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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
Rosestedt, M., Andersson, K. G., Mitran, B., Rinne, S. S., Tolmachev, V., Löfblom, J., . . . Ståhl, S. (2017). Evaluation of a radiocobalt-labelled affibody molecule for imaging of human epidermal growth factor receptor 3 expression. International Journal of Oncology, 51(6), 1765-1774
Open this publication in new window or tab >>Evaluation of a radiocobalt-labelled affibody molecule for imaging of human epidermal growth factor receptor 3 expression
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2017 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 51, no 6, p. 1765-1774Article in journal (Refereed) Published
Abstract [en]

The human epidermal growth factor receptor 3 (HER3) is involved in the development of cancer resistance towards tyrosine kinase-Targeted therapies. Several HER3-Targeting therapeutics are currently under clinical evaluation. Non-invasive imaging of HER3 expression could improve patient management. Affibody molecules are small engineered scaffold proteins demonstrating superior properties as targeting probes for molecular imaging compared with monoclonal antibodies. Feasibility of in vivo HER3 imaging using affibody molecules has been previously demonstrated. Preclinical studies have shown that the contrast when imaging using anti-HER3 affibody molecules can be improved over time. We aim to develop an agent for PET imaging of HER3 expression using the long-lived positron-emitting radionuclide cobalt-55 (55Co) (T1/2=17.5 h). A long-lived cobalt isotope 57Co was used as a surrogate for 55Co in this study. The anti-HER3 affibody molecule HEHEHE-ZHER3-NOTA was labelled with radiocobalt with high yield, purity and stability. Biodistribution of 57Co-HEHEHE-ZHER3-NOTA was measured in mice bearing DU145 (prostate carcinoma) and LS174T (colorectal carcinoma) xenografts at 3 and 24 h post injection (p.i.). Tumour-To-blood ratios significantly increased between 3 and 24 h p.i. (p<0.05). At 24 h p.i., tumour-To-blood ratios were 6 for DU145 and 8 for LS174T xenografts, respectively. HER3-expressing xenografts were clearly visualized in a preclinical imaging setting already 3 h p.i., and contrast further improved at 24 h p.i. In conclusion, the radiocobalt-labelled anti-HER3 affibody molecule, HEHEHE-ZHER3-NOTA, is a promising tracer for imaging of HER3 expression in tumours.

Place, publisher, year, edition, pages
Spandidos Publications, 2017
Keywords
A ffibody, Cobalt-55/57, H ER3, NOTAchelator, PET i maging
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:kth:diva-218113 (URN)10.3892/ijo.2017.4152 (DOI)2-s2.0-85033500587 (Scopus ID)
Note

QC 20171127

Available from: 2017-11-27 Created: 2017-11-27 Last updated: 2017-12-18Bibliographically 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
Bass, T., Rosestedt, M., Mitran, B., Frejd, F. Y., Löfblom, J., Tolmachev, V., . . . Orlova, A. (2017). In vivo evaluation of a novel format of a bivalent HER3-targeting and albumin- binding therapeutic affibody construct. Scientific Reports, 7, Article ID 43118.
Open this publication in new window or tab >>In vivo evaluation of a novel format of a bivalent HER3-targeting and albumin- binding therapeutic affibody construct
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 43118Article in journal (Refereed) Published
Abstract [en]

