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Edfors, F., Hober, A., Linderbäck, K., Maddalo, G., Azimi, A., Sivertsson, Å., . . . Uhlén, M. (2018). Enhanced validation of antibodies for research applications. Nature Communications, 9, Article ID 4130.
Open this publication in new window or tab >>Enhanced validation of antibodies for research applications
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2018 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 4130Article in journal (Refereed) Published
Abstract [en]

There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation pillars were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:kth:diva-237096 (URN)10.1038/s41467-018-06642-y (DOI)000446566000016 ()30297845 (PubMedID)2-s2.0-85054574300 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut and Alice Wallenberg Foundation
Note

QC 20181030

Available from: 2018-10-30 Created: 2018-10-30 Last updated: 2018-10-30Bibliographically approved
Lindbo, S., Garousi, J., Mitran, B., Vorobyeva, A., Oroujeni, M., Orlova, A., . . . Tolmachev, V. (2018). Optimized molecular design of ADAPT-based HER2-imaging probes labelled with 111In and 68Ga. Molecular Pharmaceutics, 15(7), 2674-2683
Open this publication in new window or tab >>Optimized molecular design of ADAPT-based HER2-imaging probes labelled with 111In and 68Ga
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2018 (English)In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 15, no 7, p. 2674-2683Article in journal (Refereed) Published
Abstract [en]

Radionuclide molecular imaging is a promising tool for visualization of cancer associated molecular abnormalities in vivo and stratification of patients for specific therapies. ADAPT is a new type of small engineered proteins based on the scaffold of an albumin binding domain of protein G. ADAPTs have been utilized to select and develop high affinity binders to different proteinaceous targets. ADAPT6 binds to human epidermal growth factor 2 (HER2) with low nanomolar affinity and can be used for its in vivo visualization. Molecular design of 111In-labeled anti-HER2 ADAPT has been optimized in several earlier studies. In this study, we made a direct comparison of two of the most promising variants, having either a DEAVDANS or a (HE)3DANS sequence at the N-terminus, conjugated with a maleimido derivative of DOTA to a GSSC amino acids sequence at the C-terminus. The variants (designated DOTA-C59-DEAVDANS-ADAPT6-GSSC and DOTA-C61-(HE)3DANS-ADAPT6-GSSC) were stably labeled with 111In for SPECT and 68Ga for PET. Biodistribution of labeled ADAPT variants was evaluated in nude mice bearing human tumor xenografts with different levels of HER2 expression. Both variants enabled clear discrimination between tumors with high and low levels of HER2 expression. 111In-labeled ADAPT6 derivatives provided higher tumor-to-organ ratios compared to 68Ga-labeled counterparts. The best performing variant was DOTA-C61-(HE)3DANS-ADAPT6-GSSC, which provided tumor-to-blood ratios of 208 ± 36 and 109 ± 17 at 3 h for 111In and 68Ga labels, respectively.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2018
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-238508 (URN)10.1021/acs.molpharmaceut.8b00204 (DOI)000448490100018 ()2-s2.0-85048138088 (Scopus ID)
Funder
Swedish Cancer Society, CAN 2015/350 2017/425Swedish Research Council, 2015-02353 2015-02509VINNOVA, 2016-04060
Note

QC 20181213

Available from: 2018-11-04 Created: 2018-11-04 Last updated: 2018-12-13Bibliographically approved
Kanje, S., Venskutonytė, R., Scheffel, J., Nilvebrant, J., Lindkvist-Petersson, K. & Hober, S. (2018). Protein engineering allows for mild affinity-based elution of therapeutic antibodies. Journal of Molecular Biology, 430(18), 3427-3438
Open this publication in new window or tab >>Protein engineering allows for mild affinity-based elution of therapeutic antibodies
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2018 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, no 18, p. 3427-3438Article in journal (Refereed) Published
Abstract [en]

