| 1. |
- Engelmark Cassimjee, Karim
(författare)
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Tools in biocatalysis : enzyme immobilisation on silica and synthesis of enantiopure amines
- 2010
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis presents two techniques in the field of biocatalysis: An enzyme immobilisation method based on the His6-tag for attachment on modified silica oxide beads, and it’s employment in aqueous and organic medium for synthesis applications. The method functions as a one step extraction and immobilisation protocol. An equilibrium displacement system which enables complete conversion in reactions with ω-transaminases where isopropylamine is the donor, a route for synthesis of pharmaceutically interesting enantiopure amines. Biocatalysis is predicted to be a paramount technology for an environmentally sustainable chemical industry, to which every newly developed method represents a small but important step. The work done here is aimed to be a part of this development.
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| 2. |
- Grimm, Sebastian, et al.
(författare)
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Selection and characterisation of affibody molecules inhibiting the interaction between Ras and Raf in vitro
- 2010
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Ingår i: NEW BIOTECHNOL. - : Elsevier BV. - 1871-6784. ; 27:6, s. 766-773
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Tidskriftsartikel (refereegranskat)abstract
- Development of molecules with the ability to selectively inhibit particular protein-protein interactions is important in providing tools for understanding cell biology In this work, we describe efforts to select small Ras- and Raf-specific three-helix bundle affibody binding proteins capable of inhibiting the interaction between H-Ras and Raf-1, from a combinatorial library displayed on bacteriophage Target-specific variants with typically high nanomolar or low micromolar affinities (K-D) could be selected successfully against both proteins, as shown by dot blot, ELISA and real-time biospecific interaction analyses Affibody molecule variants selected against H-Ras were shown to bind epitopes overlapping each other at a site that differed from that at which H-Ras interacts with Raf-1 In contrast, an affibody molecule isolated during selection against Raf-1 was shown to effectively inhibit the interaction between H-Ras and Raf-1 in a dose-dependent manner Possible intracellular applications of the selected affibody molecules are discussed
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| 3. |
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| 4. |
- Hofström, Camilla, 1979-
(författare)
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Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Affibody molecules are small (7 kDa) affinity proteins of non-immunoglobulin origin that have been generated to specifically interact with a large number of clinically important molecular targets.In this thesis, Affibody molecules have been employed as tracers for radionuclide molecular imaging of HER2- and IGF-1R-expressing tumors, paper I-IV, and for surface knock-down of EGFR, paper V. In paper I, a tag with the amino acid sequence HEHEHE was fused to the N-terminus of a HER2-specific Affibody molecule, (ZHER2), and was shown to enable facile IMAC purification and efficient tri-carbonyl 99mTc-labeling. In vivo evaluation of radioactivity uptake in different organs showed an improved biodistribution, including a 10-fold lower radioactivity uptake in liver, compared to the same construct with a H6-tag. In paper II, it was further shown that an N-terminally placed HEHEHE-tag on ZHER2 provided lower unspecific uptake of radioactivity in liver compared to its H6-tagged counterpart even when radiolabeling was at the C-terminus using alternative chemistries to attach 99mTc, 111In or 125I. In paper III, the H6-tag’s composition and position was varied with regards to charge, hydrophobicity and its C- or N-terminal placement on ZHER2. Among the ten variants investigated, it was found that an N-terminal HEHEHE-tag provided the most favorable overall biodistribution profile and that introduction of hydrophobic and positively charged amino acids provoked liver uptake of radioactivity. In paper IV, the HEHEHE-tag was shown to enable IMAC purification and tri-carbonyl 99mTc-labeling of an IGF-1R-specific Affibody molecule and improved its overall biodistribution when compared to the same construct with a H6-tag. In paper V, the aim was to develop an intracellular receptor-entrapment system to reduce the surface levels of EGFR. An EGFR-specific Affibody molecule was expressed as a fusion to different mutants of an intracellular transport protein in SKOV-3 cells, resulting in a collection of cell lines with 50%, 60%, 80% and 96% reduced surface level of EGFR. Analysis of the proliferation rate of these cell lines showed that a modest reduction (15%) in proliferation occurs between 60% and 80% reduction of the surface level of EGFR.
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| 5. |
- Hofström, Camilla, et al.
