| 1. |
- Friedman, Mikaela, et al.
(författare)
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Engineered affinity proteins for tumour-targeting applications
- 2009
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Ingår i: Biotechnology and applied biochemistry. - : Wiley. - 0885-4513 .- 1470-8744. ; 53, s. 1-29
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Forskningsöversikt (refereegranskat)abstract
- Targeting of tumour-associated antigens is an expanding treatment modality in clinical oncology as an alternative to, or in combination with, conventional treatments, such as chemotherapy, external-radiation therapy and surgery. Targeting of antigens that are unique or more highly expressed in tumours than in normal tissues can be used to increase the specificity and reduce the cytotoxic effect on normal tissues. Several targeting agents have been studied for clinical use, where monoclonal antibodies have been the ones most widely used. More than 20 monoclonal antibodies are approved for therapy today and the largest field is oncology. Advances in genetic engineering and in vitro selection technology has enabled the feasible high-throughput generation of monoclonal antibodies, antibody derivatives [e.g. scFvs, Fab molecules, dAbs (single-domain antibodies), diabodies and minibodies] and more recently also non-immunoglobulin scaffold proteins. Several of these affinity proteins have been investigated for both in vivo diagnostics and therapy. Affinity proteins in tumour-targeted therapy can affect tumour progression by altering signal transduction or by delivering a payload of toxin, drug or radionuclide. The ErbB receptor family has been extensively studied as biomarkers in tumour targeting, primarily for therapy using monoclonal antibodies. Two receptors in the ErbB family, EGFR (epidermal growth factor receptor) and HER2 (epidermal growth factor receptor 2), are over-expressed in various malignancies and associated with poor patient prognosis and are therefore interesting targets for solid turnours. In the present review, strategies are described for tumour targeting of solid turnours using affinity proteins to deliver radionuclides, either for molecular imaging or radiotherapy. Antibodies, antibody derivatives and non-immunoglobulin scaffold proteins are discussed with a certain focus on the affibody (Affibody (R)) molecule.
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| 2. |
- Friedman, Mikaela, et al.
(författare)
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Engineering and characterization of a bispecific HER2 x EGFR-binding affibody molecule
- 2009
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Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 54, s. 121-131
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Tidskriftsartikel (refereegranskat)abstract
- HER2 (human epidermal-growth-factor receptor-2; ErbB2) and EGFR (epidermal-growth-factor receptor) are overexpressed in various forms of cancer, and the co-expression of both HER2 and EGFR has been reported in a number of studies. The simultaneous targeting of HER2 and EGFR has been discussed as a strategy with which to potentially increase efficiency and selectivity in molecular imaging and therapy of certain cancers. In an effort to generate a molecule capable of bispecifically targeting HER2 and EGFR, a gene fragment encoding a bivalent HER2-binding affibody molecule was genetically fused in-frame with a bivalent EGFR-binding affibody molecule via a (G(4)S)(3) [(Gly(4)-Ser)(3)]-encoding gene fragment. The encoded 30 kDa affibody construct (Z(HER2))(2)-(G(4)S)(3)-(Z(EGFR))(2), with potential for bs (bispecific) binding to HER2 and EGFR, was expressed in Escherichia coli and characterized in terms of its binding capabilities. The retained ability to bind HER2 and EGFR separately was demonstrated using both biosensor technology and flow-cytometric analysis, the latter using HER2- and EGFR-overexpressing cells. Furthermore, simultaneous binding to HER2 and EGFR was demonstrated in: (i) a sandwich format employing real-time biospecific interaction analysis where the bs affibody molecule bound immobilized EGFR and soluble HER2; (ii) immunofluorescence microscopy, where the bs affibody molecule bound EGFR-overexpressing cells and soluble HER2; and (iii) a cell-cell interaction analysis where the bs affibody molecule bound HER2-overexpressing SKBR-3 cells and EGFR-overexpressing A-431 cells. This is, to our knowledge, the first reported bs affinity protein with potential ability for the simultaneous targeting of HER2 and EGFR. The potential future use of this and similar constructs, capable of bs targeting of receptors to increase the efficacy and selectivity in imaging and therapy, is discussed.
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| 3. |
- Grönwall, Caroline, et al.
