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Bondza, S., Foy, E., Brooks, J., Andersson, K., Robinson, J., Richalet, P. & Buijs, J. (2017). Real-time Characterization of Antibody Binding to Receptors on Living Immune Cells. Frontiers in Immunology, 8, Article ID 455.
Open this publication in new window or tab >>Real-time Characterization of Antibody Binding to Receptors on Living Immune Cells
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2017 (English)In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 8, 455Article in journal (Refereed) Published
Abstract [en]

Understanding molecular interactions on immune cells is crucial for drug development to treat cancer and autoimmune diseases. When characterizing molecular interactions, the use of a relevant living model system is important, as processes such as receptor oligomerization and clustering can influence binding patterns. We developed a protocol to enable time-resolved analysis of ligand binding to receptors on living suspension cells. Different suspension cell lines and weakly adhering cells were tethered to Petri dishes with the help of a biomolecular anchor molecule, and antibody binding was analyzed using LigandTracer. The protocol and assay described in this report were used to characterize interactions involving eight cell lines. Experiments were successfully conducted in three different laboratories, demonstrating the robustness of the protocol. For various antibodies, affinities and kinetic rate constants were obtained for binding to CD20 on both Daudi and Ramos B-cells, the T-cell co-receptor CD3 on Jurkat cells, and the Fc gamma receptor CD32 on transfected HEK293 cells, respectively. Analyzing the binding of Rituximab to B-cells resulted in an affinity of 0.7-0.9 nM, which is similar to values reported previously for living B-cells. However, we observed a heterogeneous behavior for Rituximab interacting with B-cells, which to our knowledge has not been described previously. The understanding of complex interactions will be facilitated with the possibility to characterize binding processes in real-time on living immune cells. This provides the chance to broaden the understanding of how binding kinetics relate to biological function.

Keyword
affinity, kinetics, therapeutic antibody, B-cells, T-cells, CD20, Fc gamma receptor
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:uu:diva-322794 (URN)10.3389/fimmu.2017.00455 (DOI)000399836900001 ()28484455 (PubMedID)
Available from: 2017-06-14 Created: 2017-06-14 Last updated: 2017-11-29Bibliographically approved
Multia, E., Sirén, H., Andersson, K., Samuelsson, J., Forssén, P., Fornstedt, T., . . . Riekkola, M.-L. (2017). Thermodynamic and kinetic approaches for evaluation of monoclonal antibody - Lipoprotein interactions. Analytical Biochemistry, 518, 25-34.
Open this publication in new window or tab >>Thermodynamic and kinetic approaches for evaluation of monoclonal antibody - Lipoprotein interactions
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2017 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 518, 25-34 p.Article in journal (Refereed) Published
Abstract [en]

Two complementary instrumental techniques were used, and the data generated was processed with advanced numerical tools to investigate the interactions between anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) and apoB-100 containing lipoproteins. Partial Filling Affinity Capillary Electrophoresis (PF-ACE) combined with Adsorption Energy Distribution (AED) calculations provided information on the heterogeneity of the interactions without any a priori model assumptions. The AED calculations evidenced a homogenous binding site distribution for the interactions. Quartz Crystal Microbalance (QCM) studies were used to evaluate thermodynamics and kinetics of the Low-Density Lipoprotein (LDL) and anti-apoB-100 Mab interactions. High affinity and selectivity were observed, and the emerging data sets were analysed with so called Interaction Maps. In thermodynamic studies, the interaction between LDL and anti-apoB-100 Mab was found to be predominantly enthalpy driven. Both techniques were also used to study antibody interactions with Intermediate-Density (IDL) and Very Low Density (VLDL) Lipoproteins. By screening affinity constants for IDL-VLDL sample in a single injection we were able to distinguish affinity constants for both subpopulations using the numerical Interaction Map tool.

