Scientific Reports 7, Article number: 6607 (2017) Novel strategies to mimic transmembrane tumor necrosis factor-dependent activation of tumor necrosis factor receptor 2Roman Fischer, Jessica Marsal, Cristiano Guttà, Stephan A. Eisler, Nathalie Peters, John R. Bethea, Klaus Pfizenmaier & Roland E. KontermannAbstract
Tumor necrosis factor receptor 2 (TNFR2) is known to mediate immune suppression and tissue regeneration. Interestingly, the transmembrane form of tumor necrosis factor (tmTNF) is necessary to robustly activate TNFR2. To characterize the stoichiometry and composition of tmTNF during TNFR2 activation, we constructed differently oligomerized single chain TNF ligands (scTNF) comprised of three TNF homology domain (THD) protomers that mimic tmTNF. Using a variety of cellular and in vivo assays, we can show that higher oligomerization of the scTNF trimers results in more efficient TNF/TNFR2 clustering and subsequent signal transduction. Importantly, the three-dimensional orientation of the scTNF trimers impacts the bioactivity of the oligomerized scTNF ligands. Our data unravel the organization of tmTNF-mimetic scTNF ligands capable of robustly activating TNFR2 and introduce novel TNFR2 agonists that hold promise as therapeutics to treat a variety of diseases.
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.Nature: Sci. Rep. 7, 43006; doi: 10.1038/srep43006 (2017) Cellular glycosylation affects Herceptin binding and sensitivity of breast cancer cells to doxorubicin and growth factorsDiluka Peiris, Alexander F. Spector, Hannah Lomax-Browne, TayebehAzimi, Bala Ramesh, Marilena Loizidou, Hazel Welch and Miriam V. DwekAbstract
Alterations in protein glycosylation are a key feature of oncogenesis and have been shown to affect cancer cell behaviour perturbing cell adhesion, favouring cell migration and metastasis. This study investigated the effect of N-linked glycosylation on the binding of Herceptin to HER2 protein in breast cancer and on the sensitivity of cancer cells to the chemotherapeutic agent doxorubicin (DXR) and growth factors (EGF and IGF-1). The interaction between Herceptin and recombinant HER2 protein and cancer cell surfaces (on-rate/off-rate) was assessed using a quartz crystal microbalance biosensor revealing an increase in the accessibility of HER2 to Herceptin following deglycosylation of cell membrane proteins (deglycosylated cells Bmax: 6.83Hz; glycosylated cells Bmax: 7.35Hz). The sensitivity of cells to DXR and to growth factors was evaluated using an MTT assay. Maintenance of SKBR-3 cells in tunicamycin (an inhibitor of N-linked glycosylation) resulted in an increase in sensitivity to DXR (0.1μM DXR P<0.001) and a decrease in sensitivity to IGF-1 alone and to IGF-1 supplemented with EGF (P<0.001). This report illustrates the importance of N-linked glycosylation in modulating the response of cancer cells to chemotherapeutic and biological treatments and highlights the potential of glycosylation inhibitors as future combination treatments for breast cancer.
© The Authors 2017 under license from Springer NatureAnalytical Chemistry, May 6, 2016 Combined Bacteria Microarray and Quartz Crystal Microbalance Approach for Exploring Glycosignatures of Nontypeable Haemophilus influenzae and Recognition by Host LectinsIoanna Kalograiaki, Begoña Euba, Davide Proverbio, María A. Campanero-Rhodes, Teodor Aastrup, Junkal Garmendia and Dolores SolísAbstract
Recognition of bacterial surface epitopes by host receptors plays an important role in the infectious process and is intimately associated with bacterial virulence. Delineation of bacteria−host interactions commonly relies on the detection of binding events between purified bacteria- and host-target molecules. In this work, we describe a combined microarray and quartz crystal microbalance (QCM) approach for the analysis of carbohydrate-mediated interactions directly on the bacterial surface, thus preserving the native environment of the bacterial targets. Nontypeable Haemophilus influenzae (NTHi) was selected as a model pathogenic species not displaying a polysaccharide capsule or O-antigen-containing lipopolysaccharide, a trait commonly found in several important respiratory pathogens. Here, we demonstrate the usefulness of NTHi microarrays for exploring the presence of carbohydrate structures on the bacterial surface. Furthermore, the microarray approach is shown to be efficient for detecting strain-selective binding of three innate immune lectins, namely, surfactant protein D, human galectin-8, and Siglec-14, to different NTHi clinical isolates. In parallel, QCM bacteria-chips were developed for the analysis of lectin-binding kinetics and affinity. This novel QCM approach involves capture of NTHi on lectin-derivatized chips followed by formaldehyde fixation, rendering the bacteria an integrated part of the sensor chip, and subsequent binding assays with label-free lectins. The binding parameters obtained for selected NTHi-lectin pairs provide further insights into the interactions occurring at the bacterial surface.
