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Hober, S., Lindbo, S. & Nilvebrant, J. (2019). Bispecific applications of non-immunoglobulin scaffold binders. Methods, 154, 143-152
Open this publication in new window or tab >>Bispecific applications of non-immunoglobulin scaffold binders
2019 (English)In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 154, p. 143-152Article in journal (Refereed) Published
Abstract [en]

Non-immunoglobulin scaffolds represent a proven group of small affinity proteins that can be engineered in vitro to similar affinity and potency as monoclonal antibodies. Several novel candidate biotherapeutics that exploit the potential advantages scaffold proteins hold over larger and more complex antibodies have been developed over the past decade. The ease of using small and robust binding proteins as flexible and modular building blocks has led to the development of a wide range of innovative approaches to combine them in various bi- and multispecific formats. This progress is expected to aid the ongoing challenge of identifying niche applications where clear differentiation from antibody-based molecules will be key to success. Given the many engineering options that are available for non-immunoglobulin scaffold proteins, they have potential to not only complement but probably also surpass antibodies in certain applications.

Place, publisher, year, edition, pages
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2019
Keywords
Non-immunoglobulin scaffold, In vitro evolution, Protein engineering, Bispecific, Biparatopic
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-244538 (URN)10.1016/j.ymeth.2018.09.010 (DOI)000457813100017 ()30287281 (PubMedID)2-s2.0-85054456517 (Scopus ID)
Note

QC 20190403

Available from: 2019-04-03 Created: 2019-04-03 Last updated: 2019-08-30Bibliographically approved
Garousi, J., Lindbo, S., Borin, J., von Witting, E., Vorobyeva, A., Oroujeni, M., . . . Hober, S. (2019). Comparative evaluation of dimeric and monomeric forms of ADAPT scaffold protein for targeting of HER2-expressing tumours. European journal of pharmaceutics and biopharmaceutics, 134, 37-48
Open this publication in new window or tab >>Comparative evaluation of dimeric and monomeric forms of ADAPT scaffold protein for targeting of HER2-expressing tumours
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2019 (English)In: European journal of pharmaceutics and biopharmaceutics, ISSN 0939-6411, E-ISSN 1873-3441, Vol. 134, p. 37-48Article in journal (Refereed) Published
Abstract [en]

ADAPTs are small engineered non-immunoglobulin scaffold proteins, which have demonstrated very promising features as vectors for radionuclide tumour targeting. Radionuclide imaging of human epidermal growth factor 2 (HER2) expression in vivo might be used for stratification of patients for HER2-targeting therapies. ADAPT6, which specifically binds to HER2, has earlier been shown to have very promising features for in vivo targeting of HER2 expressing tumours. In this study we tested the hypothesis that dimerization of ADAPT6 would increase the apparent affinity to HER2 and accordingly improve tumour targeting. To find an optimal molecular design of dimers, a series of ADAPT dimers with different linkers, -SSSG- (DiADAPT6L1), -(SSSG)(2)- (DiADAPT6L2), and -(SSSG)(3)- (DiADAPT6L3) was evaluated. Dimers in combination with optimal linker lengths demonstrated increased apparent affinity to HER2. The best variants, DiADAPT6L2 and DiADAPT6L3 were site-specifically labelled with In-111 and I-125, and compared with a monomeric ADAPT6 in mice bearing HER2-expressing tumours. Despite higher affinity, both dimers had lower tumour uptake and lower tumour-to-organ ratios compared to the monomer. We conclude that improved affinity of a dimeric form of ADAPT does not compensate the disadvantage of increased size. Therefore, increase of affinity should be obtained by affinity maturation and not by dimerization.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE BV, 2019
Keywords
ADAPT, HER2, Dimer, Radionuclide molecular imaging, Indium-111, Iodine-125
National Category
Pharmaceutical Biotechnology
Identifiers
urn:nbn:se:kth:diva-243967 (URN)10.1016/j.ejpb.2018.11.004 (DOI)000456225000004 ()30408518 (PubMedID)2-s2.0-85056893627 (Scopus ID)
Note

QC 20190301

Available from: 2019-03-01 Created: 2019-03-01 Last updated: 2019-03-01Bibliographically approved
Scheffel, J., Kanje, S., Borin, J. & Hober, S. (2019). Optimization of a calcium-dependent Protein A-derived domain for mild antibody purification. mAbs
Open this publication in new window or tab >>Optimization of a calcium-dependent Protein A-derived domain for mild antibody purification
2019 (English)In: mAbs, ISSN 1942-0862, E-ISSN 1942-0870Article in journal (Refereed) Published
Abstract [en]

