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  • 1.
    Akpe, Victor
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Vernet, Erik
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Characterization studies of aluminum phthalocyanine binding to antibodies from SKBR 3 cell line2008Report (Other academic)
  • 2. Akter, Farhima
    et al.
    Mie, Masayasu
    Grimm, Sebastian
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Kobatake, Eiry
    Detection of Antigens Using a Protein-DNA Chimera Developed by Enzymatic Covalent Bonding with phiX Gene A2012In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 84, no 11, p. 5040-5046Article in journal (Refereed)
    Abstract [en]

    The chemical reactions used to make antibody DNA conjugates in many immunoassays diminish antigen-binding activity and yield heterogeneous products. Here, we address these issues by developing an antibody-based rolling circle amplification (RCA) strategy using a fusion of phi X174 gene A* protein and Z(mab2s) (A*-Zmab). The phi X174 gene A* protein is an enzyme that can covalently link with DNA, while the Z(mab2s) protein moiety can bind to specific species of antibodies. The DNA in an A*-Zmab conjugate was attached to the A* protein at a site chosen to not interfere with protein function, as determined by enzyme-linked immunosorbent assay (ELISA) and gel mobility shift analysis. The novel A*-Zmab-DNA conjugate retained its binding capabilities to a specific class of murine immunoglobulin gamma 1 (IgG1) but not to rabbit IgG. This indicates the generality of the A*-Zmab-based immuno-RCA assay that can be used in-sandwich ELISA format. Moreover, the enzymatic covalent method dramatically increased the yields of A*-Zmab-DNA conjugates up to 80% after a 15 min reaction. Finally, sensitive detection of human interferon-gamma (IFN-gamma) was achieved by immuno-RCA using our fusion protein in sandwich ELISA format. This new approach of the use of site-specific enzymatic DNA conjugation to proteins should be applicable to fabrication of novel immunoassays for biosensing.

  • 3. Allerbring, E. B.
    et al.
    Duru, A. D.
    Uchtenhagen, H.
    Madhurantakam, C.
    Tomek, M. B.
    Grimm, Sebastian
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Mazumdar, P. A.
    Friemann, R.
    Uhlin, M.
    Sandalova, T.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Achour, A.
    Unexpected T-cell recognition of an altered peptide ligand is driven by reversed thermodynamics2012In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 42, no 11, p. 2990-3000Article in journal (Refereed)
    Abstract [en]

    The molecular basis underlying T-cell recognition of MHC molecules presenting altered peptide ligands is still not well-established. A hierarchy of T-cell activation by MHC class I-restricted altered peptide ligands has been defined using the T-cell receptor P14 specific for H-2D b in complex with the immunodominant lymphocytic choriomeningitis virus peptide gp33 (KAVYNFATM). While substitution of tyrosine to phenylalanine (Y4F) or serine (Y4S) abolished recognition by P14, the TCR unexpectedly recognized H-2D b in complex with the alanine-substituted semiagonist Y4A, which displayed the most significant structural modification. The observed functional hierarchy gp33 > Y4A > Y4S = Y4F was neither due to higher stabilization capacity nor to differences in structural conformation. However, thermodynamic analysis demonstrated that while recognition of the full agonist H-2D b/gp33 was strictly enthalpy driven, recognition of the weak agonist H-2D b/Y4A was instead entropy driven with a large reduction in the favorable enthalpy term. The fourfold larger negative heat capacity derived for the interaction of P14 with H-2D b/gp33 compared with H-2D b/Y4A can possibly be explained by higher water entrapment at the TCR/MHC interface, which is also consistent with the measured opposite entropy contributions for the interactions of P14 with both MHCs. In conclusion, this study demonstrates that P14 makes use of different strategies to adapt to structural modifications in the MHC/peptide complex.

  • 4. Altai, M.
    et al.
    Perols, Anna
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Sandström, M.
    Boschetti, F.
    Orlova, A.
    Tolmachev, V.
    Evaluation of a maleimido derivative of NODAGA for site-specific In-111-labeling of Affibody molecules2011In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 38, p. S146-S146Article in journal (Other academic)
  • 5. Altai, M.
    et al.
    Strand, J.
    Rosik, Daniel
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Selvaraju, R.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Orlova, A.
    Tolmachev, V.
    Comparative evaluation of anti-HER2 affibody molecules labeled with 68Ga and 111In using maleimido derivatives of DOTA and NODAGA.2012In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 39, p. S299-S299Article in journal (Refereed)
  • 6. Altai, Mohamed
    et al.
    Perols, Anna
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Sandström, Mattias
    Boschetti, Frederic
    Orlova, Anna
    Tolmachev, Vladimir
    Preclinical evaluation of anti-HER2 Affibody molecules site-specifically labeled with In-111 using a maleimido derivative of NODAGA2012In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 39, no 4, p. 518-529Article in journal (Refereed)
    Abstract [en]

    Introduction: Affibody molecules have demonstrated potential for radionuclide molecular imaging. The aim of this study was to synthesize and evaluate a maleimido derivative of the 1,4,7-triazacyclononane-l-glutaric acid-4,7-diacetic acid (NODAGA) for site-specific labeling of anti-HER2 Affibody molecule. Methods: The maleimidoethylmonoamide NODAGA (MMA-NODAGA) was synthesized and conjugated to Z(HER2:2395) Affibody molecule having a C-terminal cysteine. Labeling efficiency, binding specificity to and cell internalization by HER2-expressing cells of [In-111-MMA-NODAGA-Cys(61)]-Z(HER2:2395) were studied. Biodistribution of [In-111-MMA-NODAGA-Cys(61)]-Z(HER2:2395) and [In-111-MMA-DOTA-Cys(61)]-Z(HER2:2395) was compared in mice. Results: The affinity of [MMA-NODAGA-Cys(61)]-Z(HER2:2395) binding to HER2 was 67 pM. The In-1111-labeling yield was 99.6%+/- 0.5% after 30 min at 60 degrees C. [In-111-MMA-NODAGA-Cys(61)]-Z(HER2:2395) bound specifically to HER2-expressing cells in vitro and in vivo. Tumor uptake of [In-111-MMA-NODAGA-Cys(61)]-ZHER(2:2395) in mice bearing DU-145 xenografts (4.7%+/- 0.8% ID/g) was lower than uptake of [In-111-MMA-DOTA-Cys(61)]-Z(HER2:2395) (7.5%+/- 1.6% ID/g). However, tumor-to-organ ratios were higher for [In-111-MMA-NODAGA-Cys(61)]-Z(HER2:2395) due to higher clearance rate from normal tissues. Conclusions: MMA-NODAGA is a promising chelator for site-specific labeling of targeting proteins containing unpaired cysteine. Appreciable influence of chelators on targeting properties of Affibody molecules was demonstrated.

  • 7. Altai, Mohamed
    et al.
    Strand, Joanna
    Rosik, Daniel
    Selvaraju, Ram Kumar
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Orlova, Anna
    Tolmachev, Vladimir
    Influence of Nuclides and Chelators on Imaging Using Affibody Molecules: Comparative Evaluation of Recombinant Affibody Molecules Site-Specifically Labeled with Ga-68 and In-111 via Maleimido Derivatives of DOTA and NODAGA2013In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 24, no 6, p. 1102-1109Article in journal (Refereed)
    Abstract [en]

    Accurate detection of cancer-associated molecular abnormalities in tumors could make cancer treatment more of personalized. Affibody molecules enable high contrast imaging of tumor-associated protein expression shortly after injection. The use should increase sensitivity of HER2 imaging. The chemical nature of the generator-produced positron-emitting radionuclide Ga-68 of radionuclides and chelators influences the biodistribution of Affibody molecules, providing an opportunity to further increase the imaging contrast. The aim of the study was to compare maleimido derivatives of DOTA and NODAGA for site-specific labeling of a recombinant Z(HER2:2395) HER2-binding Affibody molecule with Ga-68. DOTA and NODAGA were site-specifically conjugated to the Z(HER2:2395) Affibody molecule having a C-terminal cysteine and labeled with Ga-68 and In-111. All labeled conjugates retained specificity to HER2 in vitro. Most of the cell-associated activity was membrane-bound with a minor difference in internalization rate. All variants demonstrated specific targeting of xenografts and a high tumor uptake. The xenografts were dearly visualized using all conjugates. The influence of chelator on the biodistribution and targeting properties was much less pronounced for Ga-68 than for In-111. The tumor uptake of Ga-68-NODAGA-Z(HER2:2395) and Ga-68-NODAGA-Z(HER2:2395) and tumor-to-blood ratios at 2 h p.i. did not differ significantly. However, the tumor-to-liver ratio was significantly higher for Ga-68-NODAGA- Z(HER2:2395) (8 +/- 2 vs 5.0 +/- 0.3) offering the advantage of better liver metastases visualization. In conclusion, influence of chelators on biodistribution of Affibody molecules depends on the radionuclides and reoptimization of labeling chemistry is required when a radionuclide label is changed.

