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  • 1. 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.

  • 2.
    Allerbring, E. B.
    et al.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Duru, A. D.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Uchtenhagen, H.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Madhurantakam, C.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Tomek, M. B.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Grimm, Sebastian
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Mazumdar, P. A.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Friemann, R.
    Univ Gothenburg, Dept Chem Biochem & Biophys, Gothenburg, Sweden..
    Sandalova, T.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Uhlin, M.
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Stockholm, Sweden..
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Achour, A.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    The unexpected T-cell recognition of an altered peptide ligand is driven by reversed thermodynamics and an alternative structural hotspot2011In: Immunology, ISSN 0019-2805, E-ISSN 1365-2567, Vol. 135, p. 70-70Article in journal (Other academic)
  • 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.
    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.

  • 5.
    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.

  • 6.
    Grimm, Sebastian
    et al.
    KTH, School of Biotechnology (BIO).
    Lundberg, Emma
    KTH, School of Biotechnology (BIO).
    Shibasaki, Seiji
    KTH, School of Biotechnology (BIO).
    Vernet, Erik
    KTH, School of Biotechnology (BIO).
    Skogs, Marie
    KTH, School of Biotechnology (BIO).
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO).
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO).
    Selection and characterization of affibody molecules interfering with the interaction between Ras and RafManuscript (Other academic)
  • 7.
    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

  • 8.
    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.

  • 9.
    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.

  • 10.
    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.

  • 11.
    Tolmachev, Vladimir
    et al.
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Gronroos, Tove J.
    Univ Turku, Turku PET Ctr, Turku, Finland.;Univ Turku, MediCity Res Lab, Turku, Finland.;Turku Univ Hosp, Dept Oncol & Radiotherapy, Turku, Finland..
    Yim, Cheng-Bin
    Univ Turku, Turku PET Ctr, Turku, Finland.;Abo Akad Univ, Turku PET Ctr, Turku, Finland..
    Garousi, Javad
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Yue, Ying
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Grimm, Sebastian
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Rajander, Johan
    Abo Akad Univ, Turku PET Ctr, Turku, Finland..
    Perols, Anna
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Haaparanta-Solin, Merja
    Univ Turku, Turku PET Ctr, Turku, Finland.;Univ Turku, Dept Chem, Turku, Finland..
    Solin, Olof
    Univ Turku, Turku PET Ctr, Turku, Finland.;Abo Akad Univ, Turku PET Ctr, Turku, Finland.;Univ Turku, Dept Chem, Turku, Finland..
    Ferdani, Riccardo
    Washington Univ, St Louis, MO USA..
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Anderson, Carolyn J.
    Univ Pittsburgh, Dept Med, Pittsburgh, PA 15203 USA.;Univ Pittsburgh, Dept Radiol, Pittsburgh, PA 15203 USA.;Univ Pittsburgh, Dept Bioengn, Pittsburgh, PA 15203 USA.;Univ Pittsburgh, Dept Pharmacol & Chem Biol, Pittsburgh, PA 15203 USA..
    Karlström, Amelie Eriksson
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Molecular design of radiocopper-labelled Affibody molecules2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 6542Article in journal (Refereed)
    Abstract [en]

    The use of long-lived positron emitters Cu-64 or Cu-61 for labelling of Affibody molecules may improve breast cancer patients' stratification for HER-targeted therapy. Previous animal studies have shown that the use of triaza chelators for Cu-64 labelling of synthetic Affibody molecules is suboptimal. In this study, we tested a hypothesis that the use of cross-bridged chelator, CB-TE2A, in combination with Gly-Glu-Glu-Glu spacer for labelling of Affibody molecules with radiocopper would improve imaging contrast. CB-TE2A was coupled to the N-terminus of synthetic Affibody molecules extended either with a glycine (designation CB-TE2A-G-ZHER2:342) or Gly-Glu-Glu-Glu spacer (CB-TE2A-GEEE-ZHER2:342). Biodistribution and targeting properties of Cu-64-CB-TE2A-G-ZHER2:342 and Cu-64-CB-TE2A-GEEE-ZHER2:342 were compared in tumor-bearing mice with the properties of Cu-64-NODAGA-ZHER2:S1, which had the best targeting properties in the previous study. Cu-64-CB-TE2A-GEEE-ZHER2:342 provided appreciably lower uptake in normal tissues and higher tumor-to-organ ratios than Cu-64-CB-TE2A-GZHER2:342 and Cu-64-NODAGA-ZHER2:S1. The most pronounced was a several-fold difference in the hepatic uptake. At the optimal time point, 6 h after injection, the tumor uptake of Cu-64-CB-TE2A-GEEE-ZHER2: 342 was 16 +/- 6% ID/g and tumor-to-blood ratio was 181 +/- 52. In conclusion, a combination of the cross-bridged CB-TE2A chelator and Gly-Glu-Glu-Glu spacer is preferable for radiocopper labelling of Affibody molecules and, possibly, other scaffold proteins having high renal re-absorption.

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