Presented here is an engineered protein domain, based on Protein A, that displays a calcium-dependent binding to antibodies. This protein, ZCa, is shown to efficiently function as an affinity ligand for mild purification of antibodies through elution with ethylenediaminetetraacetic acid. Antibodies are commonly used tools in the area of biological sciences and as therapeutics, and the most commonly used approach for antibody purification is based on Protein A using acidic elution. Although this affinity-based method is robust and efficient, the requirement for low pH elution can be detrimental to the protein being purified. By introducing a calcium-binding loop in the Protein A-derived Z domain, it has been re-engineered to provide efficient antibody purification under mild conditions. Through comprehensive analyses of the domain as well as the ZCa–Fc complex, the features of this domain are well understood. This novel protein domain provides a very valuable tool for effective and gentle antibody and Fc-fusion protein purification

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
antibody purification, calcium-dependent binding, Protein A, protein engineering, Z domain
National Category
Pharmaceutical Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-238507 (URN)10.1016/j.jmb.2018.06.004 (DOI)000444668100025 ()2-s2.0-85048734234 (Scopus ID)
Funder
VINNOVA
Note

QC 20181130

Available from: 2018-11-04 Created: 2018-11-04 Last updated: 2018-11-30Bibliographically approved
Wu, Y.-T., Qiu, X., Lindbo, S., Susumu, K., Medintz, I. L., Hober, S. & Hildebrandt, N. (2018). Quantum Dot-Based FRET Immunoassay for HER2 Using Ultrasmall Affinity Proteins. Small, 14(35), Article ID 1802266.
Open this publication in new window or tab >>Quantum Dot-Based FRET Immunoassay for HER2 Using Ultrasmall Affinity Proteins
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2018 (English)In: Small, ISSN 1613-6810, E-ISSN 1613-6829, Vol. 14, no 35, article id 1802266Article in journal (Refereed) Published
Abstract [en]

Engineered scaffold affinity proteins are used in many biological applications with the aim of replacing natural antibodies. Although their very small sizes are beneficial for multivalent nanoparticle conjugation and efficient Forster resonance energy transfer (FRET), the application of engineered affinity proteins in such nanobiosensing formats has been largely neglected. Here, it is shown that very small (approximate to 6.5 kDa) histidine-tagged albumin-binding domain-derived affinity proteins (ADAPTs) can efficiently self-assemble to zwitterionic ligand-coated quantum dots (QDs). These ADAPT-QD conjugates are significantly smaller than QD-conjugates based on IgG, Fab', or single-domain antibodies. Immediate applicability by the quantification of the human epidermal growth factor receptor 2 (HER2) in serum-containing samples using time-gated Tb-to-QD FRET detection on the clinical benchtop immunoassay analyzer KRYPTOR is demonstrated here. Limits of detection down to 40 x 10(-12)m (approximate to 8 ng mL(-1)) are in a relevant clinical concentration range and outperform previously tested assays with antibodies, antibody fragments, and nanobodies.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2018
Keywords
ADAPT, HER2, nanoparticle, nonantibody scaffold, terbium
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-234590 (URN)10.1002/smll.201802266 (DOI)000443012700020 ()30079524 (PubMedID)2-s2.0-85052370633 (Scopus ID)
Note

QC 20180914

Available from: 2018-09-14 Created: 2018-09-14 Last updated: 2018-09-14Bibliographically approved
Garousi, J., Lindbo, S., Mitran, B., Vorobyeva, A., Oroujeni, M., Orlova, A., . . . Tolmachev, V. (2018). Selection of the most optimal ADAPT6-based probe for imaging of HER2 using PET and SPECT. Paper presented at 31st Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 13-17, 2018, Dusseldorf, GERMANY. European Journal of Nuclear Medicine and Molecular Imaging, 45, S77-S78
Open this publication in new window or tab >>Selection of the most optimal ADAPT6-based probe for imaging of HER2 using PET and SPECT
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2018 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, p. S77-S78Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
SPRINGER, 2018
National Category
Basic Medicine
Identifiers
urn:nbn:se:kth:diva-239821 (URN)10.1007/s00259-018-4148-3 (DOI)000449266200131 ()2-s2.0-85056052770 (Scopus ID)
Conference
31st Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 13-17, 2018, Dusseldorf, GERMANY
Note