  • 8. Altai, Mohamed
    et al.
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Orlova, Anna
    Rosestedt, Maria
    Hosseinimehr, Seyed Jalal
    Tolmachev, Vladimir
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of Tc-99m-labeled recombinant Affibody molecules2012In: Amino Acids, ISSN 0939-4451, E-ISSN 1438-2199, Vol. 42, no 5, p. 1975-1985Article in journal (Refereed)
    Abstract [en]

    Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide Tc-99m. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of Tc-99m-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied Tc-99m-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of Tc-99m. The biodistribution of all Tc-99m-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of Tc-99m was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.

  • 9.
    Angleby, Helen
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Forensic informativity of domestic dog mtDNA control region sequences2005In: Forensic Science International, ISSN 0379-0738, E-ISSN 1872-6283, Vol. 154, no 03-feb, p. 99-110Article in journal (Refereed)
    Abstract [en]

    We have analysed the genetic information to be obtained from analysis of mitochondrial DNA (mtDNA) in domestic dogs studying the exclusion capacity in different populations and the correlation between mtDNA types and breeds or types of dogs. The exclusion capacities for a 573 bp sequence of the mitochondrial control region was between 0.86 and 0.95 for dogs in Sweden, the UK, Germany, Japan and China. The direct correlation between mtDNA type and breed, type of dog, and geographical origin of breed was generally low, but in some cases certain mtDNA types were overrepresented in one breed, and for wider groupings such as morphologically similar breeds, some mtDNA types were in many cases found in a distinct group of breeds, often originating from the same geographic region. This type of information may be used as an indication of the breed and, with some degree of probability, to include or exclude certain breeds from being the source of evidence materials.

  • 10.
    Bandmann, Nina
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Combinatorial expression vector engineering for tuning of recombinant protein production in Escherichi coli2007In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, no 5Article in journal (Refereed)
    Abstract [en]

    The complex and integrated nature of both genetic and protein level factors influencing recombinant protein production in Escherichia coli makes it difficult to predict the optimal expression strategy for a given protein. Here, two combinatorial library strategies were evaluated for their capability of tuning recombinant protein production in the cytoplasm of E. coli. Large expression vector libraries were constructed through either conservative (ExLib1) or free (ExLib2) randomization of a seven-amino-acid window strategically located between a degenerated start codon and a sequence encoding a fluorescently tagged target protein. Flow cytometric sorting and analyses of libraries, subpopulations or individual clones were followed by SDS-PAGE, western blotting, mass spectrometry and DNA sequencing analyses. For ExLib1, intracellular accumulation of soluble protein was shown to be affected by codon specific effects at some positions of the common N-terminal extension. Interestingly, for ExLib2 where the same sequence window was randomized via seven consecutive NN(G/T) tri-nucleotide repeats, high product levels (up to 24-fold higher than a reference clone) were associated with a preferential appearance of novel SID-like sequences. Possible mechanisms behind the observed effects are discussed.

  • 11. Bjorklund, Marcus Gry
    et al.
    Natanaelsson, Christian
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Hao, Yong
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Microarray analysis using disiloxyl 70mer oligonucleotides2008In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 36, no 4, p. 1334-1342Article in journal (Refereed)
    Abstract [en]

    DNA microarray technology has evolved dramatically in recent years, and is now a common tool in researchers portfolios. The scope of the technique has expanded from small-scale studies to extensive studies such as classification of disease states. Technical knowledge regarding solid phase microarrays has also increased, and the results acquired today are more reliable than those obtained just a few years ago. Nevertheless, there are various aspects of microarray analysis that could be improved. In this article we show that the proportions of full-length probes used significantly affects the results of global analyses of transcriptomes. In particular, measurements of transcripts in low abundance are more sensitive to truncated probes, which generally increase the degree of cross hybridization and loss of specific signals. In order to improve microarray analysis, we here introduce a disiloxyl purification step, which ensures that all the probes on the microarray are at full length. We demonstrate that when the features on microarrays consist of full-length probes the signal intensity is significantly increased. The overall increase in intensity enables the hybridization stringency to be increased, and thus enhance the robustness of the results.

  • 12. Bourbeillon, Julie
    et al.
    Orchard, Sandra
    Benhar, Itai
    Borrebaeck, Carl
    de Daruvar, Antoine
    Duebel, Stefan
    Frank, Ronald
    Gibson, Frank
    Gloriam, David
    Haslam, Niall
    Hiltker, Tara
    Humphrey-Smith, Ian
    Hust, Michael
    Juncker, David
    Koegl, Manfred
    Konthur, Zoltan
    Korn, Bernhard
    Krobitsch, Sylvia
    Muyldermans, Serge
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Palcy, Sandrine
    Polic, Bojan
    Rodriguez, Henry
    Sawyer, Alan
    Schlapshy, Martin
    Snyder, Michael
    Stoevesandt, Oda
    Taussig, Michael J.
    Templin, Markus
    Uhlén, Matthias
    KTH, School of Biotechnology (BIO), Proteomics. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    van der Maarel, Silvere
    Wingren, Christer
    Hermjakob, Henning
    Sherman, David
    Minimum information about a protein affinity reagent (MIAPAR)2010In: Nature Biotechnology, ISSN 1087-0156, E-ISSN 1546-1696, Vol. 28, no 7, p. 650-653Article in journal (Other academic)
  • 13. Carlsson, Jörgen
    et al.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Eriksson, Tove
    Gunnariusson, Elin
    Nilsson, Fredrik
    Polypeptides having binding affinity for HER22003Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    A polypeptide is provided, which has a binding affinity for HER2 and which is related to a domain of staphylococcal protein A (SPA) in that the sequence of the polypeptide corresponds to the sequence of the SPA domain having from 1 to about 20 substitution mutations. Nucleic acid encoding the polypeptide, as well as expression vector and host cell for expressing the nucleic acid, are also provided. Also provided is the use of such a polypeptide as a medicament, and as a targeting agent for directing substances conjugated thereto to cells overexpressing HER2. Methods, and kits for performing the methods, are also provided, which methods and kits rely on the binding of the polypeptide to HER2.

  • 14. Chen, Si
    et al.
    Jokilaakso, Nima
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Björk, Per
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Zhang, Shi-Li
    A two-terminal silicon nanoribbon field-effect pH sensor2010In: Applied Physics Letters, ISSN 0003-6951, E-ISSN 1077-3118, Vol. 97, no 26, p. 264102-Article in journal (Refereed)
    Abstract [en]

    This paper reports on a two-terminal silicon nanoribbon (SiNR) field-effect pH sensor operated in electrolyte. Observed experimentally and confirmed by modeling, the sensor is activated by self-gating with a gate bias set by the potential difference of the two terminals. The effect of this gate bias on the SiNR conductance is modulated by the potential drop over the electrical double layer (EDL) established on the SiNR surface, similarly to the threshold voltage modulation by EDL in a three-terminal SiNR field-effect transistor with an independent gate electrode. The potential drop over EDL is determined by the pH value of the electrolyte.

  • 15. Chen, Si
    et al.
    Nyholm, Leif
    Jokilaakso, Nima
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Karlström, Amelie Eriksson
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Linnros, Jan
    KTH, School of Information and Communication Technology (ICT), Material Physics.
    Smith, Ulf
    Zhang, Shi-Li
    Current Instability for Silicon Nanowire Field-Effect Sensors Operating in Electrolyte with Platinum Gate Electrodes2011In: Electrochemical and solid-state letters, ISSN 1099-0062, E-ISSN 1944-8775, Vol. 14, no 7, p. J34-J37Article in journal (Refereed)
    Abstract [en]

    Current instability is observed for silicon nanowire field-effect transistors operating in electrolytes with Pt gate electrodes. A comparative study involving an Ag/AgCl-reference gate electrode reveals that the effect results from a drift in the potential at the Pt-electrode/electrolyte interface. In a phosphate buffer saline of pH 7.4, the stabilization of the potential of the Pt electrode was found to require approximately 1000 s. A concurrent potential drift, with a comparable time constant, occurring at the electrolyte/oxidized-nanowire interface rendered a complex device current response which complicated the interpretation of the results.

