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Publications (10 of 12) Show all publications
Boutajangout, A., Lindberg, H., Awwad, A., Paul, A., Baitalmal, R., Almokyad, I., . . . Wisniewski, T. (2019). Affibody-Mediated Sequestration of Amyloid beta Demonstrates Preventive Efficacy in a Transgenic Alzheimer's Disease Mouse Model. Frontiers in Aging Neuroscience, 11, Article ID 64.
Open this publication in new window or tab >>Affibody-Mediated Sequestration of Amyloid beta Demonstrates Preventive Efficacy in a Transgenic Alzheimer's Disease Mouse Model
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2019 (English)In: Frontiers in Aging Neuroscience, ISSN 1663-4365, E-ISSN 1663-4365, Vol. 11, article id 64Article in journal (Refereed) Published
Abstract [en]

Different strategies for treatment and prevention of Alzheimer's disease (AD) are currently under investigation, including passive immunization with anti-amyloid beta (anti-A beta) monoclonal antibodies (mAbs). Here, we investigate the therapeutic potential of a novel type of A beta-targeting agent based on an affibody molecule with fundamentally different properties to mAbs. We generated a therapeutic candidate, denoted Z(SYM73)-albumin-binding domain (ABD; 16.8 kDa), by genetic linkage of the dimeric Z(SYM73) affibody for sequestering of monomeric A beta-peptides and an ABD for extension of its in vivo half-life. Amyloid precursor protein (APP)/PS1 transgenic AD mice were administered with Z(SYM73)-ABD, followed by behavioral examination and immunohistochemistry. Results demonstrated rescued cognitive functions and significantly lower amyloid burden in the treated animals compared to controls. No toxicological symptoms or immunology-related side-effects were observed. To our knowledge, this is the first reported in vivo investigation of a systemically delivered scaffold protein against monomeric A beta, demonstrating a therapeutic potential for prevention of AD.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2019
Keywords
Alzheimer's disease, affibody molecule, amyloid beta (A beta), behavior, histology, immunotherapy, transgenic mice
National Category
Geriatrics
Identifiers
urn:nbn:se:kth:diva-249880 (URN)10.3389/fnagi.2019.00064 (DOI)000462530400001 ()30967771 (PubMedID)
Note

QC 20190423

Available from: 2019-04-23 Created: 2019-04-23 Last updated: 2019-04-23Bibliographically approved
Lindberg, H., Sandersjöö, L., Meister, S. W., Uhlén, M., Löfblom, J. & Ståhl, S. (2017). Flow-cytometric screening of aggregation-inhibitors using a fluorescence-assisted intracellular method. Biotechnology Journal, 12(1), Article ID 1600364.
Open this publication in new window or tab >>Flow-cytometric screening of aggregation-inhibitors using a fluorescence-assisted intracellular method
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2017 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 12, no 1, article id 1600364Article in journal (Refereed) Published
Abstract [en]

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

Place, publisher, year, edition, pages
John Wiley & Sons, 2017
Keywords
Affibody molecules, Aggregation-inhibitor, Amyloid beta, Combinatorial protein engineering, Intracellular selection system, Cells, Cytology, Diagnosis, Fluorescence, Glycoproteins, Molecules, Neurodegenerative diseases, Peptides, Aggregation inhibitors, Amyloid betas, Protein engineering, Selection systems, Proteins, alpha synuclein, amyloid beta protein, green fluorescent protein, recombinant protein, chemistry, Escherichia coli, flow cytometry, gene vector, genetics, metabolism, preclinical study, procedures, alpha-Synuclein, Amyloid beta-Peptides, Drug Evaluation, Preclinical, Genetic Vectors, Green Fluorescent Proteins, Recombinant Proteins
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-202248 (URN)10.1002/biot.201600364 (DOI)000395638400009 ()2-s2.0-85006293410 (Scopus ID)
Note

Funding text: We thank Affibody AB for providing the genetic construct of ZHER2-mCherry. The Wallenberg Center for Protein Research and the Swedish Brain Foundation (grant FO2015-0174) are acknowledged for funding. Conceived and designed the experiments: HL LS JL SS. Performed the experiments: HL LS SM. Contributed reagents/materials/analysis tools and analyzed the data: HL LS SM MU JL SS. Wrote the paper: HL LS JL SS. The authors declare no conflicts of interest. QC 20170313