Overexpression of human epidermal growth factor receptor 3 (HER3) is involved in resistance to several therapies for malignant tumours. Currently, several anti-HER3 monoclonal antibodies are under clinical development. We introduce an alternative approach to HER3-targeted therapy based on engineered scaffold proteins, i.e. affibody molecules. We designed a small construct (22.5 kDa, denoted 3A3), consisting of two high-affinity anti-HER3 affibody molecules flanking an albumin-binding domain ABD, which was introduced for prolonged residence in circulation. In vitro, 3A3 efficiently inhibited growth of HER3-expressing BxPC-3 cells. Biodistribution in mice was measured using 3A3 that was site-specifically labelled with In-111 via a DOTA chelator. The residence time of In-111-DOTA-3A3 in blood was extended when compared with the monomeric affibody molecule. In-111-DOTA-3A3 accumulated specifically in HER3-expressing BxPC-3 xenografts in mice. However, In-111-DOTA-3A3 cleared more rapidly from blood than a size-matched control construct In-111-DOTA-TAT, most likely due to sequestering of 3A3 by mErbB3, the murine counterpart of HER3. Repeated dosing and increase of injected protein dose decreased uptake of In-111-DOTA-3A3 in mErbB3-expressing tissues. Encouragingly, growth of BxPC-3 xenografts in mice was delayed in an experimental (pilot-scale) therapy study using 3A3. We conclude that the 3A3 affibody format seems promising for treatment of HER3-overexpressing tumours.

Place, publisher, year, edition, pages
Nature Publishing Group, 2017
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-204078 (URN)10.1038/srep43118 (DOI)000394748000001 ()28230065 (PubMedID)2-s2.0-85013769093 (Scopus ID)
Note

QC 20170329

Available from: 2017-03-29 Created: 2017-03-29 Last updated: 2017-11-29Bibliographically approved
Garousi, J., Andersson, K. G., Dam, J. H., Olsen, B. B., Mitran, B., Orlova, A., . . . Tolmachev, V. (2017). The use of radiocobalt as a label improves imaging of EGFR using DOTA-conjugated Affibody molecule. Scientific Reports, 7, Article ID 5961.
Open this publication in new window or tab >>The use of radiocobalt as a label improves imaging of EGFR using DOTA-conjugated Affibody molecule
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 5961Article in journal (Refereed) Published
Abstract [en]

Several anti-cancer therapies target the epidermal growth factor receptor (EGFR). Radionuclide imaging of EGFR expression in tumours may aid in selection of optimal cancer therapy. The In-111-labelled DOTA-conjugated Z(EGFR:2377) Affibody molecule was successfully used for imaging of EGFR-expressing xenografts in mice. An optimal combination of radionuclide, chelator and targeting protein may further improve the contrast of radionuclide imaging. The aim of this study was to evaluate the targeting properties of radiocobalt-labelled DOTA-Z(EGFR:2377). DOTA-Z(EGFR:2377) was labelled with Co-57 (T-1/2 = 271.8 d), Co-55 (T-1/2 = 17.5 h), and, for comparison, with the positron-emitting radionuclide Ga-68 (T-1/2 = 67.6 min) with preserved specificity of binding to EGFR-expressing A431 cells. The long-lived cobalt radioisotope Co-57 was used in animal studies. Both Co-57-DOTA-Z(EGFR:2377) and Ga-68-DOTA-Z(EGFR:2377) demonstrated EGFR-specific accumulation in A431 xenografts and EGFR-expressing tissues in mice. Tumour-to-organ ratios for the radiocobalt-labelled DOTA-Z(EGFR:2377) were significantly higher than for the gallium-labelled counterpart already at 3 h after injection. Importantly, Co-57-DOTA-Z(EGFR:2377) demonstrated a tumour-to-liver ratio of 3, which is 7-fold higher than the tumour-to-liver ratio for (68)GaDOTA-Z(EGFR:2377). The results of this study suggest that the positron-emitting cobalt isotope 55Co would be an optimal label for DOTA-Z(EGFR:2377) and further development should concentrate on this radionuclide as a label.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2017
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-214350 (URN)10.1038/s41598-017-05700-7 (DOI)000405907800015 ()28729680 (PubMedID)2-s2.0-85025472392 (Scopus ID)
Note