  • 16.
    Dogan, Jakob
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Structural and thermodynamical basis for molecular recognition between engineered binding proteins2006Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    The structural determination of interacting proteins, both as individual proteins and in their complex, complemented by thermodynamical studies are vital in order to gain in-depth insights of the phenomena leading to the highly selective protein-protein interactions characteristic of numerous life processes. This thesis describes an investigation of the structural and thermodynamical basis for molecular recognition in two different protein-protein complexes, formed between so-called affibody proteins and their respective targets. Affibody proteins are a class of engineered binding proteins, which can be functionally selected for binding to a given target protein from large collections (libraries) constructed via combinatorial engineering of 13 surface-located positions of the 58-residue three-helix bundle Z domain derived from Staphylococcal protein (SPA).

    In a first study, an affibody:target protein pair consisting of the ZSPA-1 affibody and the parental Z domain, with a dissociation constant (Kd) of approximately 1 µM, was investigated. ZSPA-1 was in its free state shown to display molten globule-like characteristics. The enthalpy change on binding between Z and ZSPA-1 as measured by isothermal titration calorimetry, was found to be a non-linear function of temperature. This nonlinearity was found to be due to the temperature dependent folded-unfolded equilibrium of ZSPA-1 upon binding to the Z domain and, the energetics of the unfolding equilibrium of the molten globule state of ZSPA-1 could be separated from the binding thermodynamics. Further dissection of the binding entropy revealed that a significant reduction in conformational entropy resulting from the stabilization of the molten globule state of ZSPA-1 upon complex formation could be a major reason for the moderate binding affinity.

    A second studied affibody:target complex (Kd ~ 0.1 µM) consisted of the ZTaq affibody protein originally selected for binding to Taq DNA polymerase and the anti-ZTaq affibody protein, selected for selective binding to the ZTaq affibody protein, thus constituting an "anti-idiotypic" affinity protein pair. The structure of the ZTaq:anti-ZTaq affibody complex as well as the free state structures of ZTaq and anti-ZTaq were determined using NMR spectroscopy. Both ZTaq and anti-ZTaq are well defined three helix bundles in their free state and do not display the same molten globule-like behaviour of ZSPA-1. The interaction surface was found to involve all of the varied positions in helices 1 and 2 of the anti-ZTaq, the majority of the corresponding side chains in ZTaq, and also several non-mutated residues. The total buried surface area was determined to about 1670 Å2 which is well inside the range of what is typical for many protein-protein complexes, including antibody:antigen complexes. Structural rearrangements, primarily at the side chain level, were observed to take place upon binding. There are similarities between the ZTaq:anti-ZTaq and the Z:ZSPA-1 structure, for instance, the binding interface area in both complexes has a large fraction of non-polar content, the buried surface area is of similar size, and certain residues have the same positioning. However, the relative orientation between the subunits in ZTaq:anti-ZTaq is markedly different from that observed in Z:ZSPA-1. The thermodynamics of ZTaq:anti-ZTaq association were investigated by isothermal titration calorimetry. A dissection of the entropic contributions showed that a large and favourable desolvation entropy of non-polar surface is associated with the binding reaction which is in good agreement with hydrophobic nature of the binding interface, but as in the case for the Z:ZSPA-1 complex a significant loss in conformational entropy opposes complex formation.

    A comparison with complexes involving affibody proteins or SPA domains suggests that affibody proteins inherit intrinsic binding properties from the original SPA surface. The structural and biophysical data suggest that although extensive mutations are carried out in the Z domain to obtain affibody proteins, this does not necessarily affect the structural integrity or lead to a significant destabilization.

  • 17.
    Dogan, Jakob
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Lendel, Christofer
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Härd, Torleif
    Thermodynamics of folding and binding in an affibody:affibody complex2006In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 359, no 5, p. 1305-1315Article in journal (Refereed)
    Abstract [en]

    Affibody binding proteins are selected from phage-displayed libraries of variants of the 58 residue Z domain. Z(Taq) is an affibody originally selected as a binder to Taq DNA polymerase. The anti-Z(Taq) affibody was selected as a binder to Z(Taq) and the Z(Taq):anti-Z(Taq) complex is formed with a dissociation constant K-d = 0.1 mu M. We have determined the structure of the Z(Taq):anti-Z(Taq) complex as well as the free state structures of Z(Taq) and anti-Z(Taq) using NMR. Here we complement the structural data with thermodynamic studies of Z(Taq) and anti-Z(Taq) folding and complex formation. Both affibody proteins show cooperative two-state thermal denaturation at melting temperatures T-M similar to 56 degrees C. Z(Taq):anti-Z(Taq) complex formation at 25 degrees C in 50 mM NaCl and 20 mM phosphate buffer (pH 6.4) is enthalpy driven with Delta H degrees(bind) = -9.0(+/- 0.1) kcal mol(-1). The heat capacity change Delta C-P degrees,(bind) = -0.43(+/- 0.01) kcal mol(-1) K-1 is in accordance with the predominantly non-polar character of the binding surface, as judged from calculations based on changes in accessible surface areas. A further dissection of the small binding entropy at 25 degrees C (-T Delta S degrees(bind) = -0.6(+/- 0.1) kcal mol(-1)) suggests that a favourable desolvation of non-polar surface is almost completely balanced by unfavourable conformational entropy changes and loss of rotational and translational entropy. Such effects can therefore be limiting for strong binding also when interacting protein components are stable and homogeneously folded. The combined structure and thermodynamics data suggest that protein properties are not likely to be a serious limitation for the development of engineered binding proteins based on the Z domain.

  • 18.
    Ekblad, Torun
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Chemical Synthesis of Affibody Molecules for Protein Detection and Molecular Imaging2008Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Proteins are essential components in most processes in living organisms. The detection and quantification of specific proteins can be used e.g. as measures of certain physiological conditions, and are therefore of great importance. This thesis focuses on development of affinity-based bioassays for specific protein detection. The use of Affibody molecules for specific molecular recognition has been central in all studies in this thesis. Affibody molecules are affinity proteins developed by combinatorial protein engineering of the 58-residue protein A-derived Z domain scaffold. In the first paper, solid phase peptide synthesis is investigated as a method to generate functional Affibody molecules. Based on the results from this paper, chemical synthesis has been used throughout the following papers to produce Affibody molecules tailored with functional groups for protein detection applications in vitro and in vivo.

     

    In paper I, an orthogonal protection scheme was developed to enable site-specific chemical introduction of three different functional probes into synthetic Affibody molecules. Two of the probes were fluorophores that were used in a FRET-based binding assay to detect unlabeled target proteins. The third probe was biotin, which was used as an affinity handle for immobilization onto a solid support. In paper II, a panel of Affibody molecules carrying different affinity handles were synthesized and evaluated as capture ligands on microarrays. Paper III describes the synthesis of an Affibody molecule that binds to the human epidermal growth factor receptor type 2, (HER2), and the site-specific incorporation of a mercaptoacetyl-glycylglycylglycine (MAG3) chelating site in the peptide sequence to allow for radiolabeling with 99mTc. The derivatized Affibody molecule was found to retain its binding capacity, and the 99mTc-labeling was efficient and resulted in a stable chelate formation. 99mTc-labeled Affibody molecules were evaluated as in vivo HER2-targeting imaging agents in mice. In the following studies, reported in papers IV-VI, the 99mTc-chelating sequence was engineered in order to optimize the pharmacokinetic properties of the radiolabeled Affibody molecules and allow for high-contrast imaging of HER2-expressing tumors and metastatic lesions. The main conclusion from these investigations is that the biodistribution of Affibody molecules can be dramatically modified by amino acid substitutions directed to residues in the MAG3-chelator. Finally, paper VII is a report on the chemical synthesis and chemoselective ligation to generate a cross-linked HER2-binding Affibody molecule with improved thermal stability and tumor targeting capacity.

     

    Taken together, the studies presented in this thesis illustrate how peptide synthesis can be used for production and modification of small affinity proteins, such as Affibody molecules for protein detection applications.

  • 19.
    Ekblad, Torun
    et al.
    KTH, School of Biotechnology (BIO).
    Orlova, Anna
    Feldwisch, Joachim
    Wennborg, Anders
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Tolmachev, Vladimir
    Positioning of Tc-99m-chelators influences radiolabeling, stability and biodistribution of Affibody molecules2009In: Bioorganic & Medicinal Chemistry Letters, ISSN 0960-894X, E-ISSN 1090-2120, Vol. 19, no 14, p. 3912-3914Article in journal (Refereed)
    Abstract [en]

    Affibody molecules represent a novel class of affinity proteins with a high potential as tracers for radio-nuclide molecular imaging. In this comparative structure-property study, a series of Affibody molecules with the Tc-99m-chelators maGGG, maSSS, or maESE attached to the e-amine of the internally positioned K49 was prepared by peptide synthesis, for comparison to molecules with similar chelators positioned at the N-terminus. The conjugates were labeled with Tc-99m and evaluated in vitro and in vivo. It was found that both composition and position of the chelating moiety influence the label stability, biodistribution and targeting properties of HER2-binding Affibody molecules.

