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  • 1.
    Grönwall, Caroline
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Affibody molecules for proteomic and therapeutic applications2008Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    This thesis describes generation and characterization of Affibody molecules with future applications in proteomics research, protein structure determinations, therapeutic treatment of disease and medical imaging for in vivo diagnostics. Affibody molecules are engineered affinity proteins developed by combinatorial protein engineering from the 58-residue protein A-derived Z domain scaffold. Novel Affibody molecules targeting human proteins were selected from a combinatorial library using phage display technology.

    In the first two investigations, an Affibody molecule specifically targeting the high abundant human serum protein transferrin was generated. The intended future use of this Affibody ligand would be as capture ligand for depletion of transferrin from human samples in proteomics analysis. Strong and highly specific transferrin binding of the selected Affibody molecule was demonstrated by biosensor technology, dot blot analysis and affinity chromatography. Efficient Affibody-mediated depletion of transferrin in human plasma and cerebrospinal fluid (CSF) was demonstrated in combination with IgG and HSA removal. Furthermore, depletion of five high abundant proteins including transferrin from human CSF gave enhanced identification of proteins in a shotgun proteomics analysis.

    Two studies involved the selection and characterization of Affibody molecules recognizing Alzheimer’s amyloid beta (Abeta) peptides. Future prospect for the affinity ligands would primarily be for therapeutic applications in treatment of Alzheimer’s disease. The developed A-binding Affibody molecules were found to specifically bind to non-aggregated forms of Abeta and to be capable of efficiently and selectively capture Abeta peptides from spiked human serum. Interestingly, the Abeta-binding Affibody ligands were found to bind much better to Abeta as dimeric constructs, and with impressive affinity as cysteine-bridged dimers (KD~17 nM). NMR spectroscopy studies revealed that the original helix one, of the two Affibody molecules moieties of the cysteine-bridged dimers, was unfolded upon binding, forming intermolecular β-sheets that stabilized the Abeta peptide, enabling a high resolution structure of the peptide. Furthermore, the Abeta-binding Affibody molecules were found to inhibit Abeta fibrillation in vitro.

    In the last study, Affibody molecules directed to the interleukin 2 (IL-2) receptor alpha (CD25) were generated. CD25-binding Affibody molecules could potentially have a future use in medical imaging of inflammation, and possibly in therapeutic treatment of disease conditions with CD25 overexpression. The selected Affibody molecules were demonstrated to bind specifically to human CD25 with an apparent affinity of 130-240 nM. Moreover, the CD25-targeting Affibody molecules were found to have overlapping binding sites with the natural ligand IL-2 and an IL-2 blocking monoclonal antibody. Furthermore, the Affibody molecules demonstrated selective binding to CD25 expressing cells.

  • 2.
    Grönwall, Caroline
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Jonsson, Andreas
    Affibody AB, Bromma.
    Lindström, Sara
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Gunneriusson, Elin
    Affibody AB, Bromma.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Herne, Nina
    Affibody AB, Bromma.
    Selection and characterization of Affibody ligands binding to Alzheimer amyloid beta peptides2007In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 128, no 1, p. 162-183Article in journal (Refereed)
    Abstract [en]

    Affibody (Affibody) ligands specific for human amyloid beta (Abeta) peptides (40 or 42 amino acid residues in size), involved in the progress of Alzheimer's disease, were selected by phage display technology from a combinatorial protein library based on the 58-amino acid residue staphylococcal protein A-derived Z domain. Post-selection screening of 384 randomly picked clones, out of which 192 clones were subjected to DNA sequencing and clustering, resulted in the identification of 16 Affibody variants that were produced and affinity purified for ranking of their binding properties. The two most promising Affibody variants were shown to selectively and efficiently bind to Abeta peptides, but not to the control proteins. These two Affibody ligands were in dimeric form (to gain avidity effects) coupled to affinity resins for evaluation as affinity devices for capture of Abeta peptides from human plasma and serum. It was found that both ligands could efficiently capture Abeta that were spiked (100 microgml(-1)) to plasma and serum samples. A ligand multimerization problem that would yield suboptimal affinity resins, caused by a cysteine residue present at the binding surface of the Affibody ligands, could be circumvented by the generation of second-generation Affibody ligands (having cysteine to serine substitutions). In an epitope mapping effort, the preferred binding site of selected Affibody ligands was mapped to amino acids 30-36 of Abeta, which fortunately would indicate that the Affibody molecules should not bind the amyloid precursor protein (APP). In addition, a significant effort was made to analyze which form of Abeta (monomer, dimer or higher aggregates) that was most efficiently captured by the selected Affibody ligand. By using Western blotting and a dot blot assay in combination with size exclusion chromatography, it could be concluded that selected Affibody ligands predominantly bound a non-aggregated form of analyzed Abeta peptide, which we speculate to be dimeric Abeta. In conclusion, we have successfully selected Affibody ligands that efficiently capture Abeta peptides from human plasma and serum. The potential therapeutic use of these optimized ligands for extracorporeal capture of Abeta peptides in order to slow down or reduce amyloid plaque formation, is discussed.

