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  • 51. Hansson, M.
    et al.
    Nygren, Per-Åke
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Design and production of recombinant subunit vaccines2000Inngår i: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 32, s. 95-107Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    The development of subunit vaccines is presently the main strategy being evaluated for prevention of infectious diseases. The use of recombinant-DNA techniques has facilitated the development of new principles for design and production of subunit vaccines. First of all, the properties of a target protein immunogen can be improved by the use of gene-fusion technology or by the creation of specific changes, to generate 'second-generation protein vaccines', Properties that can be modified include protein solubility, protein stability, in vivo half-lives, etc. In addition, for subunit protein vaccine candidates, the immunogenic properties can be significantly augmented by the addition of immunopotentiating tags or by means of targeting to immunoreactive sites. The recombinant subunit vaccine can furthermore be adapted by gene-fusion technology, to be efficiently incorporated into immunopotentiating adjuvant systems. Also in passive vaccination strategies, i.e. the use of antibodies or antibody fragments for prevention of infectious diseases, the recombinant strategies have become increasingly important. Humanized antibodies and antibody fusion. proteins represent common present anti-infectious-disease agents. The selected examples will indicate that recombinant strategies will indeed have an impact on the design, selection and production of recombinant proteins to be used in the prevention of infectious diseases.

  • 52. Hansson, M.
    et al.
    Samuelson, Patrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Gunneriusson, E.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Surface display on gram positive bacteria2001Inngår i: Combinatorial chemistry & high throughput screening, ISSN 1386-2073, E-ISSN 1875-5402, Vol. 4, nr 2, s. 171-184Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Heterologous surface display on Gram-positive bacteria was first described almost a decade ago and has since then developed into an active research area. Gram-positive bacterial surface display has today found a range of applications, in immunology, microbiology and biotechnology. Live bacterial vaccine delivery vehicles are being developed through the surface display of selected foreign antigens on the bacterial surfaces. In this field, second generation vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered Gram-positive bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other variants of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. This article explains the basis of Grampositive bacterial surface display, and discusses current uses and possible future trends of this emerging technology.

  • 53. Hansson, M.
    et al.
    Samuelson, Patrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Nguyen, T. N.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    General expression vectors for Staphylococcus carnosus enabled efficient production of the outer membrane protein A of Klebsiella pneumoniae2002Inngår i: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 210, nr 2, s. 263-270Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    General expression vectors, designed for intracellular expression or secretion of recombinant proteins in the non-pathogenic Staphylococcus carnosus, were constructed. Both vector systems encode two different affinity tags, an upstream albumin binding protein and a downstream hexahistidyl peptide, and are furnished with cleavage sites for two site-specific proteases for optional affinity tag removal. To evaluate the novel vectors, the gene encoding the outer membrane protein A (OmpA) of Klebsiella pneumoniae was introduced into the vectors. Efficient production was demonstrated in both systems, although, as expected for OmpA fusions, somewhat better intracellularly, and the fusion proteins could be recovered as full-length products by affinity chromatography.

  • 54.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Fernandez, Carmen Diez
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Johannesson, Henrik
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Uhlen, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Exploring epitopes of antibodies toward the human tryptophanyl-tRNA synthetase2010Inngår i: NEW BIOTECHNOL, ISSN 1871-6784, Vol. 27, nr 2, s. 129-137Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    There is a need to characterize the epitopes of affinity reagents to develop high quality affinity reagents for research, diagnostics and therapy. Here, we describe the analysis of epitopes of antibodies generated toward human tryptophanyl-tRNA synthetase (WARS) using both combinatorial bacterial display and suspension bead array. The bacterial display revealed that the polyclonal antibody binds to three separate epitopes and peptide scanning using 15-mers revealed binding to a 13 amino acid consensus sequence (ELINRIERATGQR). A mouse monoclonal antibody was generated and the mapping approach revealed binding toward a slightly shifted position of the same epitope. Structural analysis showed that the antibodies bind to a-helical regions on the surface of the target protein. An alanine-scanning experiment showed binding to four specific residues. The implications for the systematic analysis of antibody epitopes on the basis of these results are discussed.

  • 55.
    Hoyer, Wolfgang
    et al.
    Department of Medical Biochemistry, Swedish Nuclear Magnetic Resonance Center, University of Gothenburg.
    Grönwall, Caroline
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Jonsson, Andreas
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Härd, Torleif
    Department of Medical Biochemistry, Swedish Nuclear Magnetic Resonance Center, University of Gothenburg.
    Stabilization of a beta-hairpin in monomeric Alzheimer´s amyloid beta-peptide inhibits amyloid formation2008Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, nr 13, s. 5099-5104Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    According to the amyloid hypothesis, the pathogenesis of Alzheimer's disease is triggered by the oligomerization and aggregation of the amyloid-β (Aβ) peptide into protein plaques. Formation of the potentially toxic oligomeric and fibrillar Aβ assemblies is accompanied by a conformational change toward a high content of β-structure. Here, we report the solution structure of Aβ(1–40) in complex with the phage-display selected affibody protein ZAβ3, a binding protein of nanomolar affinity. Bound Aβ(1–40) features a β-hairpin comprising residues 17–36, providing the first high-resolution structure of Aβ in β conformation. The positions of the secondary structure elements strongly resemble those observed for fibrillar Aβ. ZAβ3 stabilizes the β-sheet by extending it intermolecularly and by burying both of the mostly nonpolar faces of the Aβ hairpin within a large hydrophobic tunnel-like cavity. Consequently, ZAβ3 acts as a stoichiometric inhibitor of Aβ fibrillation. The selected Aβ conformation allows us to suggest a structural mechanism for amyloid formation based on soluble oligomeric hairpin intermediates.

  • 56. Jonasson, P.
    et al.
    Liljeqvist, S.
    Nygren, Per-Åke
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli2002Inngår i: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 35, s. 91-105Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Genetic strategies have been used for more than two decades to improve bacterial bioprocesses and to simplify recovery procedures. Such strategies include the design of efficient expression vectors and the improvement of bacterial production strains in different ways, e.g. by deletion of protease genes or engineering for overexpression of rare-codon tRNAs, foldases or chaperones. Gene multimerization is another such principle that has proved beneficial to improve production yields. Genetic strategies have furthermore been exploited to facilitate recovery processes by adapting the product for a particular purification principle. In this area, affinity fusions have been commonly used, but other principles, such as modified isoelectric point (pI) or hydrophobic properties have also been successfully investigated. A recent drastic step forward in the use of gene technology to improve recovery processes for recombinant proteins is the introduction of combinatorial protein engineering to generate tailor-made product-specific affinity ligands. This strategy, which allows efficient recovery of a recombinant protein in its native form, is likely to be increasingly used also in industrial-scale bioprocesses, since novel protein ligands have been described that can be sanitized using common industrial cleaning-in-p lace procedures. The examples presented in this review make it evident that genetic strategies will be of utmost importance in the future for facilitating production and recovery of recombinant proteins.

  • 57. Jonasson, P.
    et al.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Jornvall, H.
    Johansson, B. L.
    Wahren, J.
    Uhlen, M.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein2000Inngår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 76, nr 03-feb, s. 215-226Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    An integrated bioprocess has been developed suitable for production of recombinant peptides using a gene multimerization strategy and site-specific cleavage of the resulting gene product. The process has been used for production in E. coli of the human proinsulin C-peptide via a fusion protein BB-C7 containing seven copies of the 31-residues C-peptide monomer. The fusion protein BB-C7 was expressed at high level, 1.8 g l(-1), as a soluble gene product in the cytoplasm. A heat treatment procedure efficiently released the BB-C7 fusion protein into the culture medium. This step also served as an initial purification step by precipitating the majority of the host cell proteins, resulting in a 70% purity of the BB-C7 fusion protein. Following cationic polyelectrolyte precipitation of the nucleic acids and anion exchange chromatography, native C-peptide monomers were obtained by enzymatic cleavage at flanking arginine residues. The released C-peptide material was further purified by reversed-phase chromatography and size exclusion chromatography. The overall yield of native C-peptide at a purity exceeding 99% was 400 mg l(-1) culture, corresponding to an overall recovery of 56%. The suitability of this process also for the production of other recombinant proteins is discussed.