© 2016 American Chemical SocietySensing and Bio-Sensing Research 9 (2016) 23–30 Real-time and label free determination of ligand binding-kinetics to primary cancer tissue specimens; a novel tool for the assessment of biomarker targetingThomas Mandel Clausen, Marina Ayres Pereira, Htoo Zarni Oo, Mafalda Resende, Tobias Gustavson, Yang Mao, Nobuo Sugiura, Janet Liew, Ladan Fazli, Thor G. Theander, Mads Daugaard, Ali SalantiAbstract
In clinical oncology, diagnosis and evaluation of optimal treatment strategies are mostly based on histopathological examination combined with immunohistochemical (IHC) expression analysis of cancer-associated antigens in formalin fixed paraffin-embedded (FFPE) tissue biopsies. However, informative IHC analysis depends on both the specificity and affinity of the binding reagent, which are inherently difficult to quantify in situ. Here we describe a label-free method that allows for the direct and real-time assessment of molecular binding kinetics in situ on FFPE tissue specimens using quartz crystal microbalance (QCM) enabled biosensor technology. We analysed the interaction between the rVAR2 protein and its placental-like chondroitin sulfate (pl-CS) receptor in primary human placenta tissue and in breast and prostate tumour specimens in situ. rVAR2 interacted with FFPE human placenta and cancer tissue with an affinity in the nanomolar range, and showed no detectable interaction with pl-CS negative normal tissue. We further validated the method by including analysis with the androgen receptor N-20 antibody (anti-AR). As the KD value produced by this method is independent of the number of epitopes available, this readout offers a quantitative and unbiased readout for in situ binding-avidity and amount of binding epitopes. In summary, this method adds a new and important dimension to classical IHC-based molecular pathology by adding information about the binding characteristics in biologically relevant conditions. This can potentially be used to select optimal biologics for diagnostic and for therapeutic applications as well as guide the development of novel high affinity binding drugs.
© 2016 The Authors. Published by Elsevier B.V.Polymer Chemistry, DOI: 10.1039/c5py01954k Facile fabrication of glycopolymer-based iron oxide nanoparticles and their applications in the carbohydrate–lectin interaction and targeted cell imagingChen Shao, Xueming Li, Zhichao Pei, Dongdong Liu, Lin Wang, Hai Dong and
A novel method for facile fabrication of glycopolymer-based iron oxide nanoparticles (GIONs) is developed. Via perfluorophenylazide photochemically induced C–H insertion, alkynyl groups were introduced onto the polymer which was precoated on the iron oxide nanoparticle surface. GIONs were then prepared by conjugating the azide-functionalized carbohydrate to the introduced alkynyl groups via click chemistry. Polyvinyl alcohol-coated and dextran-coated iron oxide NPs were chosen as scaffolds to attach two different carbohydrates, α-D-mannose and β-D-glucose, to fabricate multivalent GIONs, respectively. The multivalent GIONs demonstrated high binding affinities towards the corresponding lectins in both protein and cell chips. As a proof of concept, fluorescent GIONs (Gal-RhB-IONPs) were fabricated, which showed selective and efficient internalization by ASGP-R overexpressing HepG2 cells targeted.