(författare)
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HAHAHA, HEHEHE, HIHIHI, or HKHKHK : Influence of Position and Composition of Histidine Containing Tags on Biodistribution of [Tc-99m(CO)(3)](+)-Labeled Affibody Molecules
- 2013
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 56:12, s. 4966-4974
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Tidskriftsartikel (refereegranskat)abstract
- Engineered affibody molecules can be used for high contrast in vivo molecular imaging. Extending a recombinantly produced HER2 binding affibody molecule with a hexa-histidine tag allows for convenient purification by immobilized metal-ion affinity chromatography and labeling with [Tc-99m(CO)(3)](+) but increases radioactivity uptake in the liver. To investigate the impact of charge, lipophilicity, and position on biodistribution, 10 variants of a histidine-based tag was attached to a HER2 binding affibody molecule. The biochemical properties and the HER2 binding affinity appeared to be similar for all variants. In vivo, positive charge promoted liver uptake. For N-terminally placed tags, promoted liver uptake and decreased kidney uptake. Kidney uptake was higher for C-terminally placed tags compared to their N-terminal counterparts. The variant with the amino acid composition HEHEHE placed in the N-terminus gave the lowest nonspecific uptake.
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| 6. |
- Hofström, Camilla, et al.
(författare)
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Use of a HEHEHE Purification Tag Instead of a Hexahistidine Tag Improves Biodistribution of Affibody Molecules Site-Specifically Labeled with Tc-99m, In-111 and I-125
- 2011
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 54:11, s. 3817-3826
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are a class of small (similar to 7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging in vivo. The attachment of a hexahistidine (His(6))-tag to the Affibody molecule allows facile purification by immobilized metal ion affinity chromatography (IMAC) but leads to high accumulation of radioactivity in the liver. Earlier, we have demonstrated that replacement of the His(6)- tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of Affibody molecules by IMAC, enables labeling with [Tc-99m(CO)(3)](+), and provides low hepatic accumulation of radioactivity. In this study, we compared the biodistribution of cysteine-containing Affibody molecules site-specifically labeled with In-111, Tc-99m, and I-125 at the C-terminus, having a His(6)-tag at the N- or C-terminus or a HEHEHE-tag at the N-terminus. We show that the use of a HEHEHE-tag provides appreciable reduction of hepatic radioactivity, especially for radiometal labels. We hope that this information can also be useful for development of other scaffold protein-based imaging agents.
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| 7. |
- Larsson, Karin, et al.
(författare)
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Novel antigen design for the generation of antibodies to G-protein-coupled receptors
- 2011
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 370:1-2, s. 14-23
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies are important tools for the study of G-protein-coupled receptors, key proteins in cellular signaling. Due to their large hydrophobic membrane spanning regions and often very short loops exposed on the surface of the cells, generation of antibodies able to recognize the receptors in the endogenous environment has been difficult. Here, we describe an antigen-design method where the extracellular loops and N-terminus are combined to a single antigen for generation of antibodies specific to three selected GPCRs: NPY5R, B2ARN and GLP1R. The design strategy enabled straightforward antigen production and antibody generation. Binding of the antibodies to intact receptors was analyzed using flow cytometry and immunofluorescence based confocal microscopy on A-431 cells overexpressing the respective GPCR. The antibody-antigen interactions were characterized using epitope mapping, and the antibodies were applied in immunohistochemical staining of human tissues. Most of the antibodies showed specific binding to their respective overexpressing cell line but not to the non-transfected cells, thus indicating binding to their respective target receptor. The epitope mapping showed that sub-populations within the purified antibody pool recognized different regions of the antigen. Hence, the genetic combination of several different epitopes enables efficient generation of specific antibodies with potential use in several applications for the study of endogenous receptors.
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| 8. |
- Li, Jingjing, et al.
