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Publications (10 of 93) Show all publications
Yu, F., Alesand, V. & Nygren, P.-Å. (2018). Site-Specific Photoconjugation of Beta-Lactamase Fragments to Monoclonal Antibodies Enables Sensitive Analyte Detection via Split-Enzyme Complementation. Biotechnology Journal, 13(7), Article ID 1700688.
Open this publication in new window or tab >>Site-Specific Photoconjugation of Beta-Lactamase Fragments to Monoclonal Antibodies Enables Sensitive Analyte Detection via Split-Enzyme Complementation
2018 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 13, no 7, article id 1700688Article in journal (Refereed) Published
Abstract [en]

Protein fragment complementation assays (PCA) rely on a proximity-driven reconstitution of a split reporter protein activity, typically via interaction between bait and prey units separately fused to the reporter protein halves. The PCA principle can also be formatted for use in immunossays for analyte detection, e.g., via the use of small immunoglobulin binding proteins (IgBp) as fusion partners to split-reporter protein fragments for conversion of pairs of antibodies into split-protein half-probes. However, the non-covalent binding between IgBp and antibodies is not ideal for development of robust assays. Here, the authors describe how split-enzyme reporter halves can be both site-specifically and covalently photoconjugated at antibody Fc-parts for use in homogeneous dual-antibody in vitro immunoassays based on analyte-dependent split-enzyme fragment complementation. The half-probes consist of parts of a beta-lactamase split-protein reporter fused to an immunoglobulin Fc binding domain equipped with a unique cysteine residue at which a photoactivable maleimide benzophenone group (MBP) is attached. Using such antibody conjugates the authors obtain an analyte-driven complementation of the reporter enzyme fragments monitored via conversion of a chromogenic substrate. Results from detection of human interferon-gamma and the extracellular domain of HER2 is shown. The described principles for site-specific conjugation of proteins to antibodies should be broadly applicable.

Place, publisher, year, edition, pages
WILEY-V C H VERLAG GMBH, 2018
Keywords
affibody, antibody, complementation, homogeneous assay, nitrocefin, photoconjugation, split-beta-lactamase
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-232401 (URN)10.1002/biot.201700688 (DOI)000437281600007 ()29485240 (PubMedID)2-s2.0-85043698701 (Scopus ID)
Funder
VINNOVA
Note

QC 20180726

Available from: 2018-07-26 Created: 2018-07-26 Last updated: 2018-07-26Bibliographically approved
Ståhl, S., Gräslund, T., Eriksson Karlström, A., Frejd, F. Y., Nygren, P.-Å. & Löfblom, J. (2017). Affibody Molecules in Biotechnological and Medical Applications. Trends in Biotechnology, 35(8), 691-712
Open this publication in new window or tab >>Affibody Molecules in Biotechnological and Medical Applications
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2017 (English)In: Trends in Biotechnology, ISSN 0167-7799, E-ISSN 1879-3096, Vol. 35, no 8, p. 691-712Article, review/survey (Refereed) Published
Abstract [en]

Affibody molecules are small (6.5-kDa) affinity proteins based on a three-helix bundle domain framework. Since their introduction 20 years ago as an alternative to antibodies for biotechnological applications, the first therapeutic affibody molecules have now entered clinical development and more than 400 studies have been published in which affibody molecules have been developed and used in a variety of contexts. In this review, we focus primarily on efforts over the past 5 years to explore the potential of affibody molecules for medical applications in oncology, neurodegenerative, and inflammation disorders, including molecular imaging, receptor signal blocking, and delivery of toxic payloads. In addition, we describe recent examples of biotechnological applications, in which affibody molecules have been exploited as modular affinity fusion partners.

Place, publisher, year, edition, pages
Elsevier, 2017
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-211597 (URN)10.1016/j.tibtech.2017.04.007 (DOI)000405847200005 ()
Note

QC 20170815

Available from: 2017-08-15 Created: 2017-08-15 Last updated: 2017-08-15Bibliographically approved
Allerbring, E. B., Duru, A. D., Chadderton, J., Markov, N., Uchtenhagen, H., Popov, A., . . . Achour, A. (2015). Structural and thermodynamic basis underlying in vivo reestablishment of T-cell recognition of a viral escape mutant. Paper presented at 8th International Workshop on Antigen Processing and Presentation, JUN 10-13, 2014, Philadelphia, PA. Molecular Immunology, 68(2), 151-151
Open this publication in new window or tab >>Structural and thermodynamic basis underlying in vivo reestablishment of T-cell recognition of a viral escape mutant
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2015 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 68, no 2, p. 151-151Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Elsevier, 2015
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-180623 (URN)000366767700073 ()
Conference
8th International Workshop on Antigen Processing and Presentation, JUN 10-13, 2014, Philadelphia, PA
Note