QC 20181217

Available from: 2018-12-18 Created: 2018-12-18 Last updated: 2018-12-18Bibliographically approved
Uhlén, M., Zhang, C., Lee, S., Sjöstedt, E., Fagerberg, L., Bidkhori, G., . . . Ponten, F. (2017). A pathology atlas of the human cancer transcriptome. Science, 357(6352), 660-+
Open this publication in new window or tab >>A pathology atlas of the human cancer transcriptome
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2017 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 357, no 6352, p. 660-+Article in journal (Refereed) Published
Abstract [en]

Cancer is one of the leading causes of death, and there is great interest in understanding the underlying molecular mechanisms involved in the pathogenesis and progression of individual tumors. We used systems-level approaches to analyze the genome-wide transcriptome of the protein-coding genes of 17 major cancer types with respect to clinical outcome. A general pattern emerged: Shorter patient survival was associated with up-regulation of genes involved in cell growth and with down-regulation of genes involved in cellular differentiation. Using genome-scale metabolic models, we show that cancer patients have widespread metabolic heterogeneity, highlighting the need for precise and personalized medicine for cancer treatment. All data are presented in an interactive open-access database (www.proteinatlas.org/pathology) to allow genome-wide exploration of the impact of individual proteins on clinical outcomes.

Place, publisher, year, edition, pages
American Association for the Advancement of Science, 2017
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-214334 (URN)10.1126/science.aan2507 (DOI)000407793600028 ()2-s2.0-85028362951 (Scopus ID)
Funder
Swedish Cancer SocietyScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut and Alice Wallenberg FoundationSwedish Research Council
Note

QC 20170913

Available from: 2017-09-13 Created: 2017-09-13 Last updated: 2018-10-17Bibliographically approved
Thul, P. J., Åkesson, L., Wiking, M., Mahdessian, D., Geladaki, A., Ait Blal, H., . . . Lundberg, E. (2017). A subcellular map of the human proteome. Science, 356(6340), Article ID 820.
Open this publication in new window or tab >>A subcellular map of the human proteome
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2017 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 356, no 6340, article id 820Article in journal (Refereed) Published
Abstract [en]

Resolving the spatial distribution of the human proteome at a subcellular level can greatly increase our understanding of human biology and disease. Here we present a comprehensive image-based map of subcellular protein distribution, the Cell Atlas, built by integrating transcriptomics and antibody-based immunofluorescence microscopy with validation by mass spectrometry. Mapping the in situ localization of 12,003 human proteins at a single-cell level to 30 subcellular structures enabled the definition of the proteomes of 13 major organelles. Exploration of the proteomes revealed single-cell variations in abundance or spatial distribution and localization of about half of the proteins to multiple compartments. This subcellular map can be used to refine existing protein-protein interaction networks and provides an important resource to deconvolute the highly complex architecture of the human cell.

Place, publisher, year, edition, pages
American Association for the Advancement of Science, 2017
Keywords
antibody, proteome, biology, cells and cell components, disease incidence, image analysis, physiological response, protein, proteomics, spatial distribution, Article, cell organelle, cellular distribution, human, human cell, immunofluorescence microscopy, mass spectrometry, priority journal, protein analysis, protein localization, protein protein interaction, single cell analysis, transcriptomics
National Category
Cell Biology
Identifiers
urn:nbn:se:kth:diva-216588 (URN)10.1126/science.aal3321 (DOI)000401957900032 ()2-s2.0-85019201137 (Scopus ID)
Note