Available from: 2017-03-13 Created: 2017-03-13 Last updated: 2017-11-29Bibliographically approved
Lindberg, H., Härd, T., Löfblom, J. & Ståhl, S. (2015). A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity. Biotechnology Journal, 10(11), pp. 1707-1718
Open this publication in new window or tab >>A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity
2015 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 10, no 11, p. 1707-1718Article in journal, News item (Refereed) Published
Abstract [en]

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

Keywords
Affibody molecules, Affinity maturation, Amyloid beta, Bacterial display, Combinatorial protein engineering
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-173855 (URN)10.1002/biot.201500131 (DOI)000365129600007 ()2-s2.0-84947041962 (Scopus ID)
Note

Updated from E-pub to Published. QC 20151218

Available from: 2015-09-21 Created: 2015-09-21 Last updated: 2017-12-04Bibliographically approved
Lindberg, H. (2015). Engineering of Affibody molecules targeting the Alzheimer’s-related amyloid β peptide. (Doctoral dissertation). Stockholm: KTH Royal Institute of Technology
Open this publication in new window or tab >>Engineering of Affibody molecules targeting the Alzheimer’s-related amyloid β peptide
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. p. x, 107
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:14
Keywords
Affibody molecules, Alzheimer’s disease, AD, amyloid beta, Aß, combinatorial protein engineering, staphylococcal surface display
National Category
Other Natural Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-173864 (URN)978-91-7595-663-3 (ISBN)
Public defence
2015-10-09, D3, Lindstedtsvägen 5, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20150922

Available from: 2015-09-22 Created: 2015-09-21 Last updated: 2015-09-22Bibliographically approved
Orlova, A., Malm, M., Lindberg, H., Varasteh, Z., Rosestedt, M., Tolmachev, V., . . . Löfblom, J. (2013). Feasibility of radionuclide imaging of HER3-expressing tumors using affibody molecules. Journal of labelled compounds & radiopharmaceuticals, 56, S11-S11
Open this publication in new window or tab >>Feasibility of radionuclide imaging of HER3-expressing tumors using affibody molecules
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2013 (English)In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 56, p. S11-S11Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Wiley-Blackwell, 2013
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-124297 (URN)000318694100012 ()
Note

QC 20130701

Available from: 2013-07-01 Created: 2013-06-28 Last updated: 2017-12-06Bibliographically approved
Orlova, A., Malm, M., Lindberg, H., Varasteh, Z., Selvaraju, R. K., Kronqvist, N., . . . Tolmachev, V. (2013). Feasibility of radionuclide imaging of HER3-expressing tumours using technetium-99m labeled affibody molecules. Paper presented at Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 19-23, 2013, Lyon, France. European Journal of Nuclear Medicine and Molecular Imaging, 40, S185-S186
Open this publication in new window or tab >>Feasibility of radionuclide imaging of HER3-expressing tumours using technetium-99m labeled affibody molecules
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2013 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, p. S185-S186Article in journal, Meeting abstract (Other academic) Published
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:kth:diva-134576 (URN)000325853400293 ()
Conference
Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 19-23, 2013, Lyon, France
Note

QC 20131127

Available from: 2013-11-27 Created: 2013-11-25 Last updated: 2017-12-06Bibliographically approved
Malm, M., Kronqvist, N., Lindberg, H., Gudmundsdotter, L., Bass, T., Frejd, F. Y., . . . Löfblom, J. (2013). Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification. PLoS ONE, 8(5), e62791
Open this publication in new window or tab >>Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 5, p. e62791-Article in journal (Refereed) Published
Abstract [en]

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

Place, publisher, year, edition, pages
Public Library Science, USA, 2013
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-124294 (URN)10.1371/journal.pone.0062791 (DOI)000318852400011 ()2-s2.0-84877609907 (Scopus ID)
Funder
Swedish Research CouncilSwedish Cancer Society
Note

QC 20130701

Available from: 2013-07-01 Created: 2013-06-28 Last updated: 2017-12-06Bibliographically approved
Lindberg, H., Johansson, A., Härd, T., Ståhl, S. & Löfblom, J. (2013). Staphylococcal display for combinatorial protein engineering of a head-to-tail affibody dimer binding the Alzheimer amyloid-ss peptide. Biotechnology Journal, 8(1), 139-145
Open this publication in new window or tab >>Staphylococcal display for combinatorial protein engineering of a head-to-tail affibody dimer binding the Alzheimer amyloid-ss peptide
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2013 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 8, no 1, p. 139-145Article in journal (Refereed) Published
Abstract [en]