QC 20160120

Available from: 2016-01-20 Created: 2016-01-19 Last updated: 2017-11-30Bibliographically approved
Sekiguchi, M., Iwasaki, T., Shibasaki, S., Nygren, P.-Å., Gräslund, T. & Sano, H. (2014). Affibody Molecules Inhibiting The Interaction Between Ras And Raf Suppress The Proliferation And The Production Of Inflammatory Mediators By Synovial Cells. Paper presented at 15th Annual European Congress of Rheumatology (EULAR), JUN 11-14, 2014, Paris, France. Annals of the Rheumatic Diseases, 73, 848-848
Open this publication in new window or tab >>Affibody Molecules Inhibiting The Interaction Between Ras And Raf Suppress The Proliferation And The Production Of Inflammatory Mediators By Synovial Cells
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2014 (English)In: Annals of the Rheumatic Diseases, ISSN 0003-4967, E-ISSN 1468-2060, Vol. 73, p. 848-848Article in journal, Meeting abstract (Other academic) Published
National Category
Rheumatology and Autoimmunity
Identifiers
urn:nbn:se:kth:diva-169164 (URN)10.1136/annrheumdis-2014-eular.2553 (DOI)000346919804447 ()
Conference
15th Annual European Congress of Rheumatology (EULAR), JUN 11-14, 2014, Paris, France
Note

QC 20150612

Available from: 2015-06-12 Created: 2015-06-11 Last updated: 2017-12-04Bibliographically approved
Yu, F., Gudmundsdotter, L., Akal, A., Gunneriusson, E., Frejd, F. & Nygren, P.-Å. (2014). An affibody-adalimumab hybrid blocks combined IL-6 and TNF-triggered serum amyloid A secretion in vivo. mAbs, 6(6), 1598-1607
Open this publication in new window or tab >>An affibody-adalimumab hybrid blocks combined IL-6 and TNF-triggered serum amyloid A secretion in vivo
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2014 (English)In: mAbs, ISSN 1942-0862, Vol. 6, no 6, p. 1598-1607Article in journal (Refereed) Published
Abstract [en]

In inflammatory disease conditions, the regulation of the cytokine system is impaired, leading to tissue damages. Here, we used protein engineering to develop biologicals suitable for blocking a combination of inflammation driving cytokines by a single construct. From a set of interleukin (IL)-6-binding affibody molecules selected by phage display, five variants with a capability of blocking the interaction between complexes of soluble IL-6 receptor a (sIL-6R alpha) and IL6 and the co-receptor gp130 were identified. In cell assays designed to analyze any blocking capacity of the classical or the alternative (trans) signaling IL-6 pathways, one variant, Z(IL-6_13) with an affinity (K-D) for IL-6 of similar to 500 pM, showed the best performance. To construct fusion proteins ("AffiMabs") with dual cytokine specificities, Z(IL-6_13) was fused to either the N-or C-terminus of both the heavy and light chains of the anti-tumor necrosis factor (TNF) monoclonal antibody adalimumab (Humira (R)). One AffiMab construct with Z(IL-6_13) positioned at the N-terminus of the heavy chain, denoted Z(IL-6_13)-HCAda, was determined to be the most optimal, and it was subsequently evaluated in an acute Serum Amyloid A (SAA) model in mice. Administration of the AffiMab or adalimumab prior to challenge with a mix of IL-6 and TNF reduced the levels of serum SAA in a dose-dependent manner. Interestingly, the highest dose (70 mg/kg body weight) of adalimumab only resulted in a 50% reduction of SAA-levels, whereas the corresponding dose of the Z(IL-6_13)-HCAda AffiMab with combined IL-6/TNF specificity, resulted in SAA levels below the detection limit.

Keywords
AffiMab, Antibody, IL-6, TNF, adalimumab, affibody, affinity, fusion, inflammation
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-159385 (URN)10.4161/mabs.36089 (DOI)000346878600024 ()2-s2.0-84920827599 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20150203

Available from: 2015-02-03 Created: 2015-01-29 Last updated: 2015-04-16Bibliographically approved
Shibasaki, S., Karasaki, M., Gräslund, T., Nygren, P.-Å., Sano, H. & Iwasaki, T. (2014). Inhibitory effects of H-Ras/Raf-1–binding affibody molecules on synovial cell function. AMB Express, 4(82)
Open this publication in new window or tab >>Inhibitory effects of H-Ras/Raf-1–binding affibody molecules on synovial cell function
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2014 (English)In: AMB Express, ISSN 2191-0855, E-ISSN 2191-0855, Vol. 4, no 82Article in journal (Refereed) Published
Abstract [en]