QC 20171208

Available from: 2017-12-08 Created: 2017-12-08 Last updated: 2017-12-08Bibliographically approved
Garousi, J., Lindbo, S., Mitran, B., Buijs, J., Vorobyeva, A., Orlova, A., . . . Hober, S. (2017). Comparative evaluation of tumor targeting using the anti-HER2 ADAPT scaffold protein labeled at the C-terminus with indium-111 or technetium-99m. Scientific Reports, 7, Article ID 14780.
Open this publication in new window or tab >>Comparative evaluation of tumor targeting using the anti-HER2 ADAPT scaffold protein labeled at the C-terminus with indium-111 or technetium-99m
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 14780Article in journal (Refereed) Published
Abstract [en]

ABD-Derived Affinity Proteins (ADAPTs) is a novel class of engineered scaffold proteins derived from an albumin-binding domain of protein G. The use of ADAPT6 derivatives as targeting moiety have provided excellent preclinical radionuclide imaging of human epidermal growth factor 2 (HER2) tumor xenografts. Previous studies have demonstrated that selection of nuclide and chelator for its conjugation has an appreciable effect on imaging properties of scaffold proteins. In this study we performed a comparative evaluation of the anti-HER2 ADAPT having an aspartate-glutamate-alanine-valine-aspartate-alanine-asparagine-serine (DEAVDANS) N-terminal sequence and labeled at C-terminus with (99)mTc using a cysteine-containing peptide based chelator, glycine-serine-serine-cysteine (GSSC), and a similar variant labeled with In-111 using a maleimido derivative of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. Both (99)mTc-DEAVDANS-ADAPT6-GSSC and In-111-DEAVDANS-ADAPT6-GSSC-DOTA accumulated specifically in HER2-expressing SKOV3 xenografts. The tumor uptake of both variants did not differ significantly and average values were in the range of 19-21% ID/g. However, there was an appreciable variation in uptake of conjugates in normal tissues that resulted in a notable difference in the tumor-to-organ ratios. The In-111-DOTA label provided 2-6 fold higher tumor-to-organ ratios than (99)mTc-GSSC and is therefore the preferable label for ADAPTs.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2017
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-217934 (URN)10.1038/s41598-017-15366-w (DOI)000414569900003 ()2-s2.0-85033458225 (Scopus ID)
Note

QC 20171121

Available from: 2017-11-21 Created: 2017-11-21 Last updated: 2018-03-15Bibliographically approved
Lindbo, S., Garousi, J., Åstrand, M., Honarvar, H., Orlova, A., Hober, S. & Tolmachev, V. (2016). Influence of Histidine-Containing Tags on the Biodistribution of ADAPT Scaffold Proteins.. Bioconjugate chemistry, 27(3), 716-726
Open this publication in new window or tab >>Influence of Histidine-Containing Tags on the Biodistribution of ADAPT Scaffold Proteins.
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2016 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 27, no 3, p. 716-726Article in journal (Refereed) Published
Abstract [en]

Engineered scaffold proteins (ESP) are high-affinity binders that can be used as probes for radionuclide imaging. Histidine-containing tags enable both efficient purification of ESP and radiolabeling with (99m)Tc(CO)3. Earlier studies demonstrated that the use of a histidine-glutamate-histidine-glutamate-histidine-glutamate (HE)3-tag instead of the commonly used hexahistidine (H6)-tag reduces hepatic uptake of radiolabeled ESP and short peptides. Here, we investigated the influence of histidine-containing tags on the biodistribution of a novel type of ESP, ADAPTs. A series of anti-HER2 ADAPT probes having H6- or (HE)3-tags in the N-termini were prepared. The constructs, (HE)3-ADAPT6 and H6-ADAPT6, were labeled with two different nuclides, (99m)Tc or (111)In. The labeling with (99m)Tc(CO)3 utilized the histidine-containing tags, while (111)In was attached through a maleimido derivative of DOTA conjugated to the N-terminus. For (111)In-labeled ADAPTs, the use of (HE)3 provided a significantly (p < 0.05) lower hepatic uptake at 1 h after injection, but there was no significant difference in hepatic uptake of (111)In-(HE)3-ADAPT6 and H6-ADAPT6 at later time points. Interestingly, in the case of (99m)Tc, (99m)Tc(CO)3-H6-ADAPT6 provided significantly (p < 0.05) lower uptake in a number of normal tissues and was more suitable as an imaging probe. Thus, the influence of histidine-containing tags on the biodistribution of the novel ADAPT scaffold proteins was different compared to its influence on other ESPs studied so far. Apparently, the effect of a histidine-containing tag on the biodistribution is highly dependent on the scaffold composition of the ESP.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2016
National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-184209 (URN)10.1021/acs.bioconjchem.5b00677 (DOI)000372478600026 ()26781756 (PubMedID)2-s2.0-84962209236 (Scopus ID)
Funder
Swedish Cancer Society, CAN 2015/350Swedish Research Council, 2015-02353 621-2012-5088
Note