  • 20.
    Ekblad, Torun
    et al.
    KTH, School of Biotechnology (BIO).
    Tolmachev, Vladimir
    Uppsala Univ, Rudbeck Lab, Unit Biomed Radiat Sci.
    Orlova, Anna
    Uppsala Univ, Rudbeck Lab, Unit Biomed Radiat Sci.
    Lendel, Christofer
    Univ Cambridge, Dept Chem.
    Abrahmsén, Lars
    Affibody AB.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Synthesis and chemoselective intramolecular crosslinking of a HER2-binding Affibody2009In: Biopolymers, ISSN 0006-3525, E-ISSN 1097-0282, Vol. 92, no 2, p. 116-123Article in journal (Refereed)
    Abstract [en]

    The human epidermal growth factor receptor HER2 has emerged as an important target for molecular imaging of breast cancer. This article presents the design and synthesis of a HER2-targeting affibody molecule with improved stability and tumor targeting capacity and with potential use as an imaging agent. The 58 aa three-helix bundle protein was assembled using solid-phase peptide synthesis, and a chemoselective ligation strategy was used to establish an intramolecular thioether bond between the side chain thiol group of a cysteine residue, positioned in the loop between helices I and II, and a chloroacetyl group on the side chain amino group of the C-terminal lysine residue. The tethered protein offered an increased thermal stability, with a melting temperature of 64 degrees C, compared to 54 degrees C for the linear control. The ligation did not have a major influence on the HER2 binding affinity, which was 320 and 380 pM for the crosslinked and linear molecules, respectively. Biodistribution studies were performed both in normal and tumor-bearing mice to evaluate the impact of the crosslinking on the in vivo behavior and on the tumor targeting performance. The distribution pattern was characterized by a low uptake in all organs except kidney, and rapid clearance from blood and normal tissue. Crosslinking of the protein resulted in a significantly increased tumor accumulation, rendering the tethered HER2-binding affibody molecule a valuable lead in the development of superior HER2 imaging agents.

  • 21. Ekerljung, L.
    et al.
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Sohrabian, A.
    Andersson, K.
    Friedman, M.
    Frejd, F. Y.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Gedda, L.
    Generation and evaluation of bispecific affibody molecules for simultaneous targeting of EGFR and HER22012In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 23, no 9, p. 1802-1811Article in journal (Refereed)
    Abstract [en]

    Coexpression of several ErbB receptors has been found in many cancers and has been linked with increased aggressiveness of tumors and a worse patient prognosis. This makes the simultaneous targeting of two surface receptors by using bispecific constructs an increasingly appreciated strategy. Here, we have generated six such bispecific targeting proteins, each comprising two monomeric affibody molecules with specific binding to either of the two human epidermal growth factor receptors, EGFR and HER2, respectively. The bispecific constructs were designed with (i) alternative positioning (N- or C-terminal) of the different affibody molecules, (ii) two alternative peptide linkers (Gly 4Ser) 3 or (Ser 4Gly) 3, and (iii) affibody molecules with different affinity (nanomolar or picomolar) for HER2. Using both Biacore technology and cell binding assays, it was demonstrated that all six constructs could bind simultaneously to both their target proteins. N-terminal positioning of the inherent monomeric affibody molecules was favorable to promote the binding to the respective target. Interestingly, bispecific constructs containing the novel (Ser 4Gly) 3 linker displayed a higher affinity in cell binding, as compared to constructs containing the more conventional linker, (Gly 4Ser) 3. It could further be concluded that bispecific constructs (but not the monomeric affibody molecules) induced dimer formation and phosphorylation of EGFR in SKBR3 cells, which express fairly high levels of both receptors. It was also investigated whether the bispecific binding would influence cell growth or sensitize cells for ionizing radiation, but no such effects were observed.

  • 22.
    Engfeldt, Torun
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Orlova, Anna
    Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Tran, Thuy
    Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Bruskin, Alexander
    Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Widström, Charles
    Department of Hospital Physics, Uppsala University Hospital.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Tolmachev, Vladimir
    Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Imaging of HER2-expressing tumours using a synthetic Affibody molecule containing the 99mTc-chelating mercaptoacetyl-glycyl-glycyl-glycyl (MAG3) sequence2007In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 34, no 5, p. 722-733Article in journal (Refereed)
    Abstract [en]

    Purpose  Expression of human epidermal growth factor receptor type 2 (HER2) in malignant tumours possesses well-documented prognostic and predictive value. Non-invasive imaging of expression can provide valuable diagnostic information, thereby influencing patient management. Previously, we reported a phage display selection of a small (about 7 kDa) protein, the Affibody molecule ZHER2:342, which binds HER2 with subnanomolar affinity, and demonstrated the feasibility of targeting of HER2-expressing xenografts using radioiodinated ZHER2:342. The goal of this study was to develop a method for 99mTc labelling of ZHER2:342 using the MAG3 chelator, which was incorporated into ZHER2:342 using peptide synthesis, and evaluate the targeting properties of the labelled conjugate. Methods  MAG3-ZHER2:342 was assembled using Fmoc/tBu solid phase peptide synthesis. Biochemical characterisation of the agent was performed using RP-HPLC, ESI-MS, biosensor studies and circular dichroism. A procedure for 99mTc labelling in the presence of sodium/potassium tartrate was established. Tumour targeting was evaluated by biodistribution study and gamma camera imaging in xenograft-bearing mice. Biodistribution of 99mTc-MAG3-ZHER2:342 and 125I-para-iodobenzoate -ZHER2:342 was compared 6 h p.i. Results  Synthetic MAG3-ZHER2:342 possessed an affinity of 0.2 nM for HER2 receptors. The peptide was labelled with 99mTc with an efficiency of about 75–80%. Labelled 99mTc-MAG3-ZHER2:342 retained capacity to bind specifically HER2-expressing SKOV-3 cells in vitro. 99mTc-MAG3-ZHER2:342 showed specific tumour targeting with a contrast similar to a radioiodinated analogue in mice bearing LS174T xenografts. Gamma camera imaging demonstrated clear and specific visualisation of HER2 expression. Conclusion  Incorporation of a mercaptoacetyl-containing chelating sequence during chemical synthesis enabled site-specific 99mTc labelling of the ZHER2:342 Affibody molecule with preserved targeting capacity.

  • 23. Falk, Ronny
    et al.
    Ramstrom, Margareta
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Approaches for systematic proteome exploration2007In: Biomolecular Engineering, ISSN 1389-0344, E-ISSN 1878-559X, Vol. 24, no 2, p. 155-168Article, review/survey (Refereed)
    Abstract [en]

    With the completion of the human genome project (HUGO) during recent years, gene function, protein abundance and expression patterns in tissues and cell types have emerged as central areas for the scientific community. A mapped human proteome will extend the value of the genome sequence and large-scale efforts aiming at elucidating protein localization, abundance and function are invaluable for biomarker and drug discovery. This research area, termed proteomics, is more demanding than any genome sequencing effort and to perform this on a wide scale is a highly diverse task. Therefore, the proteornics field employs a range of methods to examine different aspects of proteomics including protein localization, protein-protein interactions, posttranslational modifications and alteration of protein composition (e.g. differential expression) in tissues and body fluids. Here, some of the most commonly used methods, including chromatographic separations together with mass spectrometry and a number of affinity proteomics concepts are discussed and exemplified.