(författare)
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Selection of affibody molecules to the ligand-binding site of the insulin-like growth factor-1 receptor
- 2010
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Ingår i: Biotechnology and applied biochemistry. - : Wiley. - 0885-4513 .- 1470-8744. ; 55, s. 99-109
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules binding to the site of hormone interaction in IGF-IR (insulin-like growth factor-I receptor) were successfully selected by phage-display technology employing a competitive-elution strategy during biopanning, whereby release of receptor-bound phagemids was accomplished by competition with IGFI (insulin-like growth factor-I). In non-competitive selections, the elution of receptor-bound phagemids was performed by imidazole or low-pH incubation, which also resulted in the isolation of affibody molecules that could bind to the receptor. An ELISA-based assay showed that the affibody molecules generated by IGF-I competition during elution, in addition to affibody molecules generated in the noncompetitive selections, could compete with IGF-I for binding to the receptor. The affinities of the isolated variants to IGF-IR-overexpressing MCF-7 cells were determined and ranged from high nanomolar to 2.3 nM. The most promising variant, Z(4;40), was shown to recognize IGF- IR efficiently in several different contexts: in analyses based on flow cytometry, fluorescence microscopy and receptor pull-down from cell extracts. In addition, when Z, was added to the medium of MCF-7 cells that were dependent on IGF-I for efficient growth, it was found to have a dose-dependent growth-inhibitory effect on the cells. Applications of affibody-based reagents for quantitative and qualitative analyses of IGF- I R status, as well as applications of affibody-based reagents for therapy, are discussed.
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| 9. |
- Lindberg, Hanna, et al.
(författare)
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Evaluation of a HER2-targeting affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for Tc-99m-labelling at the C terminus
- 2012
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Ingår i: Tumor Biology. - : Springer. - 1010-4283 .- 1423-0380. ; 33:3, s. 641-651
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are a class of small (ca.7 kDa) robust scaffold proteins with high potential as tracers for radionuclide molecular imaging in vivo. Incorporation of a cysteine-containing peptide-based chelator at the C terminus provides an opportunity for stable labelling with the radionuclide Tc-99m. The use of a GGGC chelator at the C terminus has provided the lowest renal radioactivity retention of the previously investigated peptide-based chelators. Previously, it has also been demonstrated that replacement of the His(6)-tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of affibody molecules by immobilized metal ion affinity chromatography (IMAC) and provides low hepatic accumulation of radioactivity of conjugates site-specifically labelled at the C terminus using several different nuclides. We hypothesized that the combination of a HEHEHE-tag at the N terminus and a GGGC chelator at the C terminus of an affibody molecule would be a favourable format permitting IMAC purification and providing low uptake in excretory organs. To investigate this hypothesis, a (HE)(3)-Z(HER2:342)-GGGC affibody molecule was generated. It could be efficiently purified by IMAC and stably labelled with Tc-99m. Tc-99m-(HE)(3)-Z(HER2:342)-GGGC preserved specific binding to HER2-expressing cells. In NMRI mice, hepatic uptake of Tc-99m-(HE)(3)-Z(HER2:342)-GGGC was lower than the uptake of the control affibody molecules, Tc-99m-Z(HER2:2395)-VDC and Tc-99m-Z(HER2:342)-GGGC. At 1 and 4 h after injection, the renal uptake of Tc-99m-(HE)(3)-Z(HER2:342)-GGGC was 2-3-fold lower than uptake of Tc-99m-Z(HER2:2395)-VDC, but it was substantially higher than uptake of Tc-99m-Z(HER2:342)-GGGC. Further investigation indicated that a fraction of Tc-99m was chelated by the HEHEHE-tag which caused a higher accumulation of radioactivity in the kidneys. Thus, a combination of a HEHEHE-tag and the GGGC chelator in targeting scaffold proteins was found to be undesirable in the case of Tc-99m labelling due to a partial loss of site-specificity of nuclide chelation.
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| 10. |
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| 11. |
- Orlova, Anna, et al.
(författare)
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[99mTc(CO)3]+-(HE)3-Z IGF1R45514551 : a new Affibody conjugate for visualization of insulin-like growth factor-1 receptor expression in malignant tumours.
