
Bondza, S., Björkelund, H., Nestor, M., Andersson, K. & Buijs, J. (2017). Novel Real-Time Proximity Assay for Characterizing Multiple Receptor Interactions on Living Cells. Analytical Chemistry, 89(24), 13212-13218
Open this publication in new window or tab >>Novel Real-Time Proximity Assay for Characterizing Multiple Receptor Interactions on Living Cells
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2017 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 89, no 24, p. 13212-13218Article in journal (Refereed) Published
Abstract [en]

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

National Category
Analytical Chemistry Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-339775 (URN)10.1021/acs.analchem.7b02983 (DOI)000418626300025 ()29160688 (PubMedID)
Available from: 2018-02-09 Created: 2018-02-09 Last updated: 2018-02-09Bibliographically approved
Wang, E., Björkelund, H., Mihaylova, D., Hagemann, U. B., Karlsson, J., Malmqvist, M., . . . Andersson, K. (2014). Automated functional characterization of radiolabeled antibodies: a time-resolved approach. Nuclear medicine communications, 35(7), 767-776
Open this publication in new window or tab >>Automated functional characterization of radiolabeled antibodies: a time-resolved approach
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2014 (English)In: Nuclear medicine communications, ISSN 0143-3636, E-ISSN 1473-5628, Vol. 35, no 7, p. 767-776Article in journal (Refereed) Published
Abstract [en]

Background The number of radiolabeled monoclonal antibodies (mAbs) used for medical imaging and cancer therapy is increasing. The required chemical modification for attaching a radioactive label and all associated treatment may lead to a damaged mAb subpopulation. This paper describes a novel method, concentration through kinetics (CTK), for rapid assessment of the concentration of immunoreactive mAb and the specific radioactivity, based on monitoring binding kinetics. Methods The interaction of radiolabeled mAb with either the antigen or a general mAb binder such as Protein A was monitored in real time using the instrument LigandTracer. As the curvature of the binding trace has a distinct shape based on the interaction kinetics and concentration of the functional mAb, the immunoreactive mAb concentration could be calculated through reverse kinetic fitting of the binding curves, using software developed for this project. The specific activity, describing the degree of radioactive labeling, was determined through the use of calibrated signal intensities. Results The performance of the CTK assay was evaluated on the basis of various mAb-based interaction systems and assay formats, and it was shown that the assay can provide accurate and repeatable results for immunoreactive concentration and specific activity, with both accuracy and relative SD values below 15%. Conclusion By applying reverse kinetics on real-time binding traces it is possible to estimate the functional concentration and specific activity of radiolabeled mAb. The CTK assay may in the future be included as a complement to current quality assessment methods of radiolabeled mAbs.

Keywords
antibodies, concentration, kinetics, LigandTracer, quality control, specific activity
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-228695 (URN)10.1097/MNM.0000000000000117 (DOI)000337698200011 ()
Available from: 2014-07-22 Created: 2014-07-21 Last updated: 2017-12-05Bibliographically approved
Bondza, S., Stenberg, J., Nestor, M., Andersson, K. & Björkeund, H. (2014). Conjugation Effects on Antibody-Drug Conjugates: Evaluation of Interaction Kinetics in Real Time on Living Cells. Molecular Pharmaceutics, 11(11), 4154-4163
Open this publication in new window or tab >>Conjugation Effects on Antibody-Drug Conjugates: Evaluation of Interaction Kinetics in Real Time on Living Cells
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2014 (English)In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 11, no 11, p. 4154-4163Article in journal (Refereed) Published
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.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-239389 (URN)10.1021/mp500379d (DOI)000344307700040 ()25252166 (PubMedID)
Available from: 2014-12-22 Created: 2014-12-22 Last updated: 2017-12-05Bibliographically approved
Borzecka, K., Plociennikowska, A., Björkelund, H., Sobota, A. & Kwiatkowska, K. (2013). CD14 Mediates Binding of High Doses of LPS but Is Dispensable for TNF-alpha Production. Mediators of Inflammation, 824919
Open this publication in new window or tab >>CD14 Mediates Binding of High Doses of LPS but Is Dispensable for TNF-alpha Production
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2013 (English)In: Mediators of Inflammation, ISSN 0962-9351, E-ISSN 1466-1861, p. 824919-Article in journal (Refereed) Published
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-218352 (URN)10.1155/2013/824919 (DOI)000329736000001 ()
Available from: 2014-02-11 Created: 2014-02-11 Last updated: 2017-12-06Bibliographically approved
Xu, B., Varasteh, Z., Orlova, A., Andersson, K., Larhammar, D. & Björkelund, H. (2013). Detecting ligand interactions with G protein-coupled receptors in real-time on living cells. Biochemical and Biophysical Research Communications - BBRC, 441(4), 820-824
Open this publication in new window or tab >>Detecting ligand interactions with G protein-coupled receptors in real-time on living cells
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2013 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 441, no 4, p. 820-824Article in journal (Refereed) Published
Abstract [en]