© The Royal Society of Chemistry 2016Cancer Cell, Volume 28, Issue 4, p500–514, 12 October 2015 Targeting Human Cancer by a Glycosaminoglycan Binding Malaria ProteinAli Salanti, T M Clausen, M Ø. Agerbæk, N Al Nakouzi, M Dahlbäck, H Z Oo, S Lee, T Gustavsson, J R. Rich, B J. Hedberg, Y Mao, L Barington, M A. Pereira, J LoBello, M Endo, L Fazli, J Soden, C K. Wang, A F. Sander, R Dagil, S Thrane, P J. Holst, L Meng, F Favero, G J. Weiss, M A. Nielsen, J Freeth, T O. Nielsen, J Zaia, N L. Tran, J Trent, J S. Babcook, T G. Theander, P H. Sorensen, M DaugaardAbstract
Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can be specifically targeted by recombinant VAR2CSA (rVAR2). In tumors, placental-like CS chains are linked to a limited repertoire of cancer-associated proteoglycans including CD44 and CSPG4. The rVAR2 protein localizes to tumors in vivo and rVAR2 fused to diphtheria toxin or conjugated to hemiasterlin compounds strongly inhibits in vivo tumor cell growth and metastasis. Our data demonstrate how an evolutionarily refined parasite-derived protein can be exploited to target a common, but complex, malignancy-associated glycosaminoglycan modification.
© 2015 Elsevier Inc.Scientific Reports 5, Article number: 14066 (2015) Real-time and label-free analysis of binding thermodynamics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a QCM biosensorXueming Li, Siyu Song, Qi Shuai, Yihan Pei, Teodor Aastrup, Yuxin Pei & Zhichao PeiAbstract
A novel approach to the study of binding thermodynamics and kinetics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a quartz crystal microbalance (QCM) biosensor was developed, in which binding events take place at the cell surface, more closely mimicking a biologically relevant environment. In this study, colon adenocarcinoma cells (KM-12) and ovary adenocarcinoma cells (SKOV-3) grew on the optimized polystyrene-coated biosensor chip without fixation. The association and dissociation between the cell surface carbohydrates and a range of lectins, including WGA, Con A, UEA-I, GS-II, PNA and SBA, were monitored in real time and without label for evaluation of cell surface glycosylation. Furthermore, the thermodynamic and kinetic parameters of the interaction between lectins and cell surface glycan were studied, providing detailed information about the interactions, such as the association rate constant, dissociation rate constant, affinity constant, as well as the changes of entropy, enthalpy and Gibbs free energy. This application provides an insight into the cell surface glycosylation and the complex molecular recognition on the intact cell surface, which may have impacts on disease diagnosis and drug discovery.Scientist Live; 8th June 2015 Investigating manufactured nanoparticlesTeodor Aastrup, Diluka Peiris and Daniel WallinderAbstract
Manufactured nanoparticles (MNPs) are increasingly being considered for use in biomedical applications ranging from drug delivery to cellular imaging. Thus, the understanding of MNPs’ interactions with biological systems has become vital for both their safety profile and efficient applications. The growing interest on elucidating the impact of physicochemical properties of NPs (eg, size, surface charge, hydrophobicity, or shape) on their subsequent cellular interactions necessitates the exploration of new technical tools.
