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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
Tolmachev, V., Bragina, O., von Witting, E., Garousi, J., Zelchan, R., Sinilkin, I., . . . Chernov, V. (2019). First-in-humans Evaluation of [Tc-99m]-ADAPT6, a Novel Scaffold Protein for Visualizationof HER2 Expression. Paper presented at 32nd Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 12-16, 2019, Barcelona, SPAIN. European Journal of Nuclear Medicine and Molecular Imaging, 46(SUPPL 1), S166-S166
Open this publication in new window or tab >>First-in-humans Evaluation of [Tc-99m]-ADAPT6, a Novel Scaffold Protein for Visualizationof HER2 Expression
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2019 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 46, no SUPPL 1, p. S166-S166Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
SPRINGER, 2019
National Category
Clinical Medicine
Identifiers
urn:nbn:se:kth:diva-264314 (URN)000492444401115 ()
Conference
32nd Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 12-16, 2019, Barcelona, SPAIN
Note

QC 20191202

Available from: 2019-12-02 Created: 2019-12-02 Last updated: 2019-12-19Bibliographically 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)2-s2.0-85073955572 (Scopus ID)
Note

QC 20191008

Available from: 2019-10-08 Created: 2019-10-08 Last updated: 2020-03-09Bibliographically approved
Jennbacken, K., Wagberg, F., Karlsson, U., Eriksson, J., Magnusson, L., Chimienti, M., . . . Holmberg Schiavone, L. (2019). Phenotypic Screen with the Human Secretome Identifies FGF16 as Inducing Proliferation of iPSC-Derived Cardiac Progenitor Cells. International Journal of Molecular Sciences, 20(23), Article ID 6037.
Open this publication in new window or tab >>Phenotypic Screen with the Human Secretome Identifies FGF16 as Inducing Proliferation of iPSC-Derived Cardiac Progenitor Cells
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2019 (English)In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, no 23, article id 6037Article in journal (Refereed) Published
Abstract [en]

Paracrine factors can induce cardiac regeneration and repair post myocardial infarction by stimulating proliferation of cardiac cells and inducing the anti-fibrotic, antiapoptotic, and immunomodulatory effects of angiogenesis. Here, we screened a human secretome library, consisting of 923 growth factors, cytokines, and proteins with unknown function, in a phenotypic screen with human cardiac progenitor cells. The primary readout in the screen was proliferation measured by nuclear count. From this screen, we identified FGF1, FGF4, FGF9, FGF16, FGF18, and seven additional proteins that induce proliferation of cardiac progenitor cells. FGF9 and FGF16 belong to the same FGF subfamily, share high sequence identity, and are described to have similar receptor preferences. Interestingly, FGF16 was shown to be specific for proliferation of cardiac progenitor cells, whereas FGF9 also proliferated human cardiac fibroblasts. Biosensor analysis of receptor preferences and quantification of receptor abundances suggested that FGF16 and FGF9 bind to different FGF receptors on the cardiac progenitor cells and cardiac fibroblasts. FGF16 also proliferated naive cardiac progenitor cells isolated from mouse heart and human cardiomyocytes derived from induced pluripotent cells. Taken together, the data suggest that FGF16 could be a suitable paracrine factor to induce cardiac regeneration and repair.

Place, publisher, year, edition, pages
MDPI, 2019
Keywords
human secretome, phenotypic screening, cardiac progenitor cells, fibroblast growth factors, fibroblast growth factor 16
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-266544 (URN)10.3390/ijms20236037 (DOI)000504428300219 ()31801200 (PubMedID)2-s2.0-85075880909 (Scopus ID)
Note

QC 20200131

Available from: 2020-01-31 Created: 2020-01-31 Last updated: 2020-01-31Bibliographically 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
Garousi, J., von Witting, E., Lindbo, S., Vorobyeva, A., Altai, M., Oroujeni, M., . . . Tolmachev, V. (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. Paper presented at 32nd Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 12-16, 2019, Barcelona, SPAIN. European Journal of Nuclear Medicine and Molecular Imaging, 46(SUPPL 1), S131-S131
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: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 46, no SUPPL 1, p. S131-S131Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
SPRINGER, 2019
National Category
Clinical Medicine
Identifiers
urn:nbn:se:kth:diva-264303 (URN)000492444401051 ()
Conference
32nd Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 12-16, 2019, Barcelona, SPAIN
Note

QC 20191202

Available from: 2019-12-02 Created: 2019-12-02 Last updated: 2020-01-02Bibliographically 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: 2020-01-10Bibliographically 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 ()29865791 (PubMedID)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: 2020-03-09Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-0605-8417

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