QC 20170912

Available from: 2017-09-12 Created: 2017-09-12 Last updated: 2017-09-12Bibliographically approved
Rosestedt, M., Mitran, B., Andersson, K. G., Löfblom, J., Ståhl, S., Tolmachev, V. & Orlova, A. (2016). Development and Evaluation of Radiocobalt-labelled Affibody Molecule for Next Day PET Imaging of HER3 Expression. Paper presented at Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 15-19, 2016, Barcelona, SPAIN. European Journal of Nuclear Medicine and Molecular Imaging, 43, S37-S38
Open this publication in new window or tab >>Development and Evaluation of Radiocobalt-labelled Affibody Molecule for Next Day PET Imaging of HER3 Expression
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2016 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, p. S37-S38Article in journal (Refereed) Published
Place, publisher, year, edition, pages
Springer, 2016
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-201273 (URN)000391801600070 ()
Conference
Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 15-19, 2016, Barcelona, SPAIN
Note

QC 20170215

Available from: 2017-02-15 Created: 2017-02-15 Last updated: 2017-11-29Bibliographically approved
Andersson, K. G., Oroujeni, M., Garousi, J., Mitran, B., Ståhl, S., Orlova, A., . . . Tolmachev, V. (2016). Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with Tc-99m using a peptide-based cysteine-containing chelator. International Journal of Oncology, 49(6), 2285-2293
Open this publication in new window or tab >>Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with Tc-99m using a peptide-based cysteine-containing chelator
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2016 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 49, no 6, p. 2285-2293Article in journal (Refereed) Published
Abstract [en]

The epidermal growth factor receptor (EGFR) is overexpressed in a number of malignant tumors and is a molecular target for several specific anticancer antibodies and tyrosine kinase inhibitors. The overexpression of EGFR is a predictive biomarker for response to several therapy regimens. Radionuclide molecular imaging might enable detection of EGFR overexpression by a non-invasive procedure and could be used repeatedly. Affibody molecules are engineered scaffold proteins, which could be selected to have a high affinity and selectivity to predetermined targets. The anti-EGFR ZEGFR:2377 affibody molecule is a potential imaging probe for EGFR detection. The use of the generator-produced radionuclide Tc-99m should facilitate clinical translation of an imaging probe due to its low price, availability and favorable dosimetry of the radionuclide. In the present study, we evaluated feasibility of ZEGFR:2377 labeling with Tc-99m using a peptide-based cysteine-containing chelator expressed at the C-terminus of ZEGFR:2377. The label was stable in vitro under cysteine challenge. In addition, Tc-99m-ZEGFR:2377 was capable of specific binding to EGFR-expressing cells with high affinity (274 pM). Studies in BALB/C nu/nu mice bearing A431 xenografts demonstrated that Tc-99m-ZEGFR:2377 accumulates in tumors in an EGFR-specific manner. The tumor uptake values were 3.6 1 and 2.5 0.4% ID/g at 3 and 24 h after injection, respectively. The corresponding tumor-to-blood ratios were 1.8 0.4 and 8 3. The xenografts were clearly visualized at both time-points. This study demonstrated the potential of Tc-99m-labeled ZEGFR:2377 for imaging of EGFR in vivo.

Place, publisher, year, edition, pages
SPANDIDOS, 2016
Keywords
epidermal growth factor receptor, radionuclide molecular imaging, affibody molecules, technetium-99m, A431, biodistribution
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:kth:diva-199524 (URN)10.3892/ijo.2016.3721 (DOI)000389166000011 ()2-s2.0-84996565692 (Scopus ID)
Note

QC 20170116

Available from: 2017-01-16 Created: 2017-01-09 Last updated: 2017-11-29Bibliographically approved
Mitran, B., Güler, R., Lindstrom, E., Fleetwood, F., Tolmachev, V., Ståhl, S., . . . Löfblom, J. (2016). Feasibility of in vivo imaging of VEGFR2 expression using high affinity antagonistic biparatopic affibody construct Z(VEGFR2)-Bp(2). Paper presented at Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 15-19, 2016, Barcelona, SPAIN. European Journal of Nuclear Medicine and Molecular Imaging, 43, S97-S98
Open this publication in new window or tab >>Feasibility of in vivo imaging of VEGFR2 expression using high affinity antagonistic biparatopic affibody construct Z(VEGFR2)-Bp(2)
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2016 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, p. S97-S98Article in journal (Refereed) Published
Place, publisher, year, edition, pages
Springer, 2016
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-201270 (URN)000391801600232 ()
Conference
Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 15-19, 2016, Barcelona, SPAIN
Note