G protein-coupled receptors (GPCRs) are a large group of receptors of great biological and clinical relevance. Despite this, the tools for a detailed analysis of ligand-GPCR interactions are limited. The aim of this paper was to demonstrate how ligand binding to GPCRs can be followed in real-time on living cells. This was conducted using two model systems, the radiolabeled porcine peptide YY (pPYY) interacting with transfected human Y2 receptor (hY2R) and the bombesin antagonist RM26 binding to the naturally expressed gastrin-releasing peptide receptor (GRPR). By following the interaction over time, the affinity and kinetic properties such as association and dissociation rate were obtained. Additionally, data were analyzed using the Interaction Map method, which can evaluate a real-time binding curve and present the number of parallel interactions contributing to the curve. It was found that pPYY binds very slowly with an estimated time to equilibrium of approximately 12 h. This may be problematic in standard end-point assays where equilibrium is required. The RM26 binding showed signs of heterogeneity, observed as two parallel interactions with unique kinetic properties. In conclusion, measuring binding in real-time using living cells opens up for a better understanding of ligand interactions with GPCRs.

Keywords
GPCR, Real-time, LigandTracer, Interaction Map, Kinetics, Heterogeneity
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-215943 (URN)10.1016/j.bbrc.2013.10.149 (DOI)000328434800022 ()
Note

De två första författarna delar första författarskapet.

Available from: 2014-01-17 Created: 2014-01-17 Last updated: 2017-12-06Bibliographically approved
Dubois, L., Andersson, K., Asplund, A. & Björkelund, H. (2013). Evaluating real-time immunohistochemistry on multiple tissue samples, multiple targets and multiple antibody labeling methods. BMC Research Notes, 6, 542
Open this publication in new window or tab >>Evaluating real-time immunohistochemistry on multiple tissue samples, multiple targets and multiple antibody labeling methods
2013 (English)In: BMC Research Notes, ISSN 1756-0500, E-ISSN 1756-0500, Vol. 6, p. 542-Article in journal (Refereed) Published
Abstract [en]

Background

Immunohistochemistry (IHC) is a well-established method for the analysis of protein expression in tissue specimens and constitutes one of the most common methods performed in pathology laboratories worldwide. However, IHC is a multi-layered method based on subjective estimations and differences in staining and interpretation has been observed between facilities, suggesting that the analysis of proteins on tissue would benefit from protocol optimization and standardization. Here we describe how the emerging and operator independent tool of real-time immunohistochemistry (RT-IHC) reveals a time resolved description of antibody interacting with target protein in formalin fixed paraffin embedded tissue. The aim was to understand the technical aspects of RT-IHC, regarding generalization of the concept and to what extent it can be considered a quantitative method.

Results

Three different antibodies labeled with fluorescent or radioactive labels were applied on nine different tissue samples from either human or mouse, and the results for all RT-IHC analyses distinctly show that the method is generally applicable. The collected binding curves showed that the majority of the antibody-antigen interactions did not reach equilibrium within 3 hours, suggesting that standardized protocols for immunohistochemistry are sometimes inadequately optimized. The impact of tissue size and thickness as well as the position of the section on the glass petri dish was assessed in order for practical details to be further elucidated for this emerging technique. Size and location was found to affect signal magnitude to a larger extent than thickness, but the signal from all measurements were still sufficient to trace the curvature. The curvature, representing the kinetics of the interaction, was independent of thickness, size and position and may be a promising parameter for the evaluation of e.g. biopsy sections of different sizes.