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

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2013
Keywords
Affibody molecules, Alzheimer's disease, Amyloid beta, Bacterial display, Combinatorial protein engineering
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-111860 (URN)10.1002/biot.201200228 (DOI)000312989500022 ()22987778 (PubMedID)2-s2.0-84871749509 (Scopus ID)
Funder
Swedish Research Council, 2009-5758VINNOVA, 2009-00179
Note

QC 20130207

Available from: 2013-01-14 Created: 2013-01-14 Last updated: 2017-12-06Bibliographically approved
Lindberg, H., Hofström, C., Altai, M., Honorvar, H., Wållberg, H., Orlova, A., . . . Tolmachev, V. (2012). Evaluation of a HER2-targeting affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for Tc-99m-labelling at the C terminus. Tumor Biology, 33(3), 641-651
Open this publication in new window or tab >>Evaluation of a HER2-targeting affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for Tc-99m-labelling at the C terminus
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2012 (English)In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 33, no 3, p. 641-651Article in journal (Refereed) Published
Abstract [en]

Affibody molecules are a class of small (ca.7 kDa) robust scaffold proteins with high potential as tracers for radionuclide molecular imaging in vivo. Incorporation of a cysteine-containing peptide-based chelator at the C terminus provides an opportunity for stable labelling with the radionuclide Tc-99m. The use of a GGGC chelator at the C terminus has provided the lowest renal radioactivity retention of the previously investigated peptide-based chelators. Previously, it has also been demonstrated that replacement of the His(6)-tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of affibody molecules by immobilized metal ion affinity chromatography (IMAC) and provides low hepatic accumulation of radioactivity of conjugates site-specifically labelled at the C terminus using several different nuclides. We hypothesized that the combination of a HEHEHE-tag at the N terminus and a GGGC chelator at the C terminus of an affibody molecule would be a favourable format permitting IMAC purification and providing low uptake in excretory organs. To investigate this hypothesis, a (HE)(3)-Z(HER2:342)-GGGC affibody molecule was generated. It could be efficiently purified by IMAC and stably labelled with Tc-99m. Tc-99m-(HE)(3)-Z(HER2:342)-GGGC preserved specific binding to HER2-expressing cells. In NMRI mice, hepatic uptake of Tc-99m-(HE)(3)-Z(HER2:342)-GGGC was lower than the uptake of the control affibody molecules, Tc-99m-Z(HER2:2395)-VDC and Tc-99m-Z(HER2:342)-GGGC. At 1 and 4 h after injection, the renal uptake of Tc-99m-(HE)(3)-Z(HER2:342)-GGGC was 2-3-fold lower than uptake of Tc-99m-Z(HER2:2395)-VDC, but it was substantially higher than uptake of Tc-99m-Z(HER2:342)-GGGC. Further investigation indicated that a fraction of Tc-99m was chelated by the HEHEHE-tag which caused a higher accumulation of radioactivity in the kidneys. Thus, a combination of a HEHEHE-tag and the GGGC chelator in targeting scaffold proteins was found to be undesirable in the case of Tc-99m labelling due to a partial loss of site-specificity of nuclide chelation.

Place, publisher, year, edition, pages
Springer, 2012
Keywords
Affibody molecules, Radionuclide molecular imaging, Technetium-99m, HEHEHE-tag, GGGC chelator, Biodistribution
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:kth:diva-96436 (URN)10.1007/s13277-011-0305-z (DOI)000303530200008 ()2-s2.0-84863581172 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20120607

Available from: 2012-06-07 Created: 2012-06-04 Last updated: 2017-12-07Bibliographically approved
Boutajangout, A., Lindberg, H., Awwad, A., Paul, A., Wahlberg, E., Gudmundsdotter, H., . . . Wisniewski, T.Affibody-mediated Reduction of Amyloid Burden and Improvement of Cognitive Decline in an Animal Model of Alzheimer’s disease.
Open this publication in new window or tab >>Affibody-mediated Reduction of Amyloid Burden and Improvement of Cognitive Decline in an Animal Model of Alzheimer’s disease
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-173863 (URN)
Note

QS 2015

Available from: 2015-09-21 Created: 2015-09-21 Last updated: 2015-09-22Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-5192-7362

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