  • 24.
    Falk, Ronny
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ramström, Margareta
    KTH, School of Biotechnology (BIO), Proteomics.
    Eriksson, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Wernérus, Henrik
    KTH, School of Biotechnology (BIO), Proteomics.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Targeted protein pullout from human tissue samples using competitive elution2011In: Biotechnology Journal, ISSN 1860-6768, Vol. 6, no 1, p. 28-37Article in journal (Refereed)
    Abstract [en]

    One commonly used strategy to gain information on the proteins in a cell is to isolate the proteins of interest by specific binders, often antibodies. Not only the specificity of the capturing antibodies but also the washing and elution conditions are crucial to avoid false-positive protein identifications. Eluting the target protein from the matrix, while avoiding the release of unrelated background proteins, should both provide more correct information on the target protein and its interaction partners, and minimize the effort to perform downstream analyses through the reduced number of eluted proteins. In this study, a novel approach for selective protein pullout is presented. Monospecific antibodies were used to selectively pullout target proteins from a complex biosample. Subsequently, the target proteins were competitively eluted from the affinity media with the recombinant antigen. To deplete the antigen from the eluted sample, I MAC spin columns were utilized to bind the N-terminal His-tag of the antigens. The competitive elution method was applied both to a model system, and for the extraction of a native human target protein. In the model system the recombinant target protein BBC7 was spiked into a protein extract of human liver, whereas an endogenously expressed target protein, cTAGE5, was extracted from the liver extract directly. SDS-PAGE analysis and mass spectrometry confirmed affinity isolation of expected target proteins. More selective elution was obtained using the competitive procedure as compared to elution at low pH. Competitive elution has thus been shown to offer an effective approach for wide-scale pullout experiments where proteins and their interaction partners are to be studied.

  • 25.
    Fleetwood, Filippa
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Devoogdt, Nick
    Pellis, Mireille
    Wernery, Ulrich
    Muyldermans, Serge
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Surface display of a single-domain antibody library on Gram-positive bacteria2013In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 70, no 6, p. 1081-1093Article in journal (Refereed)
    Abstract [en]

    Combinatorial protein engineering for selection of proteins with novel functions, such as enzymes and affinity reagents, is an important tool in biotechnology, drug discovery, and other biochemical fields. Bacterial display is an emerging technology for isolation of new affinity proteins from such combinatorial libraries. Cells have certain properties that are attractive for directed evolution purposes, in particular the option to use quantitative flow-cytometric cell sorting for selection of binders. Here, an immune library of around 10(7) camelid single-domain antibody fragments (Nanobodies) was displayed on both the Gram-positive bacterium Staphylococcus carnosus and on phage. As demonstrated for the first time, the antibody repertoire was found to be well expressed on the bacterial surface and flow-cytometric sorting yielded a number of Nanobodies with subnanomolar affinity for the target protein, green fluorescent protein (GFP). Interestingly, the staphylococcal output repertoire and the binders from the phage display selection contained two slightly different sets of clones, containing both unique as well as several similar variants. All of the Nanobodies from the staphylococcal selection were also shown to enhance the fluorescence of GFP upon binding, potentially due to the fluorescence-based sorting principle. Our study highlights the impact of the chosen display technology on the variety of selected binders and thus the value of having alternative methods available, and demonstrates in addition that the staphylococcal system is suitable for generation of high-affinity antibody fragments.

  • 26.
    Friedman, Mikaela
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Affibody molecules targeting the epidermal growth factor receptor for tumor imaging applications2008Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Tumor targeting and molecular imaging of protein markers specific for or overexpressed in tumors can add useful information in deciding upon treatment and assessing the response to treatment for a cancer patient. The epidermal growth factor receptor (EGFR) is one such tumor-associated receptor, which expression is abnormal or upregulated in various cancers and associated with a poor patient prognosis. It is therefore considered a good target for imaging and therapy. Monoclonal antibodies and recently also antibody fragments have been investigated for in vivo medical applications, like therapy and imaging. In molecular imaging a small sized targeting agent is favorable to give high contrast and therefore, antibody fragments and lately also small affinity proteins based on a scaffold structure constitute promising alternatives to monoclonal antibodies. Affbody molecules are such affinity proteins that are developed by combinatorial protein engineering of the 58 amino acid residue Z-domain scaffold, derived from protein A.

    In this thesis, novel Affibody molecules specific for the EGFR have been selected from a combinatorial library using phage display technology. Affibody molecules with moderate high affinity demonstrated specific binding to native EGFR on the EGFR-expressing epithelial carcinoma A431 cell line. Further cellular assays showed that the EGFR-binding Affibody molecules could be labeled with radiohalogens or radiometals with preserved specific binding to EGFR-expressing cells. In vitro, the Affibody molecule demonstrated a high uptake and good retention to EGFR-expressing cells and was found to internalize. Furthermore, successful imaging of tumors in tumor-bearing mice was demonstrated. Low nanomolar or subnanomolar affinities are considered to be desired for successful molecular imaging and a directed evolution to increase the affinity was thus performed. This resulted in an approximately 30-fold improvement in affinity, yielding EGFR-binding Affibody molecules with KD´s in the 5-10 nM range, and successful targeting of A431 tumors in tumor-bearing mice. To find a suitable format and labeling, monomeric and dimeric forms of one affinity matured binder were labeled with 125I and 111In. The radiometal-labeled monomeric construct, 111In-labeled-ZEGFR:1907, was found to provide the best tumor-to-organ ratio due to good tumor localization and tumor retention. The tumor-to-blood ratio, which is often used as a measure of contrast, was 31±8 at 24 h post injection and the tumor was clearly visualized by gamma-camera imaging.

    Altogether, the EGFR-binding Affibody molecule is considered a promising candidate for further development of tumor imaging tracers for EGFR-expressing tumors and metastases. This could simplify the stratification of patients for treatment and the assessment of the response of treatment in patients.

  • 27.
    Friedman, Mikaela
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Lindström, Sara
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Engineering and characterization of a bispecific HER2 × EGFR-binding affibody molecule2009In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 54, p. 121-131Article in journal (Refereed)
    Abstract [en]

    HER2 (human epidermal-growth-factor receptor-2; ErbB2) and EGFR (epidermal-growth-factor receptor) are overexpressed in various forms of cancer, and the co-expression of both HER2 and EGFR has been reported in a number of studies. The simultaneous targeting of HER2 and EGFR has been discussed as a strategy with which to potentially increase efficiency and selectivity in molecular imaging and therapy of certain cancers. In an effort to generate a molecule capable of bispecifically targeting HER2 and EGFR, a gene fragment encoding a bivalent HER2-binding affibody molecule was genetically fused in-frame with a bivalent EGFR-binding affibody molecule via a (G(4)S)(3) [(Gly(4)-Ser)(3)]-encoding gene fragment. The encoded 30 kDa affibody construct (Z(HER2))(2)-(G(4)S)(3)-(Z(EGFR))(2), with potential for bs (bispecific) binding to HER2 and EGFR, was expressed in Escherichia coli and characterized in terms of its binding capabilities. The retained ability to bind HER2 and EGFR separately was demonstrated using both biosensor technology and flow-cytometric analysis, the latter using HER2- and EGFR-overexpressing cells. Furthermore, simultaneous binding to HER2 and EGFR was demonstrated in: (i) a sandwich format employing real-time biospecific interaction analysis where the bs affibody molecule bound immobilized EGFR and soluble HER2; (ii) immunofluorescence microscopy, where the bs affibody molecule bound EGFR-overexpressing cells and soluble HER2; and (iii) a cell-cell interaction analysis where the bs affibody molecule bound HER2-overexpressing SKBR-3 cells and EGFR-overexpressing A-431 cells. This is, to our knowledge, the first reported bs affinity protein with potential ability for the simultaneous targeting of HER2 and EGFR. The potential future use of this and similar constructs, capable of bs targeting of receptors to increase the efficacy and selectivity in imaging and therapy, is discussed.

  • 28.
    Friedman, Mikaela
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nordberg, Erika
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Bromma.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Adams, Gregory P.
    Fox Chase Canc Ctr, Dept Med Oncol, Philadelphia.
    Nilsson, Fredrik Y.
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Carlsson, Jörgen
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor2007In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 20, no 4, p. 189-199Article in journal (Refereed)
    Abstract [en]

    Affibody molecules specific for the epidermal growth factor receptor (EGFR) have been selected by Phage display technology from a combinatorial protein library based on the 58-residue, protein A-derived Z domain. EGFR is overexpressed in various malignancies and is frequently associated with poor patient prognosis, and the information provided by targeting this receptor could facilitate both patient diagnostics and treatment. Three selected Affibody variants were shown to selectively bind to the extracellular domain of EGFR (EGFR-ECD). Kinetic biosensor analysis revealed that the three monomeric Affibody molecules bound with similar affinity, ranging from 130 to 185 nM. Head-to-tail dimers of the Affibody molecules were compared for their binding to recombinant EGFR-ECD in biosensor analysis and in human epithelial cancer A431 cells. Although the dimeric Affibody variants were found to bind in a range of 2550 nM affinities in biosensor analysis, they were found to be low nanomolar binders in the cellular assays. Competition assays using radiolabeled Affibody dimers confirmed specific EGFR-binding and demonstrated that the three Affibody molecules competed for the same epitope. Immunofluorescence microscopy demonstrated that the selected Affibody dimers were initially binding to EGFR at the cell surface of A431, and confocal microscopy analysis showed that the Affibody dimers could thereafter be internalized. The potential use of the described Affibody molecules as targeting agents; for radionuclide based imaging applications in various carcinomas ils discussed.