  • 58.
    Jonsson, Andreas
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Wållberg, Helena
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Herne, N.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Frejd, F. Y.
    Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro2009Inngår i: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 54, s. 93-103Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 59. Jorgensen, P. M.
    et al.
    Graslund, S.
    Betz, R.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Larsson, C.
    Hoog, C.
    Characterisation of the human APC1, the largest subunit of the anaphase-promoting complex2001Inngår i: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 262, nr 02-jan, s. 51-59Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Accurate segregation of sister chromatids during mitosis is necessary to avoid the aneuploidy found in many cancers. The spindle checkpoint, which monitors the metaphase to anaphase transition, has been shown to be defective in cancers with chromosomal instability. This checkpoint regulates the anaphase-promoting complex or cyclosome (APC/C), a cell cycle ubiquitin ligase regulating among other things sister chromatid separation. We have previously investigated the mouse Apc1 protein (previously also called Tsg24), the largest subunit of the APC/C. We have now sequenced a full-length human APC1 cDNA, mapped its chromosomal location, and analysed its intron-exon boundaries. We have also investigated the RNA and protein expression of the Apc1 and other APC/C components in normal and cancer cells and the relative occurrence of expressed sequence tugs (ESTs) representing APC subunits from different tissues. The different APC/C subunits are expressed in most tissues and cell types at fairly constant levels relative to each other, suggesting that they perform their functions as part of a complex. A difference from this pattern is however seen for the APC6, which in some cases is more strongly expressed, suggesting a special function for this protein in certain tissues and cell types.

  • 60.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Severa, Denise
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display2008Inngår i: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 278, nr 1, s. 128-136Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The production of candidate affinity proteins in a soluble form, for downstream characterization, is often a time-consuming step in combinatorial protein engineering methods. Here, a novel approach for efficient production of candidate clones is described based on direct cleavage of the affinity protein from the surface of Staphylococcus carnosus, followed by affinity purification. To find a suitable strategy, three new fusion protein constructs were created, introducing a protease site for specific cleavage and purification tags for affinity chromatography purifications into the staphylococcal display vector. The three modified strains were evaluated in terms of transformation frequency, surface expression level and protease cleavage efficiency. A protocol for efficient affinity purification of protease-released affinity proteins using the introduced fusion-tags was successfully used, and the functionality of protease-treated and purified proteins was verified in a biosensor assay. To evaluate the devised method, a previously selected HER2-specific affibody was produced applying the new principle and was used to analyze HER2 expression on human breast cancer cells.

  • 61.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Jonsson, Andreas
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry2008Inngår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 21, nr 4, s. 247-255Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Here we describe the first reported use of a Gram-positive bacterial system for the selection of affinity proteins from large combinatorial libraries displayed on the surface of Staphylococcus carnosus. An affibody library of 3 x 109 variants, based on a 58 residue domain from staphylococcal protein A, was pre-enriched for binding to human tumor necrosis factor-alpha (TNF-alpha) using one cycle of phage display and thereafter transferred to the staphylococcal host (106 variants). The staphylococcal-displayed library was subjected to three rounds of flow-cytometric sorting, and the selected clones were screened and ranked by on-cell analysis for binding to TNF-alpha and further characterized using biosensor analysis and circular dichroism spectroscopy. The successful sorting yielded three different high-affinity binders (ranging from 95 pM to 2.2 nM) and constitutes the first selection of a novel affinity protein using Gram-positive bacterial display. The method combines the simplicity of working with a bacterial host with the advantages of displaying recombinant proteins on robust Gram-positive bacteria as well as using powerful flow cytometry in the selection and characterization process.

  • 62.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Göstring, Lovisa
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Gunneriusson, Elin
    Nilsson, Martin
    Affibody AB, Stockholm, Sweden.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Stockholm, Sweden.
    Gedda, Lars
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala Sweden.
    Frejd, Fredrik Y.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules2011Inngår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 24, nr 4, s. 385-396Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers. ErbB3 has recently also been implicated in resistance to ErbB2-targeting therapies. Here we report the generation of high-affinity ErbB3-specific Affibody molecules intended for future molecular imaging and biotherapeutic applications. Using a high-complexity phage-displayed Affibody library, a number of ErbB3 binders were isolated and specific cell-binding activity was demonstrated in immunofluorescence microscopic studies. Subsequently, a second-generation library was constructed based on sequences of the candidates from the phage display selection. By exploiting the sensitive affinity discrimination capacity of a novel bacterial surface display technology, the affinity of candidate Affibody molecules was further increased down to subnanomolar affinity. In summary, the demonstrated specific targeting of native ErbB3 receptor on human cancer cell lines as well as competition with the heregulin/ErbB3 interaction indicates that these novel biological agents may become useful tools for diagnostic and therapeutic targeting of ErbB3-expressing cancers. Our studies also highlight the powerful approach of combining the advantages of different display technologies for generation of functional high-affinity protein-based binders. Potential future applications, such as radionuclide-based diagnosis and treatment of human cancers are discussed.

  • 63.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Staphylococcal surface display in combinatorial protein engineering and epitope mapping of antibodies2010Inngår i: Recent Patents on Biotechnology, ISSN 1872-2083, Vol. 4, nr 3, s. 171-182Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The field of combinatorial protein engineering for generation of new affinity proteins started in the mid 80s by the development of phage display. Although phage display is a prime example of a simple yet highly efficient method, manifested by still being the standard technique 25 years later, new alternative technologies are available today. One of the more successful new display technologies is cell display. Here we review the field of cell display for directed evolution purposes, with focus on a recently developed method employing Gram-positive staphylococci as display host. Patents on the most commonly used cell display systems and on different modifications as well as specific applications of these systems are also included. General strategies for selection of new affinity proteins from cell-displayed libraries are discussed, with detailed examples mainly from studies on the staphylococcal display system. In addition, strategies for characterization of recombinant proteins on the staphylococcal cell surface, with an emphasis on an approach for epitope mapping of antibodies, are included.

  • 64.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    New Ways for Discovery of Biopharmaceuticals: Emerging Techniques using Surface Display on Gram-positive Bacteria for Combinatorial Protein Engineering and Characterization2009Inngår i: Bioforum Europe, ISSN 1611-597X, Vol. 13, nr 6-7, s. 022-Artikkel i tidsskrift (Fagfellevurdert)
  • 65. Larsson, M.
    et al.
    Graslund, S.
    Li, Y. B.
    Brundell, E.
    Uhlén, Mathias
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hoog, C.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    High-throughput protein expression of cDNA products as a tool in functional genomics2000Inngår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 80, nr 2, s. 143-157Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A proteomics approach has been developed aimed to allow high throughput analysis of protein products expressed from cDNA fragments (expressed sequence tags, ESTs). The concept relies on expression of gene products to generate specific antibodies for protein analysis, such as immunolocalization of the proteins on cellular and subcellular level. To evaluate the system, 55 cDNA clones with predominantly unknown function were selected from a mouse testis cDNA-library. A bacterial expression system was designed that allowed robust expression and easy purification. Protein levels between 15 and 80 mg l(-1) were obtained for 49 of the clones. Five clones were selected for immunization and all yielded functional antibodies that gave specific staining in Western blot screening of samples from various cell types. Furthermore, extensive immunolocalization information on subcellular level was obtained for three of the five clones. All generated data were stored in a relational database, and are made available through a web-interface (http://www.biochem.kth.se/multiscale/), which also provides relevant links and allows homology searches from the original sequences. The possibility to allow analysis of gene products from whole genomes using this 'localization proteomics' approach is discussed.