Affibody molecules specific for H-Ras and Raf-1 were evaluated for their ability to inhibit synovial cell function. Affibody molecules targeting H-Ras (Zras122, Zras220, and Zras521) or Raf-1 (Zraf322) were introduced into the MH7A synovial cell line using two delivery methods: transfection with plasmids encoding the affibody molecules or direct introduction of affibody protein using a cell-penetrating peptide reagent. Interleukin-6 (IL-6) and prostaglandin E2 (PGE2) production by MH7A cells were analyzed by enzyme-linked immunosorbent assay after stimulation with tumor necrosis factor-alpha (TNF-α). Cell proliferation was also analyzed. Phosphorylation of extracellular signal-regulated kinase (ERK) was analyzed by western blot. All affibody molecules could inhibit IL-6 and PGE2 production in TNF-α-stimulated MH7A cells. The inhibitory effect was stronger when affibody molecules were delivered as proteins via a cell-penetrating peptide reagent than when plasmid-DNA encoding the affibody moelcules was transfected into the cells. Plasmid-expressed Zras220 inhibited phosphorylation of ERK in TNF-α-stimulated MH7A cells. Protein-introduced Zraf322 inhibited the production of IL-6 and PGE2 and inhibited cell proliferation in MH7A cells. These findings suggest that affibody molecules specific for H-Ras and Raf-1 can affect intracellular signal transduction through the MAP kinase pathway to inhibit cell proliferation and production of inflammatory mediators by synovial cells.

Place, publisher, year, edition, pages
Springer Berlin/Heidelberg, 2014
Keywords
affinity cancer
National Category
Biological Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-164186 (URN)10.1186/s13568-014-0082-3 (DOI)000358068500001 ()2-s2.0-84957590363 (Scopus ID)
Note

QC 20150420

Available from: 2015-04-14 Created: 2015-04-14 Last updated: 2017-12-04Bibliographically approved
Jansson, R., Thatikonda, N., Lindberg, D., Rising, A., Johansson, J., Nygren, P.-Å. & Hedhammar, M. (2014). Recombinant Spider Silk Genetically Functionalized with Affinity Domains. Biomacromolecules, 15(5), 1696-1706
Open this publication in new window or tab >>Recombinant Spider Silk Genetically Functionalized with Affinity Domains
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2014 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 15, no 5, p. 1696-1706Article in journal (Refereed) Published
Abstract [en]

Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.

Keywords
Streptococcal Protein-G, Binding-Proteins, Fusion Proteins, Serum-Albumin, Fibroin, Cell, Fibers, Biomaterials, Antibody, Bundle
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-146554 (URN)10.1021/bm500114e (DOI)000335939800016 ()2-s2.0-84900422237 (Scopus ID)
Funder
Swedish Research CouncilVinnova
Note

QC 20140612

Available from: 2014-06-12 Created: 2014-06-12 Last updated: 2017-12-05Bibliographically approved
Uchtenhagen, H., Abualrous, E. T., Stahl, E., Allerbring, E. B., Sluijter, M., Zacharias, M., . . . Achour, A. (2013). Proline substitution independently enhances H-2D(b) complex stabilization and TCR recognition of melanoma-associated peptides. European Journal of Immunology, 43(11), 3051-3060
Open this publication in new window or tab >>Proline substitution independently enhances H-2D(b) complex stabilization and TCR recognition of melanoma-associated peptides
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2013 (English)In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 43, no 11, p. 3051-3060Article in journal (Refereed) Published
Abstract [en]

The immunogenicity of H-2D(b) (D-b) restricted epitopes can be significantly increased by substituting peptide position 3 to a proline (p3P). The p3P modification enhances MHC stability without altering the conformation of the modified epitope allowing for T-cell cross-reactivity with the native peptide. The present study reveals how specific interactions between p3P and the highly conserved MHC heavy chain residue Y159 increase the stability of D-b in complex with an optimized version of the melanoma-associated epitope gp100(25-33). Furthermore, the p3P modification directly increased the affinity of the D-b/gp100(25-33)-specific T-cell receptor (TCR) pMel. Surprisingly, the enhanced TCR binding was independent from the observed increased stability of the optimized D-b/gp100(25-33) complex and from the interactions formed between p3P and Y159, indicating a direct effect of the p3P modification on TCR recognition.