(författare)
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Generation of Affibody (R) ligands binding interieukin-2 receptor alpha/CD25
- 2008
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Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 50:2, s. 97-112
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Tidskriftsartikel (refereegranskat)abstract
- Affibody (R) molecules specific for human IL-2R alpha, the IL-2 (interieukin-2) receptor a subunit, also known as CD25, were selected by phage-display technology from a combinatorial protein library based on the 58-residue Protein A-derived Z domain. The IL-2R system plays a major role in T-cell activation and the regulation of cellular immune responses. Moreover, CD25 has been found to be overexpressed in organ rejections, a number of autoimmune diseases and T-cell malignancies. The phage-display selection using Fc-fused target protein generated 16 unique Affibody (R) molecules targeting CD25. The two most promising binders were characterized in more detail using biosensor analysis and demonstrated strong and selective binding to CD25. Kinetic biosensor analysis revealed that the two monomeric Affibody (R) molecules bound to CD25 with apparent affinities of 130 and 240 nM respectively. The Affibody (R) molecules were, on biosensor analysis, found to compete for the same binding site as the natural ligand IL-2 and the IL-2 blocking monoclonal antibody 2A3. Hence the Affibody (R) molecules were assumed to have an overlapping binding site with IL-2 and antibodies targeting the IL-2 blocking Tac epitope (for example, the monoclonal antibodies Daclizumab and Basiliximab, both of which have been approved for therapeutic use). Furthermore, immunofluorescence microscopy and flow-cytometric analysis of CD25-expressing cells demonstrated that the selected Affibody (R) molecules bound to CD4(+) CD25(+) PMBCs (peripheral-blood mononuclear cells), the IL-2-dependent cell line NK92 and phytohaemagglutinin-activated PMBCs. The potential use of the CD25-binding Affibody (R) molecules as targeting agents for medical imaging and for therapeutic applications is discussed.
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| 4. |
- Jonsson, Andreas, et al.
(författare)
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Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro
- 2009
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Ingår i: Biotechnology and applied biochemistry. - : Wiley. - 0885-4513 .- 1470-8744. ; 54, s. 93-103
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules specific for human TNF-alpha (tumour necrosis factor-alpha) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-alpha is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-alpha-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine-free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilized-metal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-alpha. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-alpha. For Z(TNF alpha:185), subnanomolar affinity (K-D = 0.1-0.5 nM) for human TNF-alpha was demonstrated, as well as significant binding to TNF-alpha from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-alpha receptor, since this interaction could be efficiently blocked by the Z(TNF-alpha:185) affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-alpha binding to its receptor could be further improved by using dinners with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.
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| 5. |
- Jonsson, Andreas, et al.
(författare)
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Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro
- 2009
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Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 54, s. 93-103
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules specific for human TNF-alpha (tumour necrosis factor-alpha) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-alpha is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-alpha-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine-free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilized-metal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-alpha. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-alpha. For Z(TNF alpha:185), subnanomolar affinity (K-D = 0.1-0.5 nM) for human TNF-alpha was demonstrated, as well as significant binding to TNF-alpha from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-alpha receptor, since this interaction could be efficiently blocked by the Z(TNF-alpha:185) affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-alpha binding to its receptor could be further improved by using dinners with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.
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| 6. |
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| 7. |
- Lundberg, Emma, et al.
(författare)
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Selection and characterization of Affibody (R) ligands to the transcription factor c-Jun
- 2009
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Ingår i: Biotechnology and applied biochemistry. - : Wiley. - 0885-4513 .- 1470-8744. ; 52, s. 17-27
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Tidskriftsartikel (refereegranskat)abstract
- c-Jun is a highly oncogenic transcription factor involved in the development of different types of cancer. In the present study we have generated c-Jun-binding-affinity proteins from a phage-displayed library of so-called 'Affibody (R) ligands', developed by combinatorial engineering of a non-immunoglobulin-based scaffold protein. Homodimeric c-Jun protein was recombinantly produced in Escherichia coli and, prior to selection, the quality of the target protein was investigated by binding analyses, which indicated specific binding to a double-stranded DNA hairpin construct containing a c-Jun response element, but not to a control sequence. Isolated Affibody (R) variants from the phage selection were expressed in E. coli, purified by affinity chromatography and their interaction with c-Jun was analysed. In biosensor analyses, one Affibody (R) ligand, denoted Z(cJun518) was shown to interact with immobilized c-Jun protein with an apparent dissociation constant of 5 mu M. By constructing a head-to-tail homodimeric version of Z(cJun518), its apparent affinity for c-Jun could be increased threefold, suggesting co-operativity effects in the binding to the immobilized c-Jun protein. Further characterization of the Z(cJun518) Affibody (R) molecule demonstrated, in both affinity-capture and Western-blotting experiments, its ability to interact selectively with c-Jun, even when the c-Jun target was present in a complex protein background consisting of a bacterial cell lysate. Z(cJun518) could also be used to stain the c-Jun-overexpressing cell line C8161 visualized by confocal fluorescence microscopy. Results from competition experiments indicated that the binding epitope on c-Jun for the Z(cJun518) Affibody (R) molecule was separate from the binding sites of both a polyclonal antibody raised against the unstructured N-terminal domain and a double-stranded DNA hairpin containing a c-Jun response element. The potential intracellular use of Affibody (R) ligands directed against transcription factors and other oncogenic factors is discussed.