  • 66. Larsson, M.
    et al.
    Norrander, J.
    Graslund, S.
    Brundell, E.
    Linck, R.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hoog, C.
    The spatial and temporal expression of Tekt1, a mouse tektin C homologue, during spermatogenesis suggest that it is involved in the development of the sperm tail basal body and axoneme2000Inngår i: European Journal of Cell Biology, ISSN 0171-9335, E-ISSN 1618-1298, Vol. 79, nr 10, s. 718-725Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. Recently we described the sequence of the first mammalian tektin protein, Tekt1 (from mouse testis), which is most homologous with sea urchin tektin C. We have now investigated the temporal and spatial expression of Tekt1 during mouse male germ cell development. By in situ hybridization analysis TEKT1 RNA expression is detected in spermatocytes and in round spermatids in the mouse testis, Immunofluorescence microscopy analysis with anti-Tekt1 antibodies showed no distinct labeling of any subcellular structure in spermatocytes, whereas in round spermatids anti-Tekt1 antibodies co-localize with anti-ANA antibodies to the centrosome. At a later stage, elongating spermatids display a larger area of anti-Tekt1 staining at their caudal ends; as spermiogenesis proceeds, the anti-Tekt1 staining disappears. Together with other evidence, these results provide the first intraspecies evidence that Tekt1 is transiently associated with the centrosome, and indicates that Tekt1 is one of several tektins to participate in the nucleation of the flagellar axoneme of mature spermatozoa, perhaps being required to assemble the basal body.

  • 67. Larsson, M.
    et al.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Wennborg, A.
    Expression profile viewer (ExProView): A software tool for transcriptome analysis2000Inngår i: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 63, nr 3, s. 341-353Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A software tool, Expression Profile Viewer (ExProView), for analysis of gene expression profiles derived from expressed sequence tags (ESTs) and SAGE (serial analysis of gene expression) is presented. The software visualizes a complete set of classified transcript data in a two-dimensional array of dots, a virtual chip, in which each dot represents a known gene as characterized in the transcript databases Expressed Gene Anatomy Database or UniGene. The virtual chip display can be changed between representations of different conceptual systems for gene/protein classification and grouping. Four alternative projections are currently available: (i) cellular role, (ii) subcellular compartment, (iii) chromosome localization, and (iv) total UniGene display. However, the chip can be adapted to any other desired layout. By selecting dots, further information about the represented genes is obtained from the local database and WWW links. The software thus provides a visualization of global mRNA expression at the descriptive level and guides in the exploration of patterns of functional expression, while maintaining direct access to detailed information on each individual gene. To evaluate the software, public EST and SAGE gene expression data obtained from the Cancer Genome Anatomy Project at the National Center for Biotechnology Information were analyzed and visualized. A demonstration of the software is available at http://www.biochem.kth. se/exproview/.

  • 68. Lehtio, J.
    et al.
    Wernérus, Henrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Samuelson, Patrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Teeri, Tuula T.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Directed immobilization of recombinant staphylococci on cotton fibers by functional display of a fungal cellulose-binding domain2001Inngår i: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 195, nr 2, s. 197-204Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The immobilization of recombinant staphylococci onto cellulose fibers through surface display of a fungal cellulose-binding domain (CBD) was investigated. Chimeric proteins containing the CBD from Trichoderma reesei cellulase Cel6A were found to be correctly targeted to the cell wall of Staphylococcus carnosus cells. since full-length proteins could be extracted and affinity-purified. Furthermore. surface accessibility of the CBD was verified using a monoclonal antibody and functionality in terms of cellulose-binding was demonstrated in two different assays in which recombinant staphylococci were found to efficiently bind to cotton fibers. The implications of this strategy of directed immobilization Tor the generation of whole-cell microbial tools Fur different applications will be discussed.

  • 69.
    Lindberg, Hanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Hofström, Camilla
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Altai, Mohamed
    Honorvar, Hadis
    Wållberg, Helena
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Orlova, Anna
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Gräslund, Torbjörn
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Tolmachev, Vladimir
    Evaluation of a HER2-targeting affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for Tc-99m-labelling at the C terminus2012Inngår i: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 33, nr 3, s. 641-651Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 70.
    Lindberg, Hanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Härd, Torleif
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity2015Inngår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 10, nr 11, s. 1707-1718Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The amyloid hypothesis suggests that accumulation of amyloid β (Aβ) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited Aβ aggregation by sequestering the aggregation prone parts of the peptide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the Aβ peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the Aβ peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high-affinity Aβ-binding molecules were isolated using flow-cytometric sorting. The best performing candidate binds Aβ with a KD value of around 300 pM, corresponding to a 50-fold improvement in affinity relative to the first-generation binder. The new dimeric Affibody molecule was shown to capture Aβ1-42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Aβ peptide.

  • 71.
    Lindberg, Hanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Johansson, Anna
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Härd, Torleif
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Staphylococcal display for combinatorial protein engineering of a head-to-tail affibody dimer binding the Alzheimer amyloid-ss peptide2013Inngår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 8, nr 1, s. 139-145Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have previously generated an affibody molecule for the disease-associated amyloid beta (A beta) peptide, which has been shown to inhibit the formation of various A beta aggregates and revert the neurotoxicity of A beta in a fruit fly model of Alzheimer's disease. In this study, we have investigated a new bacterial display system for combinatorial protein engineering of the A beta-binder as a head-to-tail dimeric construct for future optimization efforts, e.g. affinity maturation. Using the bacterial display platform, we have: (i) demonstrated functional expression of the dimeric binder on the cell surface, (ii) determined the affinity and investigated the pH sensitivity of the interaction, (iii) demonstrated the importance of an intramolecular disulfide bond through selections from a cell-displayed combinatorial library, as well as (iv) investigated the effects from rational truncation of the N-terminal part of the affibody molecule on surface expression level and A beta binding. Overall, the detailed engineering and characterization of this promising A beta-specific affibody molecule have yielded valuable insights concerning its unusual binding mechanism. The results also demonstrated that our bacterial display system is a suitable technology for future protein engineering and characterization efforts of homo- or heterodimeric affinity proteins.

  • 72.
    Lindberg, Hanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Sandersjöö, Lisa
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Meister, S. W.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Flow-cytometric screening of aggregation-inhibitors using a fluorescence-assisted intracellular method2017Inngår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 12, nr 1, artikkel-id 1600364Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Aggregation of misfolded peptides and proteins is a key event in several neurodegenerative diseases. Suggested treatments of such disorders aim to inhibit the initial aggregation process. Here, we have developed an intracellular, function-based screening method, intended for isolation of aggregation-inhibitors from combinatorial protein libraries by flow-cytometric cell sorting. The method is based on fusion of aggregation-prone peptides to a fluorescent protein, functioning as a solubility reporter. Co-expression of a protein-based aggregation-inhibitor should prevent aggregation and thus increase the whole-cell fluorescence. We evaluated the method using the aggregation-prone Alzheimer's-related amyloid-β (Aβ) peptide in fusion to green-fluorescent protein (GFP), and an Aβ aggregation-inhibiting Affibody molecule. To adapt the method for library applications, the inhibitor was linked to an mCherry reporter for normalization of protein expression levels. We found that aggregation propensity correlates with fluorescence intensity, as co-expression of the Affibody-inhibitor increased the whole-cell fluorescence relative to a non-inhibitor. Employing improved cultivation parameters, we furthermore demonstrated efficient rescue from aggregation of an α-synuclein-derived protein using a different type of aggregation-inhibitor. Importantly, we also showed that the Aβ aggregation-inhibiting Affibody molecule could be isolated from a 1:10,000 background of non-inhibitors, with around 3,500-fold enrichment, in one cycle of fluorescence-based cell sorting. In conclusion, our new method represents a promising approach for generation of novel protein-based aggregation-inhibitors.