Keyword
Monoclonal anti-apoB-100 antibody, Lipoproteins, Thermodynamics, Kinetics, Interaction map
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-316011 (URN)10.1016/j.ab.2016.10.024 (DOI)000392461000004 ()27984014 (PubMedID)
Funder
Swedish Research Council, 2015-04627
Available from: 2017-02-24 Created: 2017-02-24 Last updated: 2018-01-13Bibliographically approved
Hillerdal, V., Boura, V. F., Björkelund, H., Andersson, K. & Essand, M. (2016). Avidity characterization of genetically engineered T-cells with novel and established approaches. BMC Immunology, 17, Article ID 23.
Open this publication in new window or tab >>Avidity characterization of genetically engineered T-cells with novel and established approaches
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2016 (English)In: BMC Immunology, ISSN 1471-2172, E-ISSN 1471-2172, Vol. 17, 23Article in journal (Refereed) Published
Abstract [en]

Background: Adoptive transfer of genetically engineered autologous T-cells is becoming a successful therapy for cancer. The avidity of the engineered T-cells is of crucial importance for therapy success. We have in the past cloned a T-cell receptor (TCR) that recognizes an HLA-A2 (MHC class I)-restricted peptide from the prostate and breast cancer- associated antigen TARP. Herein we perform a side-by-side comparison of the TARP-specific TCR (TARP-TCR) with a newly cloned TCR specific for an HLA-A2-restricted peptide from the cytomegalovirus (CMV) pp65 antigen. Results: Both CD8(+) T-cells and CD4(+) T-cells transduced with the HLA-A2-restricted TARP-TCR could readily be detected by multimer analysis, indicating that the binding is rather strong, since binding occured also without the CD8 co-receptor of HLA-A2. Not surprisingly, the TARP-TCR, which is directed against a self-antigen, had weaker binding to the HLA-A2/peptide complex than the CMV pp65-specific TCR (pp65-TCR), which is directed against a viral epitope. Higher peptide concentrations were needed to achieve efficient cytokine release and killing of target cells when the TARP- TCR was used. We further introduce the LigandTracer technology to study cell-cell interactions in real time by evaluating the interaction between TCR-engineered T-cells and peptide-pulsed cancer cells. We were able to successfully detect TCR-engineered T-cell binding kinetics to the target cells. We also used the xCELLigence technology to analyzed cell growth of target cells to assess the killing potency of the TCR-engineered T-cells. T-cells transduced with the pp65 - TCR exhibited more pronounced cytotoxicity, being able to kill their targets at both lower effector to target ratios and lower peptide concentrations. Conclusion: The combination of binding assay with functional assays yields data suggesting that TARP- TCR-engineered T-cells bind to their target, but need more antigen stimulation compared to the pp65-TCR to achieve full effector response. Nonetheless, we believe that the TARP- TCR is an attractive candidate for immunotherapy development for prostate and/or breast cancer.

Keyword
T-cell receptor, Affinity, Avidity, LigandTracer, xCELLigence, HLA binding, TARP, CMV pp65
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-300454 (URN)10.1186/s12865-016-0162-z (DOI)000379892000001 ()27411667 (PubMedID)
Funder
Swedish Cancer SocietySwedish Research Council
Available from: 2016-08-09 Created: 2016-08-09 Last updated: 2018-01-10Bibliographically approved
Reijmar, K., Edwards, K., Andersson, K. & Agmo Hernández, V. (2016). Characterizing and controlling the loading and release of cationic amphiphilic peptides onto and from PEG-stabilized lipodisks. Langmuir, 32(46), 12091-12099.
Open this publication in new window or tab >>Characterizing and controlling the loading and release of cationic amphiphilic peptides onto and from PEG-stabilized lipodisks
2016 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 32, no 46, 12091-12099 p.Article in journal (Refereed) Published
Abstract [en]

Recent studies have identified PEG-stabilized lipid nanodisks (lipodisks) as promising carriers for cationic amphiphilic peptides with antimicrobial and anticancer activity. Using fluorimetric and nanogravimetric methods, we have in this work characterized the parameters describing and controlling the binding of three selected peptides (melittin, LL37, and magainin 2) onto lipodisks. It was found that the affinity of melittin for lipodisks is independent of the disk size and rim charge. On the other hand, the number of binding sites is strongly dependent on both parameters, with the highest loading being obtained for small disks with a negatively charged rim. An optimized composition of the lipodisks was utilized to study the loading of antimicrobial peptides magainin 2 and human LL37. It was observed that although magainin 2 can be loaded in large amounts, it is released very fast upon dilution, which limits future therapeutic applications. In contrast, LL37 can be loaded at relevant concentrations and the formulation is stable. This opens up for applications of LL37-loaded lipodisks as antibiotics and in anticancer treatments.