© The author(s). Courtesy of Setform Limited.Sensors 2015, 15, 5884-5894; doi:10.3390/s150305884 Study of the Interaction of Trastuzumab and SKOV3 Epithelial Cancer Cells Using a Quartz Crystal Microbalance SensorLouise Elmlund, Camilla Käck, Teodor Aastrup and Ian A. NichollsAbstract
Analytical methods founded upon whole cell-based assays are of importance in early stage drug development and in fundamental studies of biomolecular recognition. Here we have studied the binding of the monoclonal antibody trastuzumab to human epidermal growth factor receptor 2 (HER2) on human ovary adenocarcinoma epithelial cancer cells (SKOV3) using quartz crystal microbalance (QCM) technology. An optimized procedure for immobilizing the cells on the chip surface was established with respect to fixation procedure and seeding density. Trastuzumab binding to the cell decorated sensor surface was studied, revealing a mean dissociation constant, KD, value of 7 ± 1 nM (standard error of the mean). This study provides a new perspective on the affinity of the antibody-receptor complex presented a more natural context compared to purified receptors. These results demonstrate the potential for using whole cell-based QCM assay in drug development, the screening of HER2 selective antibody-based drug candidates, and for the study of biomolecular recognition. This real time, label free approach for studying interactions with target receptors present in their natural environment afforded sensitive and detailed kinetic information about the binding of the analyte to the target.
©2015 MDPI AG (Basel, Switzerland)Molecular Pharmacology Fast Forward, July 7, 2015 as DOI: 10.1124/mol.115.099671 Ligand residence time at GPCRs – why we should take our time to study itC. Hoffmann, M. Castro, A. Rinken, R. Leurs, S.J. Hill, H.F. VischerAbstract
Over the past decade the kinetics of ligand binding to a receptor have received increasing interest. The concept of drug-target residence time is becoming an invaluable parameter for drug optimization. It holds great promise for drug-development and its optimization is thought to reduce off-target effects. The success of long-acting drugs like tiotropium support this hypothesis. Nonetheless, we know surprisingly little about the dynamics and the molecular detail of the drug binding process. Since protein dynamics and adaptation during the binding event will change the conformation of the protein, ligand binding will not be the static process that is often described. This can cause problems since simple mathematical models often fail to adequately describe the dynamics of the binding process. In this perspective we will discuss the current situation with an emphasis on GPCRs. This are important membrane protein drug-targets that undergo conformational changes upon agonist binding in order to communicate signalling information across the plasma membrane of cells.
Copyright ©2015 by the American Society for Pharmacology and Experimental TherapeuticsInternational Pharmaceutical Industry, 2014 Vol. 6, Issue 2, 54-57 Label-free Cell-based Assay for the Characterization of Peptide Receptor InteractionsWright, Proverbio, Valnohova, Schulte and AastrupAbstract
Both the drug development process and fundamental studies of mechanisms’ underlying biological interactions require analytical methods that approximate the in vivo situation as much as possible. This can be achieved by using label-free cell-based assays, since they eliminate non-natural treatment, e.g. purification and labelling, of both the target receptor and the drug candidate molecule.
In this paper, we present a new label-free assay that we have developed for characterising interactions between peptides and their target receptors in a cellular environment. We will describe one such interaction that was assessed by monitoring the binding profile in real-time and by quantifying its kinetic rate constants for association and dissociation, as well as its affinity. In addition, the absence of off-target interactions with the cell membrane was confirmed by analysis of the binding profile.