- 2013
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Ingår i: European Journal of Nuclear Medicine and Molecular Imaging. - : Springer Science and Business Media LLC. - 1619-7070 .- 1619-7089. ; 40:3, s. 439-449
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Tidskriftsartikel (refereegranskat)abstract
- 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 (111)In-DOTA-His(6)-Z(IGF1R:4551). The use of (99m)Tc instead of (111)In 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 His(6) tag in Z(IGF1R:4551) would permit its convenient purification using IMAC, enable labelling with [(99m)Tc(CO)(3)](+), and improve its biodistribution. METHODS: Z(IGF1R:4551) was expressed with a HEHEHE tag in the N terminus. The resulting (HE)(3)-Z(IGF1R:4551) construct was labelled with [(99m)Tc(CO)(3)](+). Targeting of IGF-1R-expressing cells using [(99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) was evaluated in vitro and in vivo. RESULTS: (HE)(3)-Z(IGF1R:4551) was stably labelled with (99m)Tc with preserved specific binding to IGF-1R-expressing DU-145 prostate cancer cells in vitro. In mice, [(99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) accumulated in IGF-1R-expressing organs (pancreas, stomach, lung and salivary gland). [(99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) demonstrated 3.6-fold lower accumulation in the liver and spleen than (111)In-DOTA-Z(IGF1R: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: (99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) is a promising candidate for visualization of IGF-1R expression in malignant tumours.
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| 12. |
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| 13. |
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| 14. |
- Orre, L. M., et al.
(författare)
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S100A4 interacts with p53 in the nucleus and promotes p53 degradation
- 2013
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Ingår i: Oncogene. - : Springer Science and Business Media LLC. - 0950-9232 .- 1476-5594. ; 32:49, s. 5531-5540
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Tidskriftsartikel (refereegranskat)abstract
- S100A4 is a small calcium-binding protein that is commonly overexpressed in a range of different tumor types, and it is widely accepted that S100A4 has an important role in the process of cancer metastasis. In vitro binding assays has shown that S100A4 interacts with the tumor suppressor protein p53, indicating that S100A4 may have additional roles in tumor development. In the present study, we show that endogenous S100A4 and p53 interact in complex samples, and that the interaction increases after inhibition of MDM2-dependent p53 degradation using Nutlin-3A. Further, using proximity ligation assay, we show that the interaction takes place in the cell nucleus. S100A4 knockdown experiments in two p53 wild-type cell lines, A549 and HeLa, resulted in stabilization of p53 protein, indicating that S100A4 is promoting p53 degradation. Finally, we demonstrate that S100A4 knockdown leads to p53-dependent cell cycle arrest and increased cisplatin-induced apoptosis. Thus, our data add a new layer to the oncogenic properties of S100A4 through its inhibition of p53-dependent processes.
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| 15. |
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| 16. |
- Seijsing, Johan, et al.
(författare)
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An engineered affibody molecule with pH-dependent binding to FcRn mediates extended circulatory half-life of a fusion protein
- 2014
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 111:48, s. 17110-17115
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Tidskriftsartikel (refereegranskat)abstract
- Proteins endocytosed from serum are degraded in the lysosomes. However, serum albumin (SA) and IgG, through its Fc part, bind to the neonatal Fc receptor (FcRn) at low pH in the endosome after endocytosis, and are transported back to the cellular surface, where they are released into the bloodstream, resulting in an extended serum circulation time. Association with Fc or SA has been used to prolong the in vivo half-life of biopharmaceuticals, using the interaction with FcRn to improve treatment regimens. This has been achieved either directly, by fusion or conjugation to Fc or SA, or indirectly, using SA-binding proteins. The present work takes this principle one step further, presenting small affinity proteins that bind directly to FcRn, mediating extension of the serum half-life of fused biomolecules. Phage display technology was used to select affibody molecules that can bind to FcRn in the pH-dependent manner required for rescue by FcRn. The biophysical and binding properties were characterized in vitro, and the affibody molecules were found to bind to FcRn more strongly at low pH than at neutral pH. Attachment of the affibody molecules to a recombinant protein, already engineered for increased halflife, resulted in a nearly threefold longer half-life in mice. These tags should have general use as fusion partners to biopharmaceuticals to extend their half-lives in vivo.