  • 73.
    Lindberg, Hanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Sandersjöö, Lisa
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Development of a fluorescence-based intracellular method for function-based isolation of protein-based aggregation inhibitorsManuskript (preprint) (Annet vitenskapelig)
  • 74. Lindholm, L
    et al.
    Andersson, K
    Boulanger, P
    Frykholm, K
    Henning, P
    Hoeben, R
    Hong, S S
    Johannisson, J
    Magnusson, M
    Myhre, S
    Nygren, Per-Åke
    KTH, Tidigare Institutioner, Bioteknologi.
    Pettersson, E
    Stahl, Stefan
    KTH, Tidigare Institutioner, Bioteknologi.
    Vellinga, J
    Wikman, Maria
    KTH, Tidigare Institutioner, Bioteknologi.
    Genetic re-targeting of adenovirus using a hyperstable scFv domain and an affibody (R) molecule against Her2/neu2004Inngår i: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 9, s. S250-S250Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    One important goal in gene therapy is to develop adenovirus (Ad) vectors that are genetically de-targeted as well as re-targeted. Genetic re-targeting of Ad using complex cell-binding ligands has previously not been possible. We have previously demonstrated that ligands for genetic re-targeting of adenoviruses must be able to fold correctly in the cytoplasm of virus producing cells, a milieu that is not conducive to the formation of disulphide bonds.

    Here, we describe functional Ad5 viruses with fibers and pIX capsid proteins genetically modified to contain two types of complex ligands. One is affibody® molecules, corresponding to small (6 kDa) binding proteins developed by combinatorial protein engineering using a single three-helix bundle staphylococcal protein A domain. The other type is hyperstable antibody scFv domains.

    The affibody molecule used here (ZH2N) is directed against Her2/neu. Recombinant viruses were constructed with ZH2N in three different positions: (i) at the C-terminus of shaft repeat 7 of de-knobbed fibers; (ii) at the C-terminus of pIX; and (iii) in the HI-loop of the fiber knob. Each of the viruses exhibited a characteristic phenotype regarding fiber content, growth and ability to infect Her2/neu expressing cells.

    In order to test the potentials of scFv liganded Ad vectors, a hyperstable antibody scFv against b-galactosidase was genetically incorporated into knobless fibers, in tandem with a mutated protein A domain reactive with IgG1 Fc that targeted the virus to Fc-expressing 293 cells. These fibers could be rescued into viable virions that retained the original antigen binding specificity of the scFv, demonstrating the basic potential of hyperstable scFvs for genetic re-targeting of Ad. Quite unexpectedly, the fiber content of Ad with knobless, scFv containing fibers was close to normal in contrast to other Ad with knobless fibers that generally has a much reduced fiber content. The hyperstable scFv was further fused to the C-terminus of the capsid protein pIX. The recombinant molecules could be rescued into viable viruses with wt fibers. The scFv retained its binding-specificity on the recombinant virions.

    The results demonstrate that, contrary to current beliefs, it is possible to construct Ad that genetically incorporates functional scFvs and other complex ligands into the virus fiber and pIX. The feasibility is demonstrated by the creation of different viruses that are re-targeted to Her2/neu. These viruses are currently in pre-clinical development.

  • 75. Luheshi, Leila M.
    et al.
    Hoyer, Wolfgang
    de Barros, Teresa Pereira
    Härd, Iris van Dijk
    Brorsson, Ann-Christin
    Macao, Bertil
    Persson, Cecilia
    Crowther, Damian C.
    Lomas, David A.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Dobson, Christopher M.
    Härd, Torleif
    Sequestration of the A beta Peptide Prevents Toxicity and Promotes Degradation In Vivo2010Inngår i: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 8, nr 3, s. e1000334-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (A beta) peptide by using a small engineered binding protein (Z(A beta 3)) that binds with nanomolar affinity to A beta, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z(A beta 3) in the brains of Drosophila melanogaster expressing either A beta(42) or the aggressive familial Alzheimer's disease (AD) associated E22G variant of A beta(42) abolishes their neurotoxic effects. Biochemical analysis indicates that monomer A beta binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of A beta aggregation and reveal that Z(A beta 3) not only inhibits the initial association of A beta monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.

  • 76.
    Löfblom, John
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Feldwisch, J.
    Tolmachev, V.
    Carlsson, J.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Frejd, F. Y.
    Affibody molecules: Engineered proteins for therapeutic, diagnostic and biotechnological applications2010Inngår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 584, nr 12, s. 2670-2680Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Affibody molecules are a class of engineered affinity proteins with proven potential for therapeutic, diagnostic and biotechnological applications. Affibody molecules are small (6.5 kDa) single domain proteins that can be isolated for high affinity and specificity to any given protein target. Fifteen years after its discovery, the Affibody technology is gaining use in many groups as a tool for creating molecular specificity wherever a small, engineering compatible tool is warranted. Here we summarize recent results using this technology, propose an Affibody nomenclature and give an overview of different HER2-specific Affibody molecules. Cumulative evidence suggests that the three helical scaffold domain used as basis for these molecules is highly suited to create a molecular affinity handle for vastly different applications.

  • 77.
    Löfblom, John
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Feldwisch, J.
    Affibody AB, Stockholm, Sweden..
    Tolmachev, V.
    Uppsala Univ, Rudbeck Lab, Dept Oncol Radiol & Clin Immunol, Uppsala, Sweden..
    Carlsson, J.
    Uppsala Univ, Rudbeck Lab, Dept Oncol Radiol & Clin Immunol, Uppsala, Sweden..
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Frejd, F. Y.
    Affibody AB, Stockholm, Sweden..
    Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications2010Inngår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, s. 31-31Artikkel i tidsskrift (Annet vitenskapelig)
  • 78.
    Löfblom, John
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Frejd, Fredrik Y.
    Uppsala University.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Non-immunoglobulin based protein scaffolds2011Inngår i: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 22, nr 6, s. 843-848Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Non-immunoglobulin based protein scaffolds have been reported as promising alternatives to traditional monoclonal antibodies for over a decade and are often mentioned as part of the next-generation immunotherapeutics. Today, this class of biologics is beginning to demonstrate its potential for therapeutic applications and several are currently in preclinical or clinical development. A common denominator for most of these new scaffolds is the attractive properties that differentiate them from monoclonal antibodies including small size, cysteine-free sequence, flexible pharmacokinetic properties, and ease of generating multispecific molecules. In addition to therapeutic applications, substantial evidence point to superior performance of several of these scaffolds in molecular imaging compared to full-length antibodies. Here we review the most recent progress using alternative protein scaffolds for therapy and medical imaging.

  • 79.
    Löfblom, John
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Kronqvist, Nina
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Optimization of electroporation-mediated transformation: Staphylococcus carnosus as model organism2007Inngår i: Journal of Applied Microbiology, ISSN 1364-5072, E-ISSN 1365-2672, Vol. 102, nr 3, s. 736-747Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The study was conducted with an aim to optimize the transformation efficiency of the Gram-positive bacterium Staphylococcus carnosus to a level that would enable the creation of cell surface displayed combinatorial protein libraries.