QC 20160405

Available from: 2016-03-30 Created: 2016-03-30 Last updated: 2018-03-15Bibliographically approved
Garousi, J., Lindbo, S., Honarvar, H., Velletta, J., Mitran, B., Altai, M., . . . Hober, S. (2016). Influence of the N -Terminal Composition on Targeting Properties of Radiometal-Labeled Anti-HER2 Scaffold Protein ADAPT6. Bioconjugate chemistry, 27(11), 2678-2688
Open this publication in new window or tab >>Influence of the N -Terminal Composition on Targeting Properties of Radiometal-Labeled Anti-HER2 Scaffold Protein ADAPT6
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2016 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 27, no 11, p. 2678-2688Article in journal (Refereed) Published
Abstract [en]

Radionuclide-imaging-based stratification of patients to targeted therapies makes cancer treatment more personalized and therefore more efficient. Albumin-binding domain derived affinity proteins (ADAPTs) constitute a novel group of imaging probes based on the scaffold of an albumin binding domain (ABD). To evaluate how different compositions of the N-terminal sequence of ADAPTS influence their biodistribution, a series of human epidermal growth factor receptor type 2 (HER2)-binding ADAPT6 derivatives with different N-terminal sequences were created: GCH(6)DANS (2), GC(HE)(3)DANS (3), GCDEAVDANS (4), and GCVD.ANS(5). These were compared with the parental variant: GCSS(HE)(3)DEAVDANS (1). All variants were site-specifically conjugated with a maleimido-derivative of a DOTA chelator and labeled with In-III. Binding to HER2-expressing cells in vitro, in vivo biodistribution as well as targeting properties of the new variants were compared with properties of the In-III-labeled parental ADAPT variant 1 (In-III-DOTA-1). The composition of the N-terminal sequence had an apparent influence on biodistribution of ADAPT6 in mice. The use of a hexahistidine tag in (InD)-In-III-OTA-2 was associated with elevated hepatic uptake compared to the (HE)(3)-containing counterpart, In-III-DOTA-3. All new variants without a hexahistidine tag demonstrated lower uptake in blood, lung, spleen, and muscle compared to uptake in the parental variant. The best new variants, In-III-DOTA-3 and In-III-DOTA-5, provided tumor uptakes of 14.6 +/- 2.4 and 12.5 +/- 1.3% ID/g at 4 h after injection, respectively. The tumor uptake of In-III-DOTA-3 was significantly higher than the uptake of the parental In-III-DOTA-1 (9.1 +/- 2.0% ID/g). The tumor-to-blood ratios of 395 +/- 75 and 419 +/- 91 at 4 h after injection were obtained for In-III-DOTA-5 and (IIII)n-DOTA-3, respectively. In conclusion, the N-terminal sequence composition affects the biodistribution and targeting properties of ADAPT-based imaging probes, and its optimization may improve imaging contrast.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2016
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-199556 (URN)10.1021/acs.bioconjchem.6b00465 (DOI)000388430700011 ()2-s2.0-84996523484 (Scopus ID)
Note

QC 20170113

Available from: 2017-01-13 Created: 2017-01-09 Last updated: 2018-03-15Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-0605-8417

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