QC 20170215

Available from: 2017-02-15 Created: 2017-02-15 Last updated: 2017-06-29Bibliographically approved
Orlova, A., Rosestedt, M., Mitran, B., Bass, T., Frejd, F. Y., Löfblom, J., . . . Ståhl, S. (2016). In vivo evaluation of pharmacokinetics, tumors targeting and therapeutic efficacy of a novel format of HER3-targeting affibody molecule with prolonged blood circulation. Paper presented at Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 15-19, 2016, Barcelona, SPAIN. European Journal of Nuclear Medicine and Molecular Imaging, 43, S237-S237
Open this publication in new window or tab >>In vivo evaluation of pharmacokinetics, tumors targeting and therapeutic efficacy of a novel format of HER3-targeting affibody molecule with prolonged blood circulation
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2016 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, p. S237-S237Article in journal (Refereed) Published
Place, publisher, year, edition, pages
Springer, 2016
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-201271 (URN)000391801600595 ()
Conference
Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 15-19, 2016, Barcelona, SPAIN
Note

QC 20170215

Available from: 2017-02-15 Created: 2017-02-15 Last updated: 2017-11-29Bibliographically approved
Fleetwood, F., Güler, R., Gordon, E., Ståhl, S., Claesson-Welsh, L. & Löfblom, J. (2016). Novel affinity binders for neutralization of vascular endothelial growth factor (VEGF) signaling. Cellular and Molecular Life Sciences (CMLS), 73(8), 1671-1683
Open this publication in new window or tab >>Novel affinity binders for neutralization of vascular endothelial growth factor (VEGF) signaling
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2016 (English)In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 73, no 8, p. 1671-1683Article in journal (Refereed) Published
Abstract [en]

Angiogenesis denotes the formation of new blood vessels from pre-existing vasculature. Progression of diseases such as cancer and several ophthalmological disorders may be promoted by excess angiogenesis. Novel therapeutics to inhibit angiogenesis and diagnostic tools for monitoring angiogenesis during therapy, hold great potential for improving treatment of such diseases. We have previously generated so-called biparatopic Affibody constructs with high affinity for the vascular endothelial growth factor receptor-2 (VEGFR2), which recognize two non-overlapping epitopes in the ligand-binding site on the receptor. Affibody molecules have previously been demonstrated suitable for imaging purposes. Their small size also makes them attractive for applications where an alternative route of administration is beneficial, such as topical delivery using eye drops. In this study, we show that decreasing linker length between the two Affibody domains resulted in even slower dissociation from the receptor. The new variants of the biparatopic Affibody bound to VEGFR2-expressing cells, blocked VEGFA binding, and inhibited VEGFA-induced signaling of VEGFR2 over expressing cells. Moreover, the biparatopic Affibody inhibited sprout formation of endothelial cells in an in vitro angiogenesis assay with similar potency as the bivalent monoclonal antibody ramucirumab. This study demonstrates that the biparatopic Affibody constructs show promise for future therapeutic as well as in vivo imaging applications.

Place, publisher, year, edition, pages
Birkhauser Verlag, 2016
Keywords
Affibody molecules, Angiogenesis, Biparatopic affinity protein, Bispecific, VEGF, VEGFR2
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-185609 (URN)10.1007/s00018-015-2088-7 (DOI)000373141700010 ()26552422 (PubMedID)2-s2.0-84962247651 (Scopus ID)
Funder
Swedish Research Council, 621-2012-5236Swedish Cancer SocietyWenner-Gren Foundations
Note

QC 20160428

Available from: 2016-04-28 Created: 2016-04-25 Last updated: 2017-11-30Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-9282-0174

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