    Methods and Results: We have thoroughly investigated a number of different parameters for: (i) the preparation of electrocompetent cells; (ii) the treatment of cells before electroporation; (iii) the electroporation step itself; and (iv) improved recovery of transformed cells. Furthermore, a method for heat-induced inactivation of the host cell restriction system was devised to allow efficient transformation of the staphylococci with DNA prepared from other species, such as Escherichia coli. Previously described protocols for S. carnosus, giving transformation frequencies of approximately 10(2) transformants per transformation could be improved to reproducible procedures giving around 10(6) transformants for a single electroporation event, using plasmid DNA prepared from either S. carnosus or E. coli. The transformed staphylococcal cells were analysed using flow cytometry to verify that the entire cell population retained the introduced plasmid DNA and expressed the recombinant protein in a functional form on the cell surface at the same level as the positive control population.

    Conclusions: The results demonstrate that the transformation frequency for S. carnosus could be dramatically increased through optimization of the entire electroporation process, and that the restriction barrier for interspecies DNA transfer, could be inactivated by heat treatment of the cells prior to electroporation.

    Significance and Impact of the Study: The generation of large combinatorial protein libraries, displayed on the surface of S. carnosus can be envisioned in the near future, thus dramatically improving the selection compared with the traditional biopanning procedure used in phage display.

  • 80.
    Löfblom, John
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Sandberg, Julia
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Evaluation of staphylococcal cell surface display and flow cytometry for postselectional characterization of affinity proteins in combinatorial protein engineering applications2007Inngår i: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 73, nr 21, s. 6714-6721Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    For efficient generation of high-affinity protein-based binding molecules, fast and reliable downstream characterization platforms are needed. In this work, we have explored the use of staphylococcal cell surface display together with How cytometry for affinity characterization of candidate affibody molecules directly on the cell surface. A model system comprising three closely related affibody molecules with different affinities for immunoglobulin G and an albumin binding domain with affinity for human serum albumin was used to investigate advantages and differences compared to biosensor technology in a side-by-side manner. Equilibrium dissociation constant (K-D) determinations as well as dissociation rate analysis were performed using both methods, and the results show that the on-cell determinations give both KD and dissociation rate values in a very fast and reproducible manner and that the relative affinities are very similar to the biosensor results. Interestingly, the results also show that there are differences between the absolute affinities determined with the two different technologies, and possible explanations for this are discussed. This work demonstrates the advantages of cell surface display for directed evolution of affinity proteins in terms of fast postselectional, on-cell characterization of candidate clones without the need for subcloning and subsequent protein expression and purification but also demonstrates that it is important to be aware that absolute affinities determined using different methods often vary substantially and that such comparisons therefore could be difficult.

  • 81.
    Löfblom, John
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO).
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO).
    Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display2005Inngår i: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 248, nr 2, s. 189-198Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have investigated a staphylococcal surface display system for its potential future use as a protein library display system ill combinatorial biochemistry. Efficient affinity-based selections require a system capable of fine affinity discrimination of closely related binders to minimize the loss of potentially improved variants. In this Study, a significant breakthrough was achieved to avoid biases due to potential cell-to-cell variations in surface expression levels, since it was found that a generic protein tag, present within the displayed recombinant surface proteins on the cells, could be successfully employed to obtain normalization of the target-binding signal. Four mutated variants of a staphylococcal protein A domain with different affinity to human IgG were successfully expressed on the surface of recombinant Staphylococcus carnosus cells. The system was evaluated for affinity-based cell sorting experiments, where cell-displayed protein A domains with an 8-fold difference in target affinity were mixed at a ratio of 1: 1000 and sorted using FACS. Enrichment factors around 140-fold were obtained from a single round of sorting under normal library sorting conditions when the top 0.1% fraction having the highest antigen binding to Surface expression level ratio was sorted. The results demonstrate that the system would have a potential as a selection system in protein library display applications, and the normalization strategy should indeed make it possible to achieve fine affinity discriminations in future library selections. (c) 2005 Federation of European Microbiological Societies.

  • 82. Magnusson, M. K.
    et al.
    Henning, P.
    Myhre, S.
    Wikman, M.
    Uil, T. G.
    Friedman, M.
    Andersson, K. M. E.
    Hong, S. S.
    Hoeben, R. C.
    Habib, N. A.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Boulanger, P.
    Lindholm, L.
    Adenovirus 5 vector genetically re-targeted by an affibody molecule with specificity for tumor antigen HER2/neu2007Inngår i: Cancer Gene Therapy, ISSN 0929-1903, E-ISSN 1476-5500, Vol. 14, nr 5, s. 468-479Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In order to use adenovirus (Ad) type 5 (Ad5) for cancer gene therapy, Ad needs to be de-targeted from its native receptors and re-targeted to a tumor antigen. A limiting factor for this has been to find a ligand that (i) binds a relevant target, (ii) is able to fold correctly in the reducing environment of the cytoplasm and (iii) when incorporated at an optimal position on the virion results in a virus with a low physical particle to plaque-forming units ratio to diminish the viral load to be administered to a future patient. Here, we present a solution to these problems by producing a genetically re-targeted Ad with a tandem repeat of the HER2/neu reactive Affibody molecule (ZH) in the HI-loop of a Coxsackie B virus and Ad receptor (CAR) binding ablated fiber genetically modified to contain sequences for flexible linkers between the ZH and the knob sequences. ZH is an Affibody molecule specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) that is overexpressed in inter alia breast and ovarian carcinomas. The virus presented here exhibits near wild-type growth characteristics, infects cells via HER2/neu instead of CAR and represents an important step toward the development of genetically re-targeted adenoviruses with clinical relevance.

  • 83.
    Malm, Magdalena
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Bass, Tarek
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Gudmundsdotter, Lindvi
    Lord, Martin
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Frejd, Fredrik Y.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    Engineering of a bispecific affibody molecule towards HER2 and HER3 by addition of an albumin-binding domain allows for affinity purification and in vivo half-life extension2014Inngår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 9, nr 9, s. 1215-1222Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Emerging strategies in cancer biotherapy include the generation and application of bispecific antibodies, targeting two tumor-associated antigens for improved tumor selectivity and potency. Here, an alternative format for bispecific molecules was designed and investigated, in which two Affibody molecules were linked by an albumin-binding domain (ABD). Affibody molecules are small (6 kDa) affinity proteins and this new format allows for engineering of molecules with similar function as full-length bispecific antibodies, but in a dramatically smaller size (around eight-fold smaller). The ABD was intended to function both as a tag for affinity purification as well as for in vivo half-life extension in future preclinical and clinical investigations. Affinity-purified bispecific Affibody molecules, targeting HER2 and HER3, showed simultaneous binding to the three target proteins (HER2, HER3, and albumin) when investigated in biosensor assays. Moreover, simultaneous interactions with the receptors and albumin were demonstrated using flow cytometry on cancer cells. The bispecific Affibody molecules were also able to block ligand-induced phosphorylation of the HER receptors, indicating an anti-proliferative effect. We believe that this compact and flexible format has great potential for developing new potent bispecific affinity proteins in the future, as it combines the benefits of a small size (e.g. improved tissue penetration and reduced cost of goods) with a long circulatory half-life.

  • 84.
    Malm, Magdalena
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Frejd, F. Y.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Targeting HER3 using mono- and bispecific antibodies or alternative scaffolds2016Inngår i: mAbs, ISSN 1942-0862, E-ISSN 1942-0870, Vol. 8, nr 7, s. 1195-1209Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The human epidermal growth factor receptor 3 (HER3) has in recent years been recognized as a key node in the complex signaling network of many different cancers. It is implicated in de novo and acquired resistance against therapies targeting other growth factor receptors, e.g., EGFR, HER2, and it is a major activator of the PI3K/Akt signaling pathway. Consequently, HER3 has attracted substantial attention, and is today a key target for drugs in clinical development. Sophisticated protein engineering approaches have enabled the generation of a range of different affinity proteins targeting this receptor, including antibodies and alternative scaffolds that are either mono- or bispecific. Here, we describe HER3 and its role as a key tumor target, and give a comprehensive review of HER3-targeted proteins currently in development, including discussions on the opportunities and challenges of targeting this receptor.