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| 8. |
- Nydert, Per, et al.
(författare)
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Chitosan as a carrier for non-viral gene transfer in a cystic-fibrosis cell line
- 2008
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Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 51:Pt 4, s. 153-7
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Tidskriftsartikel (refereegranskat)abstract
- The gene transfer mediated by chitosan in CFBE41o(-) (a cystic-fibrosis bronchial epithelial cell line) and HEK (a human embryonic kidney cell line) has been evaluated. Polyplexes based on chitosan and PEI (polyethyleneimine) using a luciferase and enhanced green fluorescent protein reporter plasmid showed that the transfection efficacy of polyplexes in the CFBE41o(-) cell line was poor compared with that in HEK cells. In the highly differentiated cystic-fibrosis bronchial epithelial cell line the narrow-size-distributed chitosan shows enhanced transfection at a low pH compared with PEI. The enhanced transfection at lower pH could be a result of damage to the cell surface or changes in the cell-surface charge, leading to better penetration of the cell membrane.
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| 9. |
- Ramström, Margareta, et al.
(författare)
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Development of affinity columns for the removal of high-abundance proteins in cerebrospinal fluid.
- 2009
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Ingår i: Biotechnology and applied biochemistry. - 1470-8744 .- 0885-4513. ; 52:Pt 2, s. 159-66
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Tidskriftsartikel (refereegranskat)abstract
- Various approaches for removal of high-abundance components in body fluids are currently available. While most methods are constructed for plasma depletion, there is a need for body-fluid-specific strategies. The aim of the present study was to design an affinity matrix suitable for the depletion of high-abundance proteins in CSF (cerebrospinal fluid). Hence, molecules with specific affinity towards proteins present at high concentration in CSF were desired. Affibody molecules are specific binders of small size that have shown high stability under various conditions and are therefore good candidates for such a matrix. The protein composition in CSF resembles that in plasma. However, 20% of the proteins are brain-derived and are therefore present in higher proportions in CSF than in plasma, whereas larger plasma-derived proteins are less abundant in CSF. Therefore five high-abundance CSF proteins were chosen for the design of a CSF-specific depletion setup. Affibody molecules with specificity towards HSA (human serum albumin), IgG, transferrin and transthyretin were combined in an affinity column. In addition, polyclonal antibodies against cystatin C were coupled to chromatographic beads and packed in a separate column. Highly reproducible and efficient removal of the five target proteins was observed. The proportion of depleted proteins were estimated to be 99, 95, 74, 92 and 83% for HSA, IgG, transferrin, transthyretin and cystatin C respectively. SDS/PAGE analysis was used for monitoring and identifying proteins in native CSF, depleted CSF samples and the captured fractions. Moreover, shotgun proteomics was used for protein identification in native as well as depleted CSF and the achieved data were compared. Enhanced identification of lower-abundance components was observed in the depleted fraction, in terms of more detected peptides per protein.
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| 10. |
- Ramström, Margareta, et al.
(författare)
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Development of affinity columns for the removal of high-abundance proteins in cerebrospinal fluid
- 2009
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Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 52:Pt 2, s. 159-166
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Tidskriftsartikel (refereegranskat)abstract
- Various approaches for removal of high-abundance components in body fluids are currently available. While most methods are constructed for plasma depletion, there is a need for body-fluid-specific strategies. The aim of the present study was to design an affinity matrix suitable for the depletion of high-abundance proteins in CSF (cerebrospinal fluid). Hence, molecules with specific affinity towards proteins present at high concentration in CSF were desired. Affibody molecules are specific binders of small size that have shown high stability under various conditions and are therefore good candidates for such a matrix. The protein composition in CSF resembles that in plasma. However, 20% of the proteins are brain-derived and are therefore present in higher proportions in CSF than in plasma, whereas larger plasma-derived proteins are less abundant in CSF. Therefore five high-abundance CSF proteins were chosen for the design of a CSF-specific depletion setup. Affibody molecules with specificity towards HSA (human serum albumin), IgG, transferrin and transthyretin were combined in an affinity column. In addition, polyclonal antibodies against cystatin C were coupled to chromatographic beads and packed in a separate column. Highly reproducible and efficient removal of the five target proteins was observed. The proportion of depleted proteins were estimated to be 99, 95, 74, 92 and 83% for HSA, IgG, transferrin, transthyretin and cystatin C respectively. SDS/PAGE analysis was used for monitoring and identifying proteins in native CSF, depleted CSF samples and the captured fractions. Moreover, shotgun proteomics was used for protein identification in native as well as depleted CSF and the achieved data were compared. Enhanced identification of lower-abundance components was observed in the depleted fraction, in terms of more detected peptides per protein.
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