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| 17. |
- Seijsing, Johan, 1986-
(författare)
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Rational and Combinatorial Engineering of Affinity Proteins Towards Therapeutical Applications
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein engineering has had an immense impact on the development of biological drugs, including replacement therapies with engineered versions of insulin or factor VIII to treat diabetes or bleeding disorders, and monoclonal antibodies to treat cancer and various other malignancies. Now, with the next generation of treatment modalities coming up, including monoclonal reagents based on alternative scaffolds, gene and cell therapies, the importance of protein engineering to tailor-make these treatments is likely to increase further.The neonatal Fc receptor (FcRn) is widely expressed in the body. One of the receptor's interesting functions is to rescue immunoglobulin G (IgG) and serum albumin (SA) from degradation by cells in contact with blood. When serum proteins are endocytosed by a cell, they are transported via the endosome to the lysosome for degradation. However, IgG and SA associate with the FcRn already at the slightly acidic pH of the endosome followed by transport back to the cell surface. There the complex encounters the neutral pH of the blood, at which the binding affinity to FcRn is lost, and IgG and SA are released back into circulation. In the main part of this thesis, efforts are described aiming to take use of, or block, the FcRn recycling mechanism to control the serum circulation half-life of proteins.In the first study (Study I), a robust expression strategy for human FcRn was designed and evaluated. The extracellular domain was produced in the human SKOV3 cell-line after facile lentiviral delivery of the expression cassettes. This lead to continuous expression of secreted protein that could be purified to homogeneity by a single affinity chromatographic step, using the intrinsic pH-dependent interaction between FcRn and IgG, where the latter was immobilized in a column. The amount of purified protein was 1.4 mg per liter medium. The protein was characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay. The results suggested a fully functional and stable protein of high purity. In addition, the gene encoding full-length FcRn as a fusion to eGFP was delivered to HeLa cells utilizing the same lentiviral system. Subsequent analysis by flow cytometry and confocal fluorescence microscopy indicated a wide distribution of eGFP/FcRn expression among the cells. Binding of IgG and HSA was found to correlate well with the amount of eGFP/FcRn expressed by the cells.In a following study (Study II), the goal was to generate affinity proteins interacting with human FcRn in a pH-dependent manner similar to that of FcRn's natural ligands. The affinity proteins used are denoted affibody molecules, a class of small alternative scaffold proteins with a three-helical structure. Affibody molecules were selected from a combinatorial library displayed on phage where binding took place at pH 5.5 and elution was performed at pH 2.2 or 8.0. Selected variants were characterized by developed in vitro and cell based assays, and some were found to have the desired pH-dependent binding to FcRn. In vivo studies in mice showed that the serum half-life of a model protein, genetically fused to the FcRn binding affibody molecules, was extended up to almost three-fold compared to a control protein (from 33 to 91 hours).In a subsequent study (Study III), the use of a FcRn binding affibody molecule to reduce, rather than prolong, the serum half-life of proteins was explored. Here, the rationale was to investigate if injection of a FcRn binding affibody could hinder endogenous IgG from being rescued by FcRn, which could lead to depletion of IgGs by lysosomal degradation. In autoimmune diseases, such depletion of IgG would include also pathogenic IgG and could thus mitigate the symptoms of the disease. Using cell based assays, it was found that one affibody molecule, selected in Study II, could readily block IgG from binding both human and murine FcRn. A following in vivo study in mice showed that systemic injection of the molecule reduced the amount of endogenous IgG by 39%.In a fourth study (Study IV), the goal was to use a different class of affinity proteins to regulate the level of an enzyme in the brain. More specifically, artificial zinc finger-based transcription factors regulating the level of GAD67, which is the rate-limiting enzyme in synthesis of gamma-aminobutyric acid (GABA), were designed. Imbalances in GABA-signaling is involved in different diseases, including Parkinson's disease and epilepsy, and regulation of GAD67 at particular sites in the brain might be a route to ameliorate symptoms associated with such diseases. ELISA-based investigation showed that one of the designed zinc fingers, denoted G3, bound selectively to its intended target DNA sequence. A construct encoding G3 fused to a general transcriptional activator (VP64) was delivered to PC12-cells, using a lentivirus-based gene delivery system, resulting in a significant up-regulation of endogenous GAD67 expression. The same construct was subsequently delivered to the striatum of rats, with an induced disease model of Parkinson's disease. Western blot of striatal samples showed a significant up-regulation of GAD67 expression in lesioned striatum compared to intact striatum, and a tendency towards up-regulation compared to lesioned striatum.Taken together, the protein engineering efforts described in this thesis concerning affinity proteins binding other proteins or DNA, has the potential to find use in drug development and may benefit patients in the future.