  • 85.
    Malm, Magdalena
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Kronqvist, Nina
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Lindberg, Hanna
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Gudmundsdotter, Lindvi
    Affibody AB, Stockholm, Sweden.
    Bass, Tarek
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Frejd, Fredrik Y.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Stockholm, Sweden.
    Varasteh, Zohreh
    Departmaent of Medical Chemistry, Preclinical PET Platform, Uppasala University, Uppsala, Sweden.
    Orlova, Anna
    Departmaent of Medical Chemistry, Preclinical PET Platform, Uppasala University, Uppsala, Sweden.
    Tolmachev, Vladimir
    Unit of Biomedical Radiation Sciences, Uppsala University, Uppsala, Sweden.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification2013Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, nr 5, s. e62791-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 PM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.

  • 86. Mitran, B.
    et al.
    Güler, Rezan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Lindstrom, E.
    Fleetwood, Filippa
    KTH.
    Tolmachev, V.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO).
    Orlova, A.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Feasibility of in vivo imaging of VEGFR2 expression using high affinity antagonistic biparatopic affibody construct Z(VEGFR2)-Bp(2)2016Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, s. S97-S98Artikkel i tidsskrift (Fagfellevurdert)
  • 87.
    Mitran, Bogdan
    et al.
    Uppsala Univ, Dept Med Chem, Dag Hammarskjoldsv 14C,3Tr, S-75183 Uppsala, Sweden..
    Güler, Rezan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Roche, Francis P.
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Lindstrom, Elin
    Uppsala Univ, Dept Med Chem, Dag Hammarskjoldsv 14C,3Tr, S-75183 Uppsala, Sweden..
    Selvaraju, Ram Kumar
    Uppsala Univ, Dept Med Chem, Dag Hammarskjoldsv 14C,3Tr, S-75183 Uppsala, Sweden.;Uppsala Univ, Dept Med Chem, Preclin PET MRI Platform, Uppsala, Sweden..
    Fleetwood, Filippa
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Rinne, Sara S.
    Uppsala Univ, Dept Med Chem, Dag Hammarskjoldsv 14C,3Tr, S-75183 Uppsala, Sweden..
    Claesson-Welsh, Lena
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, Uppsala, Sweden..
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Ståhl, Stefan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, Dag Hammarskjoldsv 14C,3Tr, S-75183 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, Uppsala, Sweden..
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Radionuclide imaging of VEGFR2 in glioma vasculature using biparatopic affibody conjugate: proof-of-principle in a murine model2018Inngår i: Theranostics, ISSN 1838-7640, E-ISSN 1838-7640, Vol. 8, nr 16, s. 4462-4476Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Vascular endothelial growth factor receptor-2 (VEGFR2) is a key mediator of angiogenesis and therefore a promising therapeutic target in malignancies including glioblastoma multiforme (GBM). Molecular imaging of VEGFR2 expression may enable patient stratification for antiangiogenic therapy. The goal of the current study was to evaluate the capacity of the novel anti-VEGFR2 biparatopic affibody conjugate (Z(VEGFR2)-Bp(2)) for in vivo visualization of VEGFR2 expression in GBM. Methods: Z(VEGFR2)-Bp(2) coupled to a NODAGA chelator was generated and radiolabeled with indium-111. The VEGFR2-expressing murine endothelial cell line MS1 was used to evaluate in vitro binding specificity and affinity, cellular processing and targeting specificity in mice. Further tumor targeting was studied in vivo in GL261 glioblastoma orthotopic tumors. Experimental imaging was performed. Results: [In-111]In-NODAGA-Z(VEGFR2)-Bp(2) bound specifically to VEGFR2 (K-D=33 +/- 18 pM). VEGFR2-mediated accumulation was observed in liver, spleen and lungs. The tumor-to-organ ratios 2 h post injection for mice bearing MS1 tumors were approximately 11 for blood, 15 for muscles and 78 for brain. Intracranial GL261 glioblastoma was visualized using SPECT/CT. The activity uptake in tumors was significantly higher than in normal brain tissue. The tumor-to-cerebellum ratios after injection of 4 mu g [In-111]In-NODAGA-Z(VEGFR2)-Bp(2) were significantly higher than the ratios observed for the 40 mu g injected dose and for the non-VEGFR2 binding size-matched conjugate, demonstrating target specificity. Microautoradiography of cryosectioned CNS tissue was in good agreement with the SPECT/CT images. Conclusion: The anti-VEGFR2 affibody conjugate [In-111]In-NODAGA-Z(VEGFR2)-Bp(2) specifically targeted VEGFR2 in vivo and visualized its expression in a murine GBM orthotopic model. Tumor-to-blood ratios for [In-111]In-NODAGA-Z(VEGFR2)-Bp(2) were higher compared to other VEGFR2 imaging probes. [In-111]In-NODAGA-Z(VEGFR2)-Bp(2) appears to be a promising probe for in vivo noninvasive visualization of tumor angiogenesis in glioblastoma.

  • 88. Myhre, S.
    et al.
    Henning, P.
    Friedman, M.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Lindholm, L.
    Magnusson, M. K.
    Re-targeted adenovirus vectors with dual specificity; binding specificities conferred by two different Affibody molecules in the fiber2009Inngår i: Gene Therapy, ISSN 0969-7128, E-ISSN 1476-5462, Vol. 16, nr 2, s. 252-261Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Vectors based on Adenovirus type 5 (Ad5) are among the most common vectors in cancer gene therapy trials to date. However, for increased efficiency and safety, Ad5 should be de-targeted from its native receptors and re-targeted to a tumor antigen. We have described earlier an Ad5 vector genetically re-targeted to the tumor antigen HER2/neu by a dimeric version of the Affibody molecule ZH inserted in the HI-loop of the fiber knob of a coxsackie and adenovirus receptor-binding ablated fiber. This virus showed almost wild-type growth characteristics and infected cells through HER2/neu. Here we generate vectors with double specificity by incorporating two different Affibody molecules, ZH (HER2/neu-binding) and ZT (Taq polymerase-binding), at different positions relative to one another in the HI-loop. Receptor-binding studies together with viral production and gene transfer assays showed that the recombinant fiber with ZT in the first position and ZH in the second position (ZTZH) bound to both its targets, whereas surprisingly, the fiber with ZHZT was devoid of binding to HER2/neu. Hence, it is possible to construct a recombinant adenovirus with dual specificity after evaluating the best position for each ligand in the fiber knob.

  • 89. Nguyen, N
    et al.
    Samuelson, Patrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Sterky, Fredrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Merle-Poitte, C
    Robert, A
    Baussant, T
    Haeuw, F
    Uhlen, Mathias
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Binz, H
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Chromosomal sequencing using a PCR-based biotin-capture method allowed isolation of the complete gene for the outer membrane protein A of Klebsiella pneumoniae1998Inngår i: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 210, nr 1, s. 93-101Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    By employing a novel biotin-and PCR-assisted capture method, which allows determination of unknown sequences on chromosomal DNA. the gene for the outer membrane protein A (OmpA) of Klebsiella pneumoniae has been isolated and sequenced to completion. The method involves linear amplification of DNA from a biotinylated primer annealing to a region with known sequence. After capture of the amplified single-stranded DNA on to paramagnetic beads, unspecifically annealing primers, i.e. arbitrary primers, were used to generate fragments with only partly determined nt sequences. The homology of the sequenced gene to ompAs of related bacteria is discussed. The ompA gene was assembled for intracellular expression in Escherichia coli, and two different fusion proteins were produced and recovered with good yields. The importance of the novel chromosomal sequencing method for gene isolation in general and the potential use of the OmpA fusion proteins are discussed. (C) 1998 Elsevier Science B.V.