Keywords
MHC, Structural Biology, T-cell epitope, TCR, Tumor antigen
National Category
Immunology
Identifiers
urn:nbn:se:kth:diva-140169 (URN)10.1002/eji.201343456 (DOI)000328074000012 ()2-s2.0-84884337225 (Scopus ID)
Funder
Swedish Research CouncilSwedish Cancer Society
Note

QC 20140120

Available from: 2014-01-20 Created: 2014-01-17 Last updated: 2017-12-06Bibliographically approved
Neiman, M., Fredolini, C., Johansson, H., Lehtiö, J., Nygren, P.-Å., Uhlén, M., . . . Schwenk, J. M. (2013). Selectivity analysis of single binder assays used in plasma protein profiling. Proteomics, 13(23-24), 3406-3410
Open this publication in new window or tab >>Selectivity analysis of single binder assays used in plasma protein profiling
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2013 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 13, no 23-24, p. 3406-3410Article in journal (Refereed) Published
Abstract [en]

The increasing availability of antibodies toward human proteins enables broad explorations of the proteomic landscape in cells, tissues, and body fluids. This includes assays with antibody suspension bead arrays that generate protein profiles of plasma samples by flow cytometer analysis. However, antibody selectivity is context dependent so it is necessary to corroborate on-target detection over off-target binding. To address this, we describe a concept to directly verify interactions from antibody-coupled beads by analysis of their eluates by Western blots and MS. We demonstrate selective antibody binding in complex samples with antibodies toward a set of chosen proteins with different abundance in plasma and serum, and illustrate the need to adjust sample and bead concentrations accordingly. The presented approach will serve as an important tool for resolving differential protein profiles from antibody arrays within plasma biomarker discoveries.

Keywords
Antibodies, Plasma profiling, Protein arrays, Suspension bead arrays, Verification
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-139859 (URN)10.1002/pmic.201300030 (DOI)000329994000005 ()2-s2.0-84890306435 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceVinnovaKnut and Alice Wallenberg Foundation
Note

QC 20140122

Available from: 2014-01-22 Created: 2014-01-15 Last updated: 2017-12-06Bibliographically approved
Yu, F., Järver, P. & Nygren, P.-Å. (2013). Tailor-Making a Protein A-Derived Domain for Efficient Site-Specific Photocoupling to Fc of Mouse IgG(1). PLoS ONE, 8(2), e56597
Open this publication in new window or tab >>Tailor-Making a Protein A-Derived Domain for Efficient Site-Specific Photocoupling to Fc of Mouse IgG(1)
2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 2, p. e56597-Article in journal (Refereed) Published
Abstract [en]

Affinity proteins binding to antibody constant regions have proved to be invaluable tools in biotechnology. Here, protein engineering was used to expand the repertoire of available immunoglobulin binding proteins via improvement of the binding strength between the widely used staphylococcal protein A-derived Z domain and the important immunoglobulin isotype mouse IgG(1) (mIgG(1)). Addressing seven positions in the 58-residue three-helix bundle Z domain by single or double amino acid substitutions, a total of 170 variants were individually constructed, produced in E. coli and tested for binding to a set of mouse IgG(1) monoclonal antibodies (mAbs). The best variant, denoted Z(F5I) corresponding to a Phe to Ile substitution at position 5, showed a typical ten-fold higher affinity than the wild-type as determined by biosensor technology. Eight amino acid positions in the Z(F5I) variant were separately mutated to cysteine for incorporation of a photoactivable maleimide-benzophenone (MBP) group as a probe for site-specific photoconjugation to Fc of mIgG(1), The best photocoupling efficiency to mIgG(1) Fc was seen when the MBP group was coupled to Cys at position 32, resulting in adduct formation to more than 60% of all heavy chains, with no observable non-selective conjugation to the light chains. A similar coupling yield was obtained for a panel of 19 different mIgG(1) mAbs, indicating a general characteristic. To exemplify functionalization of a mIgG(1) antibody via site-specific biotinylation, the Z(F5I-Q32C-MBP) protein was first biotinylated using an amine reactive reagent and subsequently photoconjugated to an anti-human interferon-gamma mIgG(1) mAb. When comparing the specific antigen binding ability of the probe-biotinylated mAb to that of the directly biotinylated mAb, a significantly h`igher bioactivity was observed for the sample biotinylated using the Z(F5I-Q32C-MBP) probe. This result indicates that the use of a site-specific and affinity probe-mediated conjugation strategy can result in antibody reagents with increased assay sensitivity.

Keywords
Antibody-Binding, Biotinylation, Substrate, Fragment
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-121127 (URN)10.1371/journal.pone.0056597 (DOI)000315965100057 ()2-s2.0-84873724699 (Scopus ID)
Note

QC 20130419

Available from: 2013-04-19 Created: 2013-04-19 Last updated: 2017-12-06Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-4214-6991

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