National Category
Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-305377 (URN)10.1021/acs.langmuir.6b03012 (DOI)000388914400012 ()
Funder
Swedish Cancer Society
Available from: 2016-10-17 Created: 2016-10-17 Last updated: 2017-11-29Bibliographically 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, 767-776 p.Article 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.

Keyword
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, 4154-4163 p.Article 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
Barta, P., Volkova, M., Dascalu, A., Spiegelberg, D., Trejtnar, F. & Andersson, K. (2014). Determination of receptor protein binding site specificity and relative binding strength using a time-resolved competition assay. Journal of pharmacological and toxicological methods, 70(2), 145-151.
Open this publication in new window or tab >>Determination of receptor protein binding site specificity and relative binding strength using a time-resolved competition assay
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2014 (English)In: Journal of pharmacological and toxicological methods, ISSN 1056-8719, E-ISSN 1873-488X, Vol. 70, no 2, 145-151 p.Article in journal (Refereed) Published
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.  

Keyword
Binding assay, Cetuximab, Competition assay, EGF receptor, Methods, Panitumumab, TGF-alpha, Tracer, Competitor
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-235187 (URN)10.1016/j.vascn.2014.07.006 (DOI)000342158000003 ()25084055 (PubMedID)
Available from: 2014-10-30 Created: 2014-10-29 Last updated: 2018-01-11Bibliographically approved
Tolmachev, V., Orlova, A. & Andersson, K. (2014). Methods for radiolabelling of monoclonal antibodies. In: Michael Steinitz (Ed.), Human Monoclonal Antibodies: Methods and Protocols (pp. 309-330). Humana Press.
Open this publication in new window or tab >>Methods for radiolabelling of monoclonal antibodies
2014 (English)In: Human Monoclonal Antibodies: Methods and Protocols / [ed] Michael Steinitz, Humana Press, 2014, 309-330 p.Chapter in book (Refereed)
Abstract [en]

The use of radionuclide labels allows to study the pharmacokinetics of monoclonal antibodies, to control the specificity of their targeting and to monitor the response to an antibody treatment with high accuracy. Selection of label depends on the processing of an antibody after binding to an antigen, and on the character of information to be derived from the study (distribution of antibody in the extracellular space, target occupancy or determination of sites of metabolism). This chapter provides protocols for labelling of antibodies with iodine-125 (suitable also for other radioisotopes of iodine) and with indium-111. Since radiolabelling might damage and reduce the immunoreactive fraction and/or affinity of an antibody, the methods for assessment of these characteristics of an antibody are provided for control.

Place, publisher, year, edition, pages
Humana Press, 2014
Series
Methods in Molecular Biology, ISSN 1064-3745 ; 1060
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-218570 (URN)10.1007/978-1-62703-586-6_16 (DOI)24037848 (PubMedID)978-1-62703-585-9 (ISBN)978-1-62703-586-6 (ISBN)
Available from: 2014-02-12 Created: 2014-02-12 Last updated: 2015-03-24Bibliographically approved
Peng, Z., Andersson, K., Lindholm, J., Bodin, I., Pramana, S., Pawitan, Y., . . . Li, C. (2014). Operator Dependent Choice of Prostate Cancer Biopsy Has Limited Impact on a Gene Signature Analysis for the Highly Expressed Genes IGFBP3 and F3 in Prostate Cancer Epithelial Cells. PLoS ONE, 9(10), e109610.
Open this publication in new window or tab >>Operator Dependent Choice of Prostate Cancer Biopsy Has Limited Impact on a Gene Signature Analysis for the Highly Expressed Genes IGFBP3 and F3 in Prostate Cancer Epithelial Cells
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 10, e109610- p.Article in journal (Refereed) Published
Abstract [en]