  • 90. Nguyen, T. N.
    et al.
    Libon, C.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Subunit vaccine candidates engineered from the central conserved region of the RSV G protein aimed for parenteral or mucosal delivery2013Inngår i: Molecular Vaccines: From Prophylaxis to Therapy-Volume 1, Springer, 2013, s. 103-118Kapittel i bok, del av antologi (Annet vitenskapelig)
    Abstract [en]

    Respiratory syncytial virus (RSV) is a major pathogen causing severe upper and lower respiratory disease in infants and in elderly worldwide. ccording to WHO, an RSV vaccine is urgently needed. Here, we describe the design of various types of subunit vaccine concepts based on molecular engineering aimed to deliver RSV antigens. Gene segment encoding parts of the conserved central region of the RSV G protein (G2Na) were prepared for various expression and delivery formats: (1) prokaryotically expressed and purifi ed G2Na alone or fused to different carrier proteins, one of them, namely, BBG2Na (Alum), has reached clinical trials in the elderly; (2) G protein-derived antigens surface displayed on lived vectors (non pathogenic bacteria) and (3) nucleic acid vectors. These subunit vaccines were administered with or without adjuvant in rodents and in non-human primates by different routes (parenteral or mucosal). We summarise and compare immunogenicity, protective effi cacy and safety conferred by each immunisation format in RSV experimental animal models. Among these, G2Na proved to be the most promising component for an RSV subunit vaccine.

  • 91.
    Nordberg, Erika
    et al.
    Rudbeck Laboratory, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences, Uppsala University.
    Friedman, Mikaela
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Göstring, Lovisa
    Rudbeck Laboratory, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences, Uppsala University.
    Adams, Gregory P.
    Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia.
    Brismar, Hjalmar
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Nilsson, Fredrik Y.
    Rudbeck Laboratory, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences, Uppsala University.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Glimelius, Bengt
    Rudbeck Laboratory, Oncology, Radiology and Clinical Immunology, Uppsala University.
    Carlsson, Jörgen
    Rudbeck Laboratory, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences, Uppsala University.
    Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule2007Inngår i: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 34, nr 6, s. 609-618Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Introduction: The cellular binding and processing of an epidermal growth factor receptor (EGFR) targeting affibody molecule, (Z(EGFR:955))(2), was studied. This new and small molecule is aimed for applications in nuclear medicine. The natural ligand epidermal growth factor (EGF) and the antibody cetuximab were studied for comparison.

    Methods: All experiments were made with cultured A431 squamous carcinoma cells. Receptor specificity, binding time patterns, retention and preliminary receptor binding site localization studies were all made after (125) I, labeling. Internalization was studied using Oregon Green 488, Alexa Fluor 488 and CypHer5E markers.

    Results: [(125) I](Z(EGFR:955))(2) and [(125) I]cetuximab gave a maximum cellular uptake of I-125 within 4 to 8 h of incubation, while [(125) I]EGF gave a maximum uptake already after 2 h. The retention studies showed that the cell-associated fraction of 125 1 after 48 It of incubation was similar to 20% when delivered as [(125) I](Z(FGFR:955))(2) and similar to 25% when delivered as [I-125] cetuximab. [(125) I]EGF-mediated delivery gave a faster (125) I release, where almost all cell-associated radioactivity had disappeared within 24 It. All three substances were internalized as demonstrated with confocal microscopy. Competitive binding studies showed that both EGF and cetuximab inhibited binding Of (Z(EGFR:955))(2) and indicated that the three substances competed for an overlapping binding site.

    Conclusion: The results gave information on cellular processing of radionuclides when delivered with (Z(EGFR:955))(2) in comparison to delivery with EGF and cetuximab. Competition assays suggested that [I-125](Z(EGFR:955))(2) bind to Domain III of EGFR. The affibody molecule (Z(EGFR:955))(2) can be a candidate for EGFR imaging applications in nuclear medicine.

  • 92.
    Nordberg, Erika
    et al.
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Orlova, Anna
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Friedman, Mikaela
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Tolmachev, Vladimir
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Nilsson, Fredrik Y.
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Glimelius, Bengt
    Uppsala Univ, Akad Hosp, Rudbeck Lab.
    Carlsson, Jörgen
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    In vivo and in vitro uptake of 111In, delivered with the affibody molecule (ZEGFR:955)2, in EGFR expressing tumour cells2008Inngår i: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 19, nr 4, s. 853-857Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The epidermal growth factor receptor, EGFR, is overexpressed in many carcinomas. Targeting this receptor with radionuclides is important for imaging and therapy applications in nuclear medicine. We investigated the in vitro and in vivo properties of a new high affinity EGFR binding affibody molecule, (Z(EGFR:955))(2), when conjugated with CHXA"-DTPA and labelled with In-111. The binding time patterns and retention studies were performed using cultured squamous carcinoma A431 cells that overexpress EGFR. In the in vivo studies, female BALB/c nu/nu mice carrying tumours from xenografted A431 cells were used. The in vitro studies showed EGFR specific binding, high uptake and good retention of In-111 when delivered as [In-111](Z(EGFR:955))(2). The retention after 72 h of incubation was 38.0 +/- 1.15% of the initial level. The biodistribution study showed a tumour specific In-111 uptake of 3.8 +/- 1.4% of injected dose per gram turnout tissue 4 h post-injection. The tumour to blood ratio was 9.1 and the tumours could easily be visualized with a gamma camera at this time-point. In-111 delivered with [In-111](Z(EGFR:955))(2) gave an EGFR specific uptake and the results indicated that the (Z(EGFR:955))(2) affibody molecule is a candidate for radionuclide-based tumour imaging. Potential therapy applications are discussed.

  • 93. Orlova, A.
    et al.
    Magnusson, M.
    Eriksson, T. L. J.
    Nilsson, M.
    Larsson, B.
    Hoiden-Guthenherg, I.
    Widstrom, C.
    Carlsson, J.
    Tolmachev, V.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Nilsson, F. Y.
    Tumor Imaging using a picomolar affinity HER2 binding affibody molecule2006Inngår i: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 66, nr 8, s. 4339-4348Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The detection of cell-bound proteins that are produced due to aberrant gene expression in malignant tumors can provide important diagnostic information influencing patient management. The use of small radiolabeled targeting proteins would enable high-contrast radionuclide imaging of cancers expressing such antigens if adequate binding affinity and specificity could he provided. Here, we describe a HER2-specific 6 kDa Affibody molecule (hereinafter denoted Affibody molecule) with 22 pmol/L affinity that can be used for the visualization of HER2 expression in tumors in vivo using gamma camera. A library for affinity maturation was constructed by re-randomization of relevant positions identified after the alignment of first-generation variants of nanomolar affinity (50 nmol/L). One selected Affibody molecule, Z(HER2:342) showed a > 2,200-fold increase in affinity achieved through a single-library affinity maturation step. When radioiodinated, the affinity-matured Affibody molecule showed clear, high-contrast visualization of HER2-expressing xenografts in mice as early as 6 hours post-injection. The tumor uptake at 4 hours post-injection was improved 4-fold (due to increased affinity) with 9% of the injected dose per gram of tissue in the tumor. Affibody molecules represent a new class of affinity molecules that can provide small sized, high affinity cancer-specific ligands, which may be well suited for tumor imaging.