Conclusions

It was found that RT-IHC can be used for the evaluation of a number of different antibodies and tissue types, rendering it a general method. We believe that by following interactions over time during the development of conventional IHC assays, it becomes possible to better understand the different processes applied in conventional IHC, leading to optimized assay protocols with improved sensitivity.

National Category
Other Basic Medicine
Identifiers
urn:nbn:se:uu:diva-223781 (URN)10.1186/1756-0500-6-542 (DOI)
Available from: 2014-04-25 Created: 2014-04-25 Last updated: 2018-01-11Bibliographically approved
Gedda, L., Björkelund, H., Lebel, L., Asplund, A., Dubois, L., Wester, K., . . . Andersson, K. (2013). Evaluation of Real-Time Immunohistochemistry and Interaction Map as an Alternative Objective Assessment of HER2 Expression in Human Breast Cancer Tissue. Applied immunohistochemistry & molecular morphology (Print), 21(6), 497-505
Open this publication in new window or tab >>Evaluation of Real-Time Immunohistochemistry and Interaction Map as an Alternative Objective Assessment of HER2 Expression in Human Breast Cancer Tissue
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2013 (English)In: Applied immunohistochemistry & molecular morphology (Print), ISSN 1541-2016, E-ISSN 1533-4058, Vol. 21, no 6, p. 497-505Article in journal (Refereed) Published
Abstract [en]

Immunohistochemical study (IHC) is a critical tool in the clinical diagnosis of breast cancer. One common assessment is the expression level of the HER2 receptor in breast cancer tissue samples with the aim of stratifying patients for applicability of the therapeutic antibody Herceptin. In this study, we aimed to investigate whether a novel assay, real-time IHC combined with Interaction Map analysis, offers the possibility of objective assessment of HER2 expression. Interaction Map presents real-time interaction data as a collection of peaks on a surface, and it was performed on 20 patient tissue samples previously scored for HER2 expression. The result shows that the relative weight of the peaks in the maps contains novel information that could discriminate between high and low HER2 expression in an operator-independent manner (P<0.001). We conclude that the real-time IHC assay has a promising potential to complement conventional IHC and may improve the precision in the future clinical diagnostics of breast cancer.

Keywords
antibody, HER2, immunohistochemistry, IHC, kinetics, real time, tissue
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-212827 (URN)10.1097/PAI.0b013e318281162d (DOI)000327212100004 ()
Available from: 2013-12-18 Created: 2013-12-16 Last updated: 2017-12-06Bibliographically approved
Björkelund, H. (2013). Novel Methods for Analysis of Heterogeneous Protein-Cell Interactions: Resolving How the Epidermal Growth Factor Binds to Its Receptor. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Novel Methods for Analysis of Heterogeneous Protein-Cell Interactions: Resolving How the Epidermal Growth Factor Binds to Its Receptor
2013 (English)Doctoral 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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. p. 65
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 854
Keywords
Heterogeneity, Kinetics, EGFR, HER2, LigandTracer, Interaction Map, Internalization, Specificity
National Category
Medical Biotechnology Cell and Molecular Biology
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-183872 (URN)978-91-554-8570-2 (ISBN)
Public defence
2013-02-15, Rudbeck Hall, Rudbeck Laboratory, Dag Hammarskjöldsväg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-01-24 Created: 2012-11-05 Last updated: 2018-01-12Bibliographically approved
Björkelund, H., Gedda, L., Malmqvist, M. & Andersson, K. (2013). Resolving the EGF-EGFR interaction characteristics through a multiple-temperature, multiple-inhibitor, real-time interaction analysis approach. Molecular and Clinical Oncology, 1(2), 343-352
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: 2013-07-24Bibliographically approved
Altschuh, D., Björkelund, H., Strandgård, J., Chouliera, L., Malmqvist, M. & Andersson, K. (2012). Deciphering complex protein interaction kinetics using Interaction Map. Biochemical and Biophysical Research Communications - BBRC, 428(1), 74-79.
Open this publication in new window or tab >>Deciphering complex protein interaction kinetics using Interaction Map
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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
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