As reported here, we developed and optimized a purification matrix based on a Protein A-derived domain, Z(Ca), displaying calcium-dependent antibody binding. It provides an alternative to the acidic elution conditions of conventional Protein A affinity chromatography for purification of sensitive antibodies and other Fc-based molecules. We describe the multimerization of Z(Ca) to generate a chromatography resin with higher binding capacity. The highest order multimeric variant, Z(Ca)TetraCys, demonstrated a considerably high dynamic binding capacity (35 mg IgG/ml resin) while preserving the specificity for IgG. High recovery was obtained and host cell protein and DNA content in purified fractions proved to be comparable to commercial MabSelect SuRe and MabSelect PrismA. Various elution conditions for use of this domain in antibody purification were investigated. The purification data presented here revealed variations in the interaction of different subclasses of human IgG with Z(Ca)TetraCys. This resulted in diverse elution properties for the different IgGs, where complete elution of all captured antibody for IgG2 and IgG4 was possible at neutral pH. This optimized protein ligand and the proposed purification method offer a unique strategy for effective and mild purification of antibodies and Fc-fusion proteins that cannot be purified under conventional acidic elution conditions due to aggregation formation or loss of function.

Place, publisher, year, edition, pages
TAYLOR & FRANCIS INC, 2019
Keywords
Protein A, purification, calcium-dependent, antibody, multimerization, elution
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-261301 (URN)10.1080/19420862.2019.1662690 (DOI)000486888100001 ()31526164 (PubMedID)
Note

QC 20191008

Available from: 2019-10-08 Created: 2019-10-08 Last updated: 2019-10-08Bibliographically approved
Liu, H., Lindbo, S., Ding, H., Altai, M., Garousi, J., Orlova, A., . . . Gräslund, T. (2019). Potent and specific fusion toxins consisting of a HER2‑binding, ABD‑derived affinity protein, fused to truncated versions of Pseudomonas exotoxin A. International Journal of Oncology, 55(1), 309-319
Open this publication in new window or tab >>Potent and specific fusion toxins consisting of a HER2‑binding, ABD‑derived affinity protein, fused to truncated versions of Pseudomonas exotoxin A
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2019 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 55, no 1, p. 309-319Article in journal (Refereed) Published
Abstract [en]

Fusion toxins consisting of an affinity protein fused to toxic polypeptides derived from Pseudomonas exotoxin A (ETA) are promising agents for targeted cancer therapy. In this study, we examined whether fusion toxins consisting of an albumin binding domain-derived affinity protein (ADAPT) interacting with human epidermal growth factor receptor 2 (HER2), coupled to the ETA-derived polypeptides PE38X8 or PE25, with or without an albumin binding domain (ABD) for half-life extension, can be used for specific killing of HER2-expressing cells. The fusion toxins could easily be expressed in a soluble form in Escherichia coli and purified to homogeneity. All constructs had strong affinity for HER2 (K-D 10 to 26 nM) and no tendency for aggregation could be detected. The fusion toxins including the ABD showed strong interaction with human and mouse serum albumin [equilibrium dissociation constant (K-D) 1 to 3 nM and 2 to 10 nM, respectively]. The in vitro investigation of the cytotoxic potential revealed IC50-values in the picomolar range for cells expressing high levels of HER2. The specificity was also demonstrated, by showing that free HER2 receptors on the target cells are required for fusion toxin activity. In mice, the fusion toxins containing the ABD exhibited an appreciably longer time in circulation. The uptake was highest in liver and kidney. Fusion with PE25 was associated with the highest hepatic uptake. Collectively, the results suggest that fusion toxins consisting of ADAPTs and ETA-derivatives are promising agents for targeted cancer therapy.

Place, publisher, year, edition, pages
Spandidos Publications, 2019
Keywords
exotoxin A, Pseudomonas, ABD-derived affinity protein, half-life extension, cancer, human epidermal growth factor receptor 2
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-255762 (URN)10.3892/ijo.2019.4814 (DOI)000476557600026 ()31180549 (PubMedID)2-s2.0-85068411992 (Scopus ID)
Note

QC 20190812

Available from: 2019-08-12 Created: 2019-08-12 Last updated: 2019-08-12Bibliographically approved
Tolmachev, V., Lindbo, S., Altai, M., von Witting, E., Vorobyeva, A., Oroujeni, M., . . . Hober, S. (2019). Selection of the optimal macrocyclic chelators for labelling with In-111 and Ga-68 improves contrast of HER2 imaging using engineered scaffold protein ADAPT6. Journal of labelled compounds & radiopharmaceuticals, 62, S100-S101
Open this publication in new window or tab >>Selection of the optimal macrocyclic chelators for labelling with In-111 and Ga-68 improves contrast of HER2 imaging using engineered scaffold protein ADAPT6
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2019 (English)In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 62, p. S100-S101Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
WILEY, 2019
National Category
Biochemicals
Identifiers
urn:nbn:se:kth:diva-254029 (URN)000468965200073 ()
Note

QC 20190814

Available from: 2019-08-14 Created: 2019-08-14 Last updated: 2019-08-14Bibliographically approved
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: 2019-01-28Bibliographically 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
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-0605-8417

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