  • 94. Orlova, A.
    et al.
    Magnusson, M.
    Eriksson, T.
    Nilsson, M.
    Carlsson, J.
    Tolmachev, V.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Nilsson, F. Y.
    Effect of affinity maturation on biodistribution of radioiodinated anti-HER2 Affibody molecules2005Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 32, s. S78-S79Artikkel i tidsskrift (Annet vitenskapelig)
  • 95. Orlova, A.
    et al.
    Malm, Malin
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Lindberg, Hanna
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Varasteh, Z.
    Selvaraju, R. K.
    Kronqvist, Nina
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Tolmachev, V.
    Feasibility of radionuclide imaging of HER3-expressing tumours using technetium-99m labeled affibody molecules2013Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, s. S185-S186Artikkel i tidsskrift (Annet vitenskapelig)
  • 96. Orlova, A.
    et al.
    Rosestedt, M.
    Mitran, B.
    Bass, T.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Frejd, F. Y.
    Löfblom, J.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Tolmachev, V.
    Ståhl, S.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    In vivo evaluation of pharmacokinetics, tumors targeting and therapeutic efficacy of a novel format of HER3-targeting affibody molecule with prolonged blood circulation2016Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, s. S237-S237Artikkel i tidsskrift (Fagfellevurdert)
  • 97.
    Orlova, Anna
    et al.
    Uppsala Univ, Dept Med Chem, SE-75283 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, Uppsala, Sweden..
    Bass, Tarek
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Rinne, Sara S.
    Uppsala Univ, Dept Med Chem, SE-75283 Uppsala, Sweden..
    Leitao, Charles Dahlsson
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Rosestedt, Maria
    Uppsala Univ, Dept Med Chem, SE-75283 Uppsala, Sweden..
    Atterby, Christina
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Gudmundsdotter, Lindvi
    Affibody AB, Solna, Sweden..
    Frejd, Fredrik Y.
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden.;Affibody AB, Solna, Sweden..
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Ståhl, Stefan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Evaluation of the Therapeutic Potential of a HER3-Binding Affibody Construct TAM-HER3 in Comparison with a Monoclonal Antibody, Seribantumab2018Inngår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 15, nr 8, s. 3394-3403Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Human epidermal growth factor receptor type 3 (HER3) is recognized to be involved in resistance to HER targeting therapies. A number of HER3-targeting monoclonal antibodies are under clinical investigation as potential cancer therapeutics. Smaller high-affinity scaffold proteins are attractive non-Fc containing alternatives to antibodies. A previous study indicated that anti-HER3 affibody molecules could delay the growth of xenografted HER3-positive tumors. Here, we designed a second-generation HER3-targeting construct (TAM-HER3), containing two HER3-specific affibody molecules bridged by an albumin-binding domain (ABD) for extension of blood circulation. Receptor blocking activity was demonstrated in vitro. In mice bearing BxPC-3 xenografts, the therapeutic efficacy of TAM-HER3 was compared to the HER3-specific monoclonal antibody seribantumab (MM-121). TAM-HER3 inhibited heregulin-induced phosphorylation in a panel of HER3-expressing cancer cells and was found to be equally as potent as seribantumab in terms of therapeutic efficacy in vivo and with a similar safety profile. Median survival times were 60 days for TAM-HER3, 54 days for seribantumab, and 41 days for the control group. No pathological changes were observed in cytopathological examination. The multimeric HER3-binding affibody molecule in fusion to ABD seems promising for further evaluation as candidate therapeutics for treatment of HER3-overexpressing tumors.

  • 98.
    Orlova, Anna
    et al.
    Affibody AB, SE-16102 Bromma, Sweden..
    Magnusson, Mikaela
    Affibody AB, SE-16102 Bromma, Sweden..
    Larsson, Barbro
    Affibody AB, SE-16102 Bromma, Sweden..
    Wikman, Maria
    KTH.
    Steffen, Ann-Charlott
    Inst Biomed Radiat Sci, Uppsala, Sweden. Fox Chase Canc Ctr, Dept Med Oncol, Philadelphia, PA 19111 USA..
    Nilsson, Martin
    Affibody AB, SE-16102 Bromma, Sweden..
    Ståhl, Stefan
    KTH, Tidigare Institutioner (före 2005), Bioteknologi. Royal Inst Technol, Stockholm, Sweden..
    Tolmachev, Vladimir
    Inst Biomed Radiat Sci, Uppsala, Sweden. Fox Chase Canc Ctr, Dept Med Oncol, Philadelphia, PA 19111 USA..
    Carlsson, Jorgen
    Inst Biomed Radiat Sci, Uppsala, Sweden. Fox Chase Canc Ctr, Dept Med Oncol, Philadelphia, PA 19111 USA..
    Nilsson, Fredrik
    Affibody AB, SE-16102 Bromma, Sweden..
    AFFIBODY (R) MOLECULES AS NEW POTENTIAL AGENTS FOR RADIONUCLIDE IMAGING AND THERAPY2004Inngår i: Anticancer Research, ISSN 0250-7005, E-ISSN 1791-7530, Vol. 24, nr 5D, s. 3686-3686Artikkel i tidsskrift (Annet vitenskapelig)
  • 99. Orlova, Anna
    et al.
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Lindberg, Hanna
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Varasteh, Zohreh
    Rosestedt, Maria
    Tolmachev, Vadimir
    Kronqvist, Nina
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Feasibility of radionuclide imaging of HER3-expressing tumors using affibody molecules2013Inngår i: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 56, s. S11-S11Artikkel i tidsskrift (Annet vitenskapelig)
  • 100. Orlova, Anna
    et al.
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Rosestedt, Maria
    Varasteh, Zohreh
    Andersson, Ken
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Selvaraju, Ram Kumar
    Altai, Mohamed
    Honarvar, Hadis
    Strand, Joanna
    Stahl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Tolmachev, Vladimir
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Imaging of HER3-expressing xenografts in mice using a Tc-99m(CO)(3)-HEHEHE-Z(HER3:08699) affibody molecule2014Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, nr 7, s. 1450-1459Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Human epidermal growth factor receptor type 3 (HER3) is a transmembrane receptor tyrosine kinase belonging to the HER (ErbB) receptor family. Membranous expression of HER3 is associated with trastuzumab resistance in breast cancer and the transition to androgen independence in prostate cancer. Imaging of HER3 expression in malignant tumors may provide important diagnostic information that can influence patient management. Affibody molecules with low picomolar affinity to HER3 were recently selected. The aim of this study was to investigate the feasibility of HER3 imaging using radiolabeled Affibody molecules. A HER3-binding Affibody molecule, Z(08699), with a HEHEHE-tag on N-terminus was labeled with Tc-99m(CO)(3) using an IsoLink kit. In vitro and in vivo binding specificity and the cellular processing of the labeled binder were evaluated. Biodistribution of Tc-99m(CO)(3)-HEHEHE-Z(08699) was studied over time in mice bearing HER3-expressing xenografts. HEHEHE-Z(08699) was labeled with Tc-99m(CO)(3) with an isolated yield of > 80 % and a purity of > 99 %. Binding of Tc-99m(CO)(3)-HEHEHE-Z(08699) was specific to BT474 and MCF7 (breast cancer), and LS174T (colon cancer) cells. Cellular processing showed rapid binding and relatively quick internalization of the receptor/Affibody molecule complex (70 % of cell-associated radioactivity was internalized after 24 h). The tumor targeting was receptor mediated and the excretion was predominantly renal. Receptor-mediated uptake was also found in the liver, lung, stomach, intestine, and salivary glands. At 4 h pi, tumor-to-blood ratios were 7 +/- 3 for BT474, and 6 +/- 2 for LS174T xenografts. LS174T tumors were visualized by microSPECT 4 h pi. The results of this study suggest the feasibility of HER3-imaging in malignant tumors using Affibody molecules.

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