  • 29.
    Friedman, Mikaela
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Orlova, Anna
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Johansson, Eva
    Affibody AB, Bromma.
    Eriksson, Tove L. J.
    Affibody AB, Bromma.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Bromma.
    Tolmachev, Vladimir
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Nilsson, Fredrik Y.
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding Affibody molecule2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 376, no 5, p. 1388-1402Article in journal (Refereed)
    Abstract [en]

    The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K-d = 5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, Kd, was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K-d = 2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection.

  • 30.
    Friedman, Mikaela
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Engineered affinity proteins for tumour-targeting applications2009In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 53, p. 1-29Article, review/survey (Refereed)
    Abstract [en]

    Targeting of tumour-associated antigens is an expanding treatment modality in clinical oncology as an alternative to, or in combination with, conventional treatments, such as chemotherapy, external-radiation therapy and surgery. Targeting of antigens that are unique or more highly expressed in tumours than in normal tissues can be used to increase the specificity and reduce the cytotoxic effect on normal tissues. Several targeting agents have been studied for clinical use, where monoclonal antibodies have been the ones most widely used. More than 20 monoclonal antibodies are approved for therapy today and the largest field is oncology. Advances in genetic engineering and in vitro selection technology has enabled the feasible high-throughput generation of monoclonal antibodies, antibody derivatives [e.g. scFvs, Fab molecules, dAbs (single-domain antibodies), diabodies and minibodies] and more recently also non-immunoglobulin scaffold proteins. Several of these affinity proteins have been investigated for both in vivo diagnostics and therapy. Affinity proteins in tumour-targeted therapy can affect tumour progression by altering signal transduction or by delivering a payload of toxin, drug or radionuclide. The ErbB receptor family has been extensively studied as biomarkers in tumour targeting, primarily for therapy using monoclonal antibodies. Two receptors in the ErbB family, EGFR (epidermal growth factor receptor) and HER2 (epidermal growth factor receptor 2), are over-expressed in various malignancies and associated with poor patient prognosis and are therefore interesting targets for solid turnours. In the present review, strategies are described for tumour targeting of solid turnours using affinity proteins to deliver radionuclides, either for molecular imaging or radiotherapy. Antibodies, antibody derivatives and non-immunoglobulin scaffold proteins are discussed with a certain focus on the affibody (Affibody (R)) molecule.

  • 31.
    Gantelius, Jesper
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Bass, Tarek
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Gundberg, Anna
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Sundberg, Mårten
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Sjöberg, Ronald
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    A ten-minute high density lateral flow protein microarray assay2011In: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011: (MicroTAS 2011), 2011, p. 1176-1178Conference paper (Refereed)
    Abstract [en]

    Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (<50ng/ml) determination of antigen specific antibodies in less than ten minutes total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy (AUC=99.4%) was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies.

  • 32.
    Gantelius, Jesper
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Bass, Tarek
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Sjöberg, Ronald
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays2011In: International Journal of Molecular Sciences, ISSN 1661-6596, Vol. 12, no 11, p. 7748-7759Article in journal (Refereed)
    Abstract [en]

    Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (<30 ng/mL) determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings.

  • 33. Gordley, Russll M.
    et al.
    Smith, Justin D.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Barbas, Carlos F., III
    Evolution of programmable zinc finger-recombinases with activity in human cells2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 367, no 3, p. 802-813Article in journal (Refereed)
    Abstract [en]

    Site-specific recombinases are important tools for genomic engineering in many living systems. Applications of recombinases are, however, constrained by the DNA targeting endemic of the recombinase used. A tremendous range of recombinase applications can be envisioned if the targeting of recombinase specificity can be made readily programmable. To address this problem we sought to generate zinc finger-recombinase fusion proteins (ReCZFS) capable of site-specific function in a diversity of genetic contexts. Our first Rec(ZF), Tn3Ch15(X2), recombined substrates derived from the native Tn3 resolvase recombination site. Substrate Linked Protein Evolution (SLiPE) was used to optimize the catalytic domains of the enzymes Hin, Gin, and Tn3 for resolution between non-homologous sites, One of the evolved clones, GinL7C7, catalyzed efficient, site-specific recombination in a variety of sequence contexts. When introduced into human cells by retroviral transduction, GinL7C7 excised a 1.4 kb EGFP cassette out of the genome, diminishing fluorescence in similar to 17% of transduced cells. Following this template of rational design and directed evolution, Rec(ZF)S may eventually mediate gene therapies, facilitate the genetic manipulation of model organisms and cells, and mature into powerful new tools for molecular biology and medicine.

  • 34.
    Grimm, Sebastian
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ribosome display for selection and evolution of affibody molecules2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Affinity proteins are invaluable tools in biotechnological and medical applications. This thesis is about combinatorial protein engineering principles for the generation of novel affinity proteins to purify mouse immunoglobulin, detect a potential cancer marker protein or inhibit a cell proliferation pathway.

    In a first study, ribosome display was for the first time applied to the selection of so-called affibody molecules, including the design of a ribosome display gene cassette, initial test enrichment experiments and the selection of binders against murine IgG1. One of the selected binders (ZMAB25) showed a highly selective binding profile to murine IgG1, which was exploited in the recovery of two different mouse monoclonal IgG1 antibodies from a bovine immunoglobulin-containing background. Ribosome display was further applied to the selection of affibody molecules binding to SATB1, a suggested marker protein for metastasizing adenocarcinoma. The study also included the selection of VHH antibody fragments from a naïve gene repertoire displayed on phage. Binders from both classes of protein scaffolds could be isolated that selectively recognized SATB1 but not its close homologue SATB2, and were used to detect endogenous SATB1 in Jurkat cells by immunofluorescence microscopy. The well-established phage display technology was used to select affibody molecules binding to H-Ras and Raf-1, both involved in the mitogen-activated protein kinase (MAPK) pathway and playing a central role in the control of cell proliferation, survival and differentiation. An isolated affibody molecule denoted ZRAF322 was found to selectively bind to Raf-1 and inhibit the interaction between H-Ras and Raf-1 in vitro. In a continued effort, ribosome display was applied to the affinity maturation of the ZRAF322 variant in a novel approach, based on repetitive cycles of diversification by error-prone PCR of the entire affibody gene and ribosome display selection, mimicking the principles of natural evolution. The method involved a monitoring of the progress of evolution and variants of ZRAF322 with 13- to 26-fold improved affinities were obtained, that contained different combinations of single or double amino acid substitutions in either previously randomized or framework positions. Implications of the substitutions for binder stability and selectivity were also investigated, showing that a higher affinity could be associated with a lower thermal melting point and that affinity-improved variants showed uncompromised binding selectivity to the hRaf-1 target.

  • 35.
    Grimm, Sebastian
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Klooster, Rinse
    Bisschop, Ilona J.M.
    Gruselius, Joel
    KTH, School of Biotechnology (BIO). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    van der Maarel, Silvère M.
    Single domain affinity proteins for the detection of the genome organizer protein SATB1Manuscript (preprint) (Other academic)
    Abstract [en]

    Affibody molecules and VHH antibody fragments are two classes of affinity proteins, both characterized by a small size and a single subunit domain structure. Here, we report the selection and characterization of affinity proteins from both classes against the special AT rich sequence binding protein SATB1, a suggested marker protein for aggressive and metastasizing breast cancer. The selected VHH antibody fragments originate from a nonimmune phage display library, while affibody molecules were selected from a library constructed in vitro and displayed on ribosomes. It was observed that selected molecules recognizing one of three conserved DNA-binding domains of SATB1, also recognized its close homologue SATB2 while several of the selected molecules from both classes binding to other regions selectively recognized SATB1. Two of these SATB1 selective molecules, VHH clone 2D2 and affibody molecule clone ZSATB1:2, performed well in differentimmunotechnology applications including ELISA, WB, IF and pull-out experiments and gave a selective staining of endogenous SATB1 in Jurkat T cells. These molecules may thus become useful tools, either alone or in combination, for the selective detection of SATB1 in breast tumor specimens. Due to their small size in comparison to immunoglobulins, such single domain binding proteins may be suitable for high resolution microscopy techniques such as Stimulated Emission Depletion (STED) microscopy, where the resolution may get constrained by the size of the affinity reagent.