Background: Predicting the prognosis of prostate cancer disease through gene expression analysis is receiving increasing interest. In many cases, such analyses are based on formalin-fixed, paraffin embedded (FFPE) core needle biopsy material on which Gleason grading for diagnosis has been conducted. Since each patient typically has multiple biopsy samples, and since Gleason grading is an operator dependent procedure known to be difficult, the impact of the operator's choice of biopsy was evaluated. Methods: Multiple biopsy samples from 43 patients were evaluated using a previously reported gene signature of IGFBP3, F3 and VGLL3 with potential prognostic value in estimating overall survival at diagnosis of prostate cancer. A four multiplex one-step qRT-PCR test kit, designed and optimized for measuring the signature in FFPE core needle biopsy samples was used. Concordance of gene expression levels between primary and secondary Gleason tumor patterns, as well as benign tissue specimens, was analyzed. Results: The gene expression levels of IGFBP3 and F3 in prostate cancer epithelial cell-containing tissue representing the primary and secondary Gleason patterns were high and consistent, while the low expressed VGLL3 showed more variation in its expression levels. Conclusion: The assessment of IGFBP3 and F3 gene expression levels in prostate cancer tissue is independent of Gleason patterns, meaning that the impact of operator's choice of biopsy is low.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-240144 (URN)10.1371/journal.pone.0109610 (DOI)000345204000087 ()25296164 (PubMedID)
Available from: 2015-01-07 Created: 2015-01-05 Last updated: 2017-12-05Bibliographically approved
Orlova, A., Hofström, C., Strand, J., Varasteh, Z., Sandström, M., Andersson, K., . . . Gräslund, T. (2013). [99mTc(CO)3]+-(HE)3-ZIGF1R:4551, a new Affibody conjugate for visualization of insulin-like growth factor-1 receptor expression in malignant tumours. European Journal of Nuclear Medicine and Molecular Imaging, 40(3), 439-449.
Open this publication in new window or tab >>[99mTc(CO)3]+-(HE)3-ZIGF1R:4551, a new Affibody conjugate for visualization of insulin-like growth factor-1 receptor expression in malignant tumours
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2013 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, no 3, 439-449 p.Article in journal (Refereed) Published
Abstract [en]

Purpose

Radionuclide imaging of insulin-like growth factor type 1 receptor (IGF-1R) expression in tumours might be used for selection of patients who would benefit from IGF-1R-targeted therapy. We have previously shown the feasibility of IGF-1R imaging using the Affibody molecule 111In-DOTA-His6-ZIGF1R:4551. The use of 99mTc instead of 111In should improve sensitivity and resolution of imaging, and reduce the dose burden to patients. We hypothesized that inclusion of a HEHEHE tag instead of a His6 tag in ZIGF1R:4551 would permit its convenient purification using IMAC, enable labelling with [99mTc(CO)3]+, and improve its biodistribution.

Methods

ZIGF1R:4551 was expressed with a HEHEHE tag in the N terminus. The resulting (HE)3-ZIGF1R:4551 construct was labelled with [99mTc(CO)3]+. Targeting of IGF-1R-expressing cells using [99mTc(CO)3]+-(HE)3-ZIGF1R:4551 was evaluated in vitro and in vivo.

Results

(HE)3-ZIGF1R:4551 was stably labelled with 99mTc with preserved specific binding to IGF-1R-expressing DU-145 prostate cancer cells in vitro. In mice, [99mTc(CO)3]+-(HE)3-ZIGF1R:4551 accumulated in IGF-1R-expressing organs (pancreas, stomach, lung and salivary gland). [99mTc(CO)3]+-(HE)3-ZIGF1R:4551 demonstrated 3.6-fold lower accumulation in the liver and spleen than 111In-DOTA-ZIGF1R:4551. In NMRI nu/nu mice with DU-145 prostate cancer xenografts, the tumour uptake was 1.32 ± 0.11 %ID/g and the tumour-to-blood ratio was 4.4 ± 0.3 at 8 h after injection. The xenografts were visualized using a gamma camera 6 h after injection.

Conclusion

99mTc(CO)3]+-(HE)3-ZIGF1R:4551 is a promising candidate for visualization of IGF-1R expression in malignant tumours.

National Category
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-196212 (URN)10.1007/s00259-012-2284-8 (DOI)000314676900015 ()23179942 (PubMedID)
Available from: 2013-03-05 Created: 2013-03-05 Last updated: 2018-01-11Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-9141-9242

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