IPI © 2014 International Pharmaceutical IndustryTHE JOURNAL OF NUCLEAR MEDICINE 2014; 55:452–459 Quantitative ImmunoPET of Prostate Cancer Xenografts with 89Zr- and 124I-Labeled Anti-PSCA A11 MinibodyScott M. Knowles, Kirstin A. Zettlitz, Richard Tavaré, Matthew M. Rochefort, Felix B. Salazar, David B. Stout, Paul J. Yazaki, Robert E. Reiter, and Anna M. WuAbstract
Prostate stem cell antigen (PSCA) is expressed on the cell surface in 83%–100% of local prostate cancers and 87%–100% of prostate cancer bone metastases. In this study, we sought to develop immunoPET agents using 124I- and 89Zr-labeled anti-PSCA A11 minibodies (scFv-CH3 dimer, 80 kDa) and evaluate their use for quantitative immunoPET imaging of prostate cancer. Methods: A11 anti-PSCA minibody was alternatively labeled with 124I- or 89Zr-desferrioxamine and injected into mice bearing either matched 22Rv1 and 22Rv1· PSCA or LAPC-9 xenografts. Small-animal PET data were obtained and quantitated with and without recovery coefficient–based partial volume correction, and the results were compared with ex vivo biodistribution. Results: Rapid and specific localization to PSCA-positive tumors and high-contrast imaging were observed with both 124I and 89Zr-labeled A11 anti-PSCA minibody. However, the differences in tumor uptake and background uptake of the radiotracers resulted in different levels of imaging contrast. The nonresidualizing 124I-labeled minibody had lower tumor uptake (3.62 ± 1.18 percentage injected dose per gram [%ID/g] 22Rv1·PSCA, 3.63 ± 0.59 %ID/g LAPC-9) than the residualizing 89Zr-labeled minibody (7.87 ± 0.52 %ID/g22Rv1·PSCA, 9.3360.87%ID/gLAPC-9,P<0.0001 for each), but the 124I-labeled minibody achieved higher imaging contrast because of lower nonspecific uptake and better tumor–to–soft-tissue ratios (22Rv1·PSCA:22Rv1 positive-to-negative tumor, 13.31 ± 5.59 124I-A11 and 4.87 ± 0.52 89Zr-A11, P=0.02). Partial-volume correction was found to greatly improve the correspondence between small-animal PET and ex vivo quantification of tumor uptake for immunoPET imaging with both radionuclides. Conclusion: Both 124I and 89Zr-labeled A11 anti-PSCA minibody showed high-contrast imaging of PSCA expression in vivo. However, the 124I-labeled A11 minibody was found to be the superior imaging agent because of lower nonspecific uptake and higher tumor–to–soft-tissue contrast. Partial volume correctionwas found to be essential for robust quantification of immunoPET imaging with both 124I- and 89Zr-labeled A11 minibody.Innovations in Pharmaceutical Technology, 2013, Issue 46, 48-51 Talking Sense – A combined biochemical and cell-based biosensorT. AastrupAbstract
The combined biochemical and cell-based biosensor enables detailed interaction characterization between biopharmaceutical molecules, such as antibodies, and target receptors on cell surfaces. This results in cell-based information on affinity and kinetic rate constants considering accessibility and clustering of receptors for the interactions, as well as revealing off-target interaction between the antibody and cell membrane and therapeutic accessibility of the receptors.
IPT online © 2013 The Pharmaceutical Technology JournalChemComm, 30 July 2013 A suspension-cell biosensor for real-time determination of binding kinetics of protein–carbohydrate interactions on cancer cell surfacesXueming Li, Yuxin Pei, Ruina Zhang, Qi Shuai, Feng Wang, Teodor Aastrup and Zhichao PeiAbstract
A novel lectin-based suspension-cell biosensor for label-free determination of binding kinetics of protein–carbohydrate interactions on cancer cell surfaces using QCM is described. This cell-biosensor facilitates evaluation of glycosylation in real time on suspension cancer cell surfaces, where binding events take place, more closely mimicking a native environment compared with traditional biosensors.
© The Royal Society of Chemistry 2013Biosensors and Bioelectronics (2012)(In Press) A novel approach to determining the affinity of protein–carbohydrate interactions employing adherent cancer cells grown on a biosensor surfaceDiluka Peiris, Anatoliy Markiv, G. Paul Curley, Miriam V. DwekAbstract
The development of biological agents for the treatment of solid tumours is an area of considerable activity. We are pursuing carbohydrate-binding proteins (lectins) in a strategy aimed at targeting cancer associated changes in glycosylation. To evaluate lectin–cancer cell interactions we developed a novel cell biosensor in which binding events take place at the cell surface, more closely mimicking an in vivo system. Metastatic, SW620, and non-metastatic, SW480, colorectal cancer cells were grown on the surface of a tissue-culture compatible polystyrene coated biosensor chip and housed in a quartz crystal microbalance (QCM) apparatus, the kinetics of binding of a diverse range of lectins was evaluated. The lectin Helix pomatia agglutinin (HPA) has been shown to bind aggressive metastatic cancer and was produced
in recombinant form (His- and RFP-tagged). The affinity of HPA was in the nanomolar range to the metastatic SW620 cells but was only in the micromolar range to the non-metastatic SW480. Overall, the dissociation constant (KD) of the lectins tested in the new cell biosensor system was an order of magnitude lower (nanomolar range) than has generally been reported with systems such as QCM/SPR. This new cell-biosensor enables molecular interactions to be studied in a more relevant environment. An intrinsic problem with developing new biological therapies is the difficulty in determining the affinity with which proteins will interact with intact cell surfaces. This methodology will be of interest to researchers developing new biological approaches for targeting cell surfaces in a wide range of diseases, including cancer.