  • 36.
    Grimm, Sebastian
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics.
    Yu, Feifan
    KTH, School of Biotechnology (BIO), Proteomics.
    Shibasaki, Seiji
    Vernet, Erik
    KTH, School of Biotechnology (BIO), Proteomics.
    Skogs, Marie
    KTH, School of Biotechnology (BIO), Proteomics.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Selection and characterisation of affibody molecules inhibiting the interaction between Ras and Raf in vitro2010In: NEW BIOTECHNOL, ISSN 1871-6784, Vol. 27, no 6, p. 766-773Article in journal (Refereed)
    Abstract [en]

    Development of molecules with the ability to selectively inhibit particular protein-protein interactions is important in providing tools for understanding cell biology In this work, we describe efforts to select small Ras- and Raf-specific three-helix bundle affibody binding proteins capable of inhibiting the interaction between H-Ras and Raf-1, from a combinatorial library displayed on bacteriophage Target-specific variants with typically high nanomolar or low micromolar affinities (K-D) could be selected successfully against both proteins, as shown by dot blot, ELISA and real-time biospecific interaction analyses Affibody molecule variants selected against H-Ras were shown to bind epitopes overlapping each other at a site that differed from that at which H-Ras interacts with Raf-1 In contrast, an affibody molecule isolated during selection against Raf-1 was shown to effectively inhibit the interaction between H-Ras and Raf-1 in a dose-dependent manner Possible intracellular applications of the selected affibody molecules are discussed

  • 37.
    Grimm, Sebastian
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Salahshour, Samaneh
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Affinity maturation of an affibody molecule binding to human Raf-1 via non-targeted in vitro evolutionManuscript (preprint) (Other academic)
    Abstract [en]

    The use of in vitro protein library technologies for the generation of high affinity binding proteins often includes an affinity maturation step, involving the construction of secondary libraries from which second generation variants with improved affinities are selected. We describe a novel approach for the affinity maturation of affibody molecules based on stepwise in vitro molecular evolution, involving cycles of whole gene error-prone PCR amplification for the introduction of diversity over the entire 58-residue three-helix bundle structure and ribosome display for selection. Starting with a human Raf-1 binding affibody molecule of an initial 2 μM dissociation constant (KD), the in vitro evolution process was followed on both gene and protein levels via DNA sequencing and a biosensor based monitoring of the collective target binding ability of expressed pools obtained after each selection cycle. After a first cycle of diversification and selection, no increase in the hRaf-1 target binding of the pool was observed, whereas after two cycles, a significant increase in the binding response was seen. DNA sequencing showed that an alanine to valine substitution in an earlier variegated position had been effectively enriched, and was present in 11% and 83% of all clones after cycle one and two, respectively, either alone or in combination with other substitutions. Further studies on a subset of individual variants isolated after cycle two showed that the observed A27V substitution alone accounted for a 13-fold increase in affinity, predominantly through increased on-rate kinetics. Additional substitutions in framework or non-framework positions typically resulted in a further two-fold increase in affinity. Interestingly, thermal melting point (Tm) analyses showed that an increased affinity could be associated with either slightly higher or lower Tm values, compared to the parental variant. All investigated variants showed excellent refolding properties, and the selectivity of the affinity matured hRaf-1 binders had not been compromised by the substitutions, as analyzed using a multiplexed bead-based binding assay involving 77 recombinant human control protein fragments.

  • 38.
    Grimm, Sebastian
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Salahshour, Samaneh
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Monitored whole gene in vitro evolution of an anti-hRaf-1 affibody molecule towards increased binding affinity2011In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, New biotechnology, ISSN 1876-4347, Vol. 29, no 5, p. 534-542Article in journal (Refereed)
    Abstract [en]

    The use of library technologies for the generation of affinity proteins often includes an affinity maturation step, based on the construction of secondary libraries from which second generation variants with improved affinities are selected. Here, we describe for the first time the affinity maturation of affibody molecules based on step-wise in vitro molecular evolution, involving cycles of error-prone PCR (epPCR) amplification for the introduction of diversity over the entire 58-residue three-helix bundle structure and ribosome display (RD) for the selection of improved variants. The model affibody molecule for the process was Z(RAF322), binding with a 1.9μm equilibrium dissociation constant (K(D)) to human Raf-1 (hRaf-1), a protein kinase of central importance in the MAPK/ERK proliferation pathway. The molecular evolution process was followed on both gene and protein levels via DNA sequencing and a biosensor-based binding analysis of pools of selected variants. After two cycles of diversification and selection, a significant increase in binding response of selected pools was seen. DNA sequencing showed that a dominant alanine to valine substitution had been effectively enriched, and was found in 83% of all selected clones, either alone or in combination with other enriched substitutions. The evolution procedure resulted in variants showing up to 26-fold increases in affinity to the hRaf-1 target. Noteworthy, for the two variants showing the highest affinities, substitutions were also found in affibody framework positions, corresponding to regions of the protein domain not addressed by traditional affibody molecule affinity maturation strategies. Interestingly, thermal melting point (T(m)) analyses showed that an increased affinity could be associated with both higher and lower T(m) values. All investigated variants showed excellent refolding properties and selective binding to hRaf-1, as analysed using a multiplexed bead-based binding assay, making them potentially valuable affinity reagents for cell biology studies.

  • 39.
    Grimm, Sebastian
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Yu, Feifan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ribosome Display Selection of a Murine IgG(1) Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies2011In: Molecular Biotechnology, ISSN 1073-6085, E-ISSN 1559-0305, Vol. 48, no 3, p. 263-276Article in journal (Refereed)
    Abstract [en]

    Affinity reagents recognizing constant parts of antibody molecules are invaluable tools in immunotechnology applications, including purification, immobilization, and detection of immunoglobulins. In this article, murine IgG(1), the primary isotype of monoclonal antibodies (mAbs) was used as target for selection of novel binders from a combinatorial ribosome display (RD) library of 10(11) affibody molecules. Four rounds of selection using three different mouse IgG(1) mAbs as alternating targets resulted in the identification of binders with broad mIgG(1) recognition and dissociation constants (K (D)) in the low nanomolar to low micromolar range. For one of the binders, denoted Z(mab25), competition in binding to full length mIgG(1) by a streptococcal protein G (SPG) fragment and selective affinity capture of mouse IgG(1) Fab fragments after papain cleavage of a full mAb suggest that an epitope functionally overlapping with the SPG-binding site in the CH1 domain of mouse IgG(1) had been addressed. Interestingly, biosensor-based binding experiments showed that neither human IgG(1) nor bovine Ig, the latter present in fetal bovine serum (FBS) was recognized by Z(mab25). This selective binding profile towards murine IgG(1) was successfully exploited in species selective recovery of two different mouse mAbs from complex samples containing FBS, resembling a hybridoma culture supernatant.

  • 40.
    Gräslund, Torbjörn
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Eriksson, Tove
    Jonsson, Andreas
    Bergman, Thomas
    Li, Jingjing
    Lendel, Christofer
    Jarstad, Anders
    IGF-1R binding polypeptides and their use2007Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    This invention relates to polypeptides which bind to IGF-1R and to applications of those polypeptides in medicine, veterinary medicine, diagnostics and imaging.

  • 41.
    Gräslund, Torbjörn
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Li, X. L.
    Magnenat, L.
    Popkov, M.
    Barbas, C. F.
    Exploring strategies for the design of artificial transcription factors2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 5, p. 3707-3714Article in journal (Refereed)
    Abstract [en]

    Artificial transcription factors can be engineered to interact with specific DNA sequences to modulate endogenous gene expression within cells. A significant hurdle to implementation of this approach is the selection of the appropriate DNA sequence for targeting. We reasoned that a good target site should be located in chromatin, where it is accessible to DNA-binding proteins, and it should be, in the close vicinity of known transcriptional regulators of the gene. Here we have explored the efficacy of these criteria to guide our selection of potential regulators of gamma-globin expression. Several zinc finger-based transcriptional activators were designed to target the sites proximal to the -117-position of the gamma-globin promoter. This region is proximal to the binding sites of known and potential natural transcription factors. Design and study of three transcription factors identified the potent transcriptional activator, ggl-VP64-RA. This transcription factor was able to interact directly with the gamma-globin promoter and up-regulate expression of reporter gene constructs as well as the endogenous gene in a selective manner. Transfection of a gg1-VP64-RA expression vector or retroviral delivery of this transcription factor into the erythroleukemia cell line K562 resulted in an increase of fetal hemoglobin. The gamma-globin content of cells expressing gg1-vp64-HA showed up to 16-fold higher levels of fetal hemoglobin than the native K562 cell line. These transcriptional activators constitute a novel class of regulators of the globin locus that may be suitable for treatment of diseases arising from mutations in this locus such as sickle cell disease and thalassemic diseases.