  • 3.
    Grönwall, Caroline
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology. KTH, School of Biotechnology (BIO), Proteomics.
    Sjöberg, Anna
    Affibody AB, Bromma.
    Ramström, Margareta
    KTH, School of Biotechnology (BIO), Molecular Biotechnology. KTH, School of Biotechnology (BIO), Proteomics.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Bromma.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Molecular Biotechnology. KTH, School of Biotechnology (BIO), Proteomics.
    Jonasson, Per
    Affibody AB, Bromma.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology. KTH, School of Biotechnology (BIO), Proteomics.
    Affibody-mediated transferrin depletion for proteomics applications2007In: Biotechnology Journal, ISSN 1860-6768, Vol. 2, no 11, p. 1389-1398Article in journal (Refereed)
    Abstract [en]

    An Affibody® (Affibody) ligand with specific binding to human transferrin was selected by phage display technology from a combinatorial protein library based on the staphylococcal protein A (SpA)-derived Z domain. Strong and selective binding of the selected Affibody ligand to transferrin was demonstrated using biosensor technology and dot blot analysis. Impressive specificity was demonstrated as transferrin was the only protein recovered by affinity chromatography from human plasma. Efficient Affibody-mediated capture of transferrin, combined with IgG- and HSA-depletion, was demonstrated for human plasma and cerebrospinal fluid (CSF). For plasma, 85% of the total transferrin content in the samples was depleted after only two cycles of transferrin removal, and for CSF, 78% efficiency was obtained in single-step depletion. These results clearly suggest a potential for the development of Affibody-based resins for the removal of abundant proteins in proteomics analyses.

  • 4.
    Grönwall, Caroline
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Snelders, Eveline
    Department of Oncology and Pathology, Cancer Center Karolinska (CCK), Karolinska Hospital.
    Jarelöv Palm, Anna
    Eriksson, Fredrik
    Department of Oncology and Pathology, Cancer Center Karolinska (CCK), Karolinska Hospital.
    Herne, Nina
    Affibody AB, Bromma.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Generation of Affibody (R) ligands binding interieukin-2 receptor alpha/CD252008In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 50, no 2, p. 97-112Article in journal (Refereed)
    Abstract [en]

    Affibody (R) molecules specific for human IL-2R alpha, the IL-2 (interieukin-2) receptor a subunit, also known as CD25, were selected by phage-display technology from a combinatorial protein library based on the 58-residue Protein A-derived Z domain. The IL-2R system plays a major role in T-cell activation and the regulation of cellular immune responses. Moreover, CD25 has been found to be overexpressed in organ rejections, a number of autoimmune diseases and T-cell malignancies. The phage-display selection using Fc-fused target protein generated 16 unique Affibody (R) molecules targeting CD25. The two most promising binders were characterized in more detail using biosensor analysis and demonstrated strong and selective binding to CD25. Kinetic biosensor analysis revealed that the two monomeric Affibody (R) molecules bound to CD25 with apparent affinities of 130 and 240 nM respectively. The Affibody (R) molecules were, on biosensor analysis, found to compete for the same binding site as the natural ligand IL-2 and the IL-2 blocking monoclonal antibody 2A3. Hence the Affibody (R) molecules were assumed to have an overlapping binding site with IL-2 and antibodies targeting the IL-2 blocking Tac epitope (for example, the monoclonal antibodies Daclizumab and Basiliximab, both of which have been approved for therapeutic use). Furthermore, immunofluorescence microscopy and flow-cytometric analysis of CD25-expressing cells demonstrated that the selected Affibody (R) molecules bound to CD4(+) CD25(+) PMBCs (peripheral-blood mononuclear cells), the IL-2-dependent cell line NK92 and phytohaemagglutinin-activated PMBCs. The potential use of the CD25-binding Affibody (R) molecules as targeting agents for medical imaging and for therapeutic applications is discussed.