Crown Copyright © 2012 Published by Elsevier B.V.Innovations in Pharmaceutical Technology - Issue 40(2012) (Online) A New Generation of Cell-BiosensorsMiriam Dwek and Teodor AastrupAbstract
A new cell-biosensor enables molecular interactions to be studied in a more relevant cellular environment and, as a model for understanding drug-cell interactions, provides a new biological approach for targeting cell surfaces in a wide range of diseases, including cancer.
The full article is available free to view online: http://edition.pagesuite-professional.co.uk/launch.aspx?eid=f7aee819-e747-4158-8104-940ccd202c61&pnum=44
IPTonline © 2012 The Pharmaceutical Technology JournalDrug Discovery and Development (05/14/2012) Quartz Crystal Microbalance Technology Provides Biologically Relevant DataTeodor Aastrup and Ingo MontenbruckAbstract
The pharmaceutical and drug discovery communities are under ever greater pressure from the rising costs of R&D, increasing regulatory hurdles, the relatively low rate of annual drug approvals, and the number of blockbuster drugs going off-patent in the next few years. As a result, many companies are refocusing their drug discovery programs to provide a broader perspective of modes of action―as well as inclusion of biomarkers and wider use of early in vivo animal model pharmacology―offering a more predictive view of which candidates might have the greatest chance of success in the preclinical process and subsequent clinical trials.
© 2012 Advantage Business MediaInternational Journal of Nanomedicine, 27 Feb. 2012, 2012:7 905–914 Functionalization of single-walled carbon nanotubes and their binding to cancer cellsS.Y. Madani, A. Tan, M. Dwek, A. SeifalianAbstract
Single-walled carbon nanotubes (SWCNTs) have novel properties including their nanoscale size and ease of cellular uptake. This makes them useful for drug delivery, and their photo-thermal effects make them potentially useful in a wide range of applications, particularly the treatment of solid tumors. The poor solubility of SWCNTs has, however, been an issue that may potentially limit the utility of SWCNTs for cancer treatment. Functionalization of the surface of the tubes may be an approach to overcome this problem.
© Madani et al, publisher and licensee Dove Medical Press Ltd. 2012Biosensors and Bioelectronics, 20 Feb. 2012 (Online) Real-time analysis of the carbohydrates on cell surfaces using a QCM biosensor: a lectin-based approachZ- Pei, J. Saint-Guirons, C. Käck, B. Ingemarsson, T. AastrupAbstract
A novel approach to the study of molecular interactions on the surface of mammalian cells using a QCM biosensor was developed. For this study, an epidermoid carcinoma cell line (A-431) and a breast adenocarcinoma cell line (MDA-MB-468) were immobilized onto polystyrene-coated quartz crystals. The binding and dissociation between the lectin Con A and the cells as well as the inhibition of the binding by monosaccharides were monitored in real time and provided an insight into the complex avidic recognition of cell glycoconjugates. The real-time lectin screening of a range of lectins, including Con A, DBA, PNA and UEA-I, enabled the accurate study of the glycosylation changes between cells, such as changes associated with cancer progression and development. Furthermore, the kinetic parameters of the interaction of Con A with MDA-MB-468 cells were studied. This application provides investigators in the field of glycobiology with a novel tool to study cell surface glycosylation and may also have impacts on drug discovery.
© Elsevier B.V. 2012