  • 42.
    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.

  • 43.
    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.

  • 44.
    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.

  • 45.
    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.

  • 46.
    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.

  • 47.
    Göstring, Lovisa
    et al.
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Malm, Magdalena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Stockholm, Sweden.
    Frejd, Fredrik Y.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Löfblom, John
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Gedda, Lars
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala Sweden.
    Cellular Effects of HER3-Specific Affibody Molecules2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 6, p. e40023-Article in journal (Refereed)
    Abstract [en]

    Recent studies have led to the recognition of the epidermal growth factor receptor HER3 as a key player in cancer, and consequently this receptor has gained increased interest as a target for cancer therapy. We have previously generated several Affibody molecules with subnanomolar affinity for the HER3 receptor. Here, we investigate the effects of two of these HER3-specific Affibody molecules, Z05416 and Z05417, on different HER3-overexpressing cancer cell lines. Using flow cytometry and confocal microscopy, the Affibody molecules were shown to bind to HER3 on three different cell lines. Furthermore, the receptor binding of the natural ligand heregulin (HRG) was blocked by addition of Affibody molecules. In addition, both molecules suppressed HRG-induced HER3 and HER2 phosphorylation in MCF-7 cells, as well as HER3 phosphorylation in constantly HER2-activated SKBR-3 cells. Importantly, Western blot analysis also revealed that HRG-induced downstream signalling through the Ras-MAPK pathway as well as the PI3K-Akt pathway was blocked by the Affibody molecules. Finally, in an in vitro proliferation assay, the two Affibody molecules demonstrated complete inhibition of HRG-induced cancer cell growth. Taken together, our findings demonstrate that Z05416 and Z05417 exert an anti-proliferative effect on two breast cancer cell lines by inhibiting HRG-induced phosphorylation of HER3, suggesting that the Affibody molecules are promising candidates for future HER3-targeted cancer therapy.

  • 48. Hedhammar, M.
    et al.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Protein engineering strategies for selective protein purification2005In: Chemical Engineering & Technology, ISSN 0930-7516, E-ISSN 1521-4125, Vol. 28, no 11, p. 1315-1325Article, review/survey (Refereed)
    Abstract [en]

    When producing and purifying recombinant proteins it is of importance to minimize the number of unit operations during the purification procedure. This is accomplished by increasing the selectivity in each step. Due to the high selectivity of affinity chromatography it has a widespread use in protein purification. However, most target proteins lack a suitable affinity ligand usable for capture oil a solid matrix. A way to circumvent this obstacle is to genetically fuse the gene encoding the target protein with a gene encoding a purification tag. When the chimeric protein is expressed, the tag allows for specific capture of the fusion protein. In industrial-scale production, extension of the target protein often is unwanted since it might interfere with the function of the target protein. Hence, a purification scheme developed for the native protein is desired. In this review, different fusion strategies used for protein purification are discussed. Also, the development of ligands for selective affinity purification of native target proteins is surveyed.

  • 49. Henning, P
    et al.
    Andersson, K M E
    Frykholm, K
    Ali, A
    Magnusson, M K
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Granio, O
    Hong, S S
    Boulanger, P
    Lindholm, L
    Tumor cell targeted gene delivery by adenovirus 5 vectors carrying knobless fibers with antibody-binding domains2005In: Gene Therapy, ISSN 0969-7128, E-ISSN 1476-5462, Vol. 12, no 3, p. 211-224Article in journal (Refereed)
    Abstract [en]

    Most human carcinoma cell lines lack the high-affinity receptors for adenovirus serotype 5 (Ad5) at their surface and are nonpermissive to Ad5. We therefore tested the efficiency of retargeting Ad5 to alternative cellular receptors via immunoglobulin (Ig)-binding domains inserted at the extremity of short-shafted, knobless fibers. The two recombinant Ad5' s constructed, Ad5/R7- Z(wt)- Z(wt) and Ad5/R7-C2-C2, carried tandem Ig-binding domains from Staphylococcal protein A ( abbreviated Z(wt)) and from Streptococcal protein G (C2), respectively. Both viruses bound their specific Ig isotypes with the expected affinity. They transduced human carcinoma cells independently of the CAR pathway, via cell surface receptors targeted by specific monoclonal antibodies, that is, EGF-R on A549, HT29 and SW1116, HER-2/ neu on SK-OV-3 and SK-BR-3, CA242 ( epitope recognized by the monoclonal antibody C242) antigen on HT29 and SW1116, and PSMA ( prostate-specific membrane antigen) expressed on HEK-293 cells, respectively. However, Colo201 and Colo205 cells were neither transduced by targeting CA242 or EGF-R nor were LNCaP cells transduced by targeting PSMA. Our results suggested that one given surface receptor could mediate transduction of certain cells but not others, indicating that factors and steps other than cell surface expression and virus - receptor interaction are additional determinants of Ad5-mediated transduction of tumor cells. Using penton base RGD mutants, we found that one of these limiting steps was virus endocytosis.

  • 50. Heskamp, Sandra
    et al.
    Laverman, Peter
    Rosik, Daniel
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Boschetti, Frederic
    van der Graaf, Winette T. A.
    Oyen, Wim J. G.
    van Laarhoven, Hanneke W. M.
    Tolmachev, Vladimir
    Boerman, Otto C.
    Imaging of Human Epidermal Growth Factor Receptor Type 2 Expression with (18)F-Labeled Affibody Molecule Z(HER2:2395) in a Mouse Model for Ovarian Cancer2012In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 53, no 1, p. 146-153Article in journal (Refereed)
    Abstract [en]

    Affibody molecules are small (7 kDa) proteins with subnanomolar targeting affinity. Previous SPECT studies in xenografts have shown that the Affibody molecule (111)In-DOTA-Z(HER2:2395) can discriminate between high and low human epidermal growth factor receptor type 2 (HER2)-expressing tumors, indicating that radiolabeled Affibody molecules have potential for patient selection for HER2-targeted therapy. Compared with SPECT, PET with positron-emitting radionuclides, such as (18)F, may improve imaging of HER2 expression because of higher sensitivity and improved quantification of PET. The aim of the present study was to determine whether the (18)F-labeled NOTA-conjugated Affibody molecule Z(HER2:2395) is a suitable agent for imaging of HER2 expression. The tumor-targeting properties of (18)F-labeled Z(HER2:2395) were compared with (111)In- and (68)Ga-labeled Z(HER2:2395) in mice with HER2-expressing SK-OV-3 xenografts. Methods: Z(HER2:2395) was conjugated with NOTA and radiolabeled with (18)F, (68)Ga, and (111)In. Radiolabeling with (18)F was based on the complexation of Al(18)F by NOTA. The 50% inhibitory concentration values for NOTA-Z(HER2:2395) labeled with (19)F, (69)Ga, and (115)In were determined in a competitive cell-binding assay using SK-OV-3 cells. Mice bearing subcutaneous SK-OV-3 xenografts were injected intravenously with radiolabeled NOTA-Z(HER2:2395). One and 4 h after injection, PET/CT or SPECT/CT images were acquired, and the biodistribution was determined by ex vivo measurement. Results: The 50% inhibitory concentration values for (19)F-, (69)Ga-, and (115)In-NOTA-Z(HER2:2395) were 5.0, 6.3, and 5.3 nM, respectively. One hour after injection, tumor uptake was 4.4 +/- 0.8 percentage injected dose per gram (% ID/g), 5.6 +/- 1.6 % ID/g, and 7.1 +/- 1.4 % ID/g for (18)F-, (68)Ga-, and (111)In-NOTA-Z(HER2:2395), respectively, and the respective tumor-to-blood ratios were 7.4 +/- 1.8, 8.0 +/- 1.3, and 4.8 +/- 1.3. Tumor uptake was specific, because uptake could be blocked efficiently by coinjection of an excess of unlabeled Z(HER2:2395). PET/CT and SPECT/CT images clearly visualized HER2-expressing SK-OV-3 xenografts. Conclusion: This study showed that (18)F-NOTA-Z(HER2:2395) is a promising new imaging agent for HER2 expression in tumors. Affibody molecules were successfully labeled with (18)F within 30 min, based on the complexation of Al(18)F by NOTA. Further research is needed to determine whether this technique can be used for patient selection for HER2-targeted therapy.

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