  • 5.
    Grönwall, Caroline
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Engineered affinity proteins-Generation and applications2009In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 140, no 3-4, p. 254-269Article, review/survey (Refereed)
    Abstract [en]

    The use of combinatorial protein engineering to design proteins with novel binding specificities and desired properties has evolved into a powerful technology, resulting in the recent advances in protein library selection strategies and the emerge of a variety of new engineered affinity proteins. The need for different protein library selection methods is due to that each target protein pose different challenges in terms of its availability and inherent properties. At present, alternative engineered affinity proteins are starting to complement and even challenge the classical immunoglobulins in different applications in biotechnology and potentially also for in vivo use as imaging agents or as biotherapeutics. This review article covers the generation and use of affinity proteins generated through combinatorial protein engineering. The most commonly used selection techniques for isolation of desired variants from large protein libraries are described. Different antibody derivatives, as well as a variety of the most validated engineered protein scaffolds, are discussed. In addition, we provide an overview of some of the major present and future applications for these engineered affinity proteins in biotechnology and medicine.

  • 6.
    Hoyer, Wolfgang
    et al.
    Department of Medical Biochemistry, Swedish Nuclear Magnetic Resonance Center, University of Gothenburg.
    Grönwall, Caroline
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Jonsson, Andreas
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    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 formation2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 13, p. 5099-5104Article in journal (Refereed)
    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.

  • 7. Lundin, Marika
    et al.
    Lindström, Hannah
    Grönwall, Caroline
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Persson, Mats A. A.
    Dual topology of the processed hepatitis C virus protein NS4B is influenced by the NS5A protein2006In: Journal of General Virology, ISSN 0022-1317, E-ISSN 1465-2099, Vol. 87, p. 3263-3272Article in journal (Refereed)
    Abstract [en]

    Among the least-known hepatitis C virus proteins is the non-structural protein 4B (NS4B). It localizes to the endoplasmic reticulum (ER) membrane and induces membrane changes, resulting in a membranous web that is reported to be the locale for virus replication. A model was presented previously for the topology of recombinant HCV NS4B of the 1 a genotype based on in vitro data. In this model, the N-terminal tail of a considerable fraction of the NS4B molecules was translocated into the ER lumen via a post-translational process, giving the protein a dual transmembrane topology. It is now reported that translocation of the N terminus also occurs for processed NS4B expressed in cells in the context of the polyprotein. In the presence of NS5A, however, a lower degree of translocation was observed, which may indicate that NS5A influences the topology of NS4B. In vitro expression studies of NS4B from all major genotypes demonstrated that translocation of the N terminus to the ER lumen is conserved across genotypes. This clearly suggests an important function for this feature. Furthermore, when disrupting a previously reported amphipathic helix (AH) in the N terminus of NS4B, translocation was inhibited. As a disrupted AH also abolished the ability of NS4B to rearrange membranes, these data indicate for the first time an association between translocation of the N terminus and membrane rearrangement. Finally, the present experiments also confirm the predicted location of the first luminal loop to be around aa 112.

  • 8.
    Ramström, Margareta
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Zuberovic, Aida
    Analytical Chemistry, Department of Physical and Analytical Chemistry, Uppsala University.
    Grönwall, Caroline
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Hanrieder, Jörg
    Analytical Chemistry, Department of Physical and Analytical Chemistry, Uppsala University.
    Bergquist, Jonas
    Analytical Chemistry, Department of Physical and Analytical Chemistry, Uppsala University.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Development of affinity columns for the removal of high-abundance proteins in cerebrospinal fluid2009In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 52, no 2, p. 159-166Article in journal (Refereed)
    Abstract [en]

    Various approaches for removal of high-abundance components in body fluids are currently available. While most methods are constructed for plasma depletion, there is a need for body-fluid-specific strategies. The aim of the present study was to design an affinity matrix suitable for the depletion of high-abundance proteins in CSIF (cerebrospinal fluid). Hence, molecules with specific affinity towards proteins present at high concentration in CSIF were desired. Affibody(R) molecules are specific binders of small size that have shown high stability under various conditions and are therefore good candidates for such a matrix. The protein composition in CSF resembles that in plasma. However, 20 % of the proteins are brain-derived and are therefore present in higher proportions in CSF than in plasma, whereas larger plasma-derived proteins are less abundant in CSF. Therefore five high-abundance CSIF proteins were chosen for the design of a CSF-specific depletion setup. Affibody(R) molecules with specificity towards HSA (human serum albumin), IgG, transferrin and transthyretin were combined in an affinity column. In addition, polyclonal antibodies against cystatin C were coupled to chromatographic beads and packed in a separate column. Highly reproducible and efficient removal of the five target proteins was observed. The proportion of depleted proteins were estimated to be 99, 95, 74, 92 and 83 % for HSA, IgG, transferrin, transthyretin and cystatin C respectively. SDS/PAGE analysis was used for monitoring and identifying proteins in native CSF, depleted CSIF samples and the captured fractions. Moreover, shotgun proteomics was used for protein identification in native as well as depleted: CSIF and the achieved data were compared. Enhanced identification of lower abundance components was observed in the depleted fraction, in terms of more detected peptides per protein.

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