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| 18. |
- Seijsing, Johan, et al.
(författare)
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Robust Expression of the Human Neonatal Fc Receptor in a Truncated Soluble Form and as a Full-Length Membrane-Bound Protein in Fusion with eGFP
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:11, s. e81350-
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Tidskriftsartikel (refereegranskat)abstract
- Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding.
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| 19. |
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| 20. |
- Shibasaki, Seiji, et al.
(författare)
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Inhibitory effects of H-Ras/Raf-1–binding affibody molecules on synovial cell function
- 2014
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Ingår i: AMB Express. - : Springer Berlin/Heidelberg. - 2191-0855. ; 4:82
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules specific for H-Ras and Raf-1 were evaluated for their ability to inhibit synovial cell function. Affibody molecules targeting H-Ras (Zras122, Zras220, and Zras521) or Raf-1 (Zraf322) were introduced into the MH7A synovial cell line using two delivery methods: transfection with plasmids encoding the affibody molecules or direct introduction of affibody protein using a cell-penetrating peptide reagent. Interleukin-6 (IL-6) and prostaglandin E2 (PGE2) production by MH7A cells were analyzed by enzyme-linked immunosorbent assay after stimulation with tumor necrosis factor-alpha (TNF-α). Cell proliferation was also analyzed. Phosphorylation of extracellular signal-regulated kinase (ERK) was analyzed by western blot. All affibody molecules could inhibit IL-6 and PGE2 production in TNF-α-stimulated MH7A cells. The inhibitory effect was stronger when affibody molecules were delivered as proteins via a cell-penetrating peptide reagent than when plasmid-DNA encoding the affibody moelcules was transfected into the cells. Plasmid-expressed Zras220 inhibited phosphorylation of ERK in TNF-α-stimulated MH7A cells. Protein-introduced Zraf322 inhibited the production of IL-6 and PGE2 and inhibited cell proliferation in MH7A cells. These findings suggest that affibody molecules specific for H-Ras and Raf-1 can affect intracellular signal transduction through the MAP kinase pathway to inhibit cell proliferation and production of inflammatory mediators by synovial cells.
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| 21. |
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| 22. |
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| 23. |
- Tolmachev, Vladimir, et al.
(författare)
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Imaging of Insulinlike Growth Factor Type 1 Receptor in Prostate Cancer Xenografts Using the Affibody Molecule (111)In-DOTA-Z(IGF1R:4551)
- 2012
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Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 53:1, s. 90-97
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Tidskriftsartikel (refereegranskat)abstract
- One of the pathways leading to androgen independence in prostate cancer involves upregulation of insulinlike growth factor type 1 receptor (IGF-1R). Radionuclide imaging of IGF-1R in tumors might be used for selection of patients who would most likely benefit from IGF-1R-targeted therapy. The goal of this study was to evaluate the feasibility of in vivo radionuclide imaging of IGF-1R expression in prostate cancer xenografts using a small nonimmunoglobulin-derived binding protein called an Affibody molecule. Methods: The IGF-1R-binding Z(IGF1R:4551) Affibody molecule was site-specifically conjugated with a maleimido derivative of DOTA and labeled with (111)In. The binding of radiolabeled Z(IGF1R:4551) to IGF-1R-expressing cells was evaluated in vitro and in vivo. Results: DOTA-Z(IGF1R:4551) can be stably labeled with (111)In with preserved specific binding to IGF-1R-expressing cells in vitro. In mice, (111)In-DOTAZ(IGF1R):(4551) accumulated in IGF-1R-expressing organs (pancreas, stomach, lung, and salivary gland). Receptor saturation experiments demonstrated that targeting of DU-145 prostate cancer xenografts in NMRI nu/nu mice was IGF-1R-specific. The tumor uptake was 1.1 +/- 0.3 percentage injected dose per gram, and the tumor-to-blood ratio was 3.2 +/- 0.2 at 8 h after injection. Conclusion: This study demonstrates the feasibility of in vivo targeting of IGF-1R-expressing prostate cancer xenografts using an Affibody molecule. Further development of radiolabeled Affibody molecules might provide a useful clinical tool for stratification of patients with prostate cancer for IGF-1R-targeting therapy.
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| 24. |
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