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Publications (10 of 94) Show all publications
Boutajangout, A., Lindberg, H., Awwad, A., Paul, A., Baitalmal, R., Almokyad, I., . . . Wisniewski, T. (2019). Affibody-Mediated Sequestration of Amyloid beta Demonstrates Preventive Efficacy in a Transgenic Alzheimer's Disease Mouse Model. Frontiers in Aging Neuroscience, 11, Article ID 64.
Open this publication in new window or tab >>Affibody-Mediated Sequestration of Amyloid beta Demonstrates Preventive Efficacy in a Transgenic Alzheimer's Disease Mouse Model
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2019 (English)In: Frontiers in Aging Neuroscience, ISSN 1663-4365, E-ISSN 1663-4365, Vol. 11, article id 64Article in journal (Refereed) Published
Abstract [en]

Different strategies for treatment and prevention of Alzheimer's disease (AD) are currently under investigation, including passive immunization with anti-amyloid beta (anti-A beta) monoclonal antibodies (mAbs). Here, we investigate the therapeutic potential of a novel type of A beta-targeting agent based on an affibody molecule with fundamentally different properties to mAbs. We generated a therapeutic candidate, denoted Z(SYM73)-albumin-binding domain (ABD; 16.8 kDa), by genetic linkage of the dimeric Z(SYM73) affibody for sequestering of monomeric A beta-peptides and an ABD for extension of its in vivo half-life. Amyloid precursor protein (APP)/PS1 transgenic AD mice were administered with Z(SYM73)-ABD, followed by behavioral examination and immunohistochemistry. Results demonstrated rescued cognitive functions and significantly lower amyloid burden in the treated animals compared to controls. No toxicological symptoms or immunology-related side-effects were observed. To our knowledge, this is the first reported in vivo investigation of a systemically delivered scaffold protein against monomeric A beta, demonstrating a therapeutic potential for prevention of AD.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2019
Keywords
Alzheimer's disease, affibody molecule, amyloid beta (A beta), behavior, histology, immunotherapy, transgenic mice
National Category
Geriatrics
Identifiers
urn:nbn:se:kth:diva-249880 (URN)10.3389/fnagi.2019.00064 (DOI)000462530400001 ()30967771 (PubMedID)
Note

QC 20190423

Available from: 2019-04-23 Created: 2019-04-23 Last updated: 2019-04-23Bibliographically approved
Andersson, K. G., Persson, J., Ståhl, S. & Löfblom, J. (2019). Autotransporter-Mediated Display of a Naive Affibody Library on the Outer Membrane of Escherichia coli. Biotechnology Journal, 14(4), Article ID 1800359.
Open this publication in new window or tab >>Autotransporter-Mediated Display of a Naive Affibody Library on the Outer Membrane of Escherichia coli
2019 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 14, no 4, article id 1800359Article in journal (Refereed) Published
Abstract [en]

Development of new affinity proteins using combinatorial protein engineering is today established for generation of monoclonal antibodies and also essential for discovery of binders that are based on non-immunoglobulin proteins. Phage display is most frequently used, but yeast display is becoming increasingly popular, partly due to the option of utilizing fluorescence-activated cell sorting (FACS) for isolation of new candidates. Escherichia coli has several valuable properties for library applications and in particular the high transformation efficiency. The use of various autotransporters and intimins for secretion and anchoring on the outer membrane have shown promising results and particularly for directed evolution of different enzymes. Here, the authors report on display of a large naive affibody library on the outer membrane of E. coli using the autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I). The expression cassette is first engineered by removing non-essential sequences, followed by introduction of an affibody library, comprising more than 10(9) variants, into the new display vector. The quality of the library and general performance of the method is assessed by FACS against five different targets, which resulted in a panel of binders with down to nanomolar affinities, suggesting that the method has potential as a complement to phage display for generation of affibody molecules.

Place, publisher, year, edition, pages
WILEY-V C H VERLAG GMBH, 2019
Keywords
affibody library, AIDA-I, autodisplay, bacterial display, directed evolution
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-249806 (URN)10.1002/biot.201800359 (DOI)000462917100020 ()30179307 (PubMedID)2-s2.0-85053871994 (Scopus ID)
Note

QC 20190423

Available from: 2019-04-23 Created: 2019-04-23 Last updated: 2019-04-23Bibliographically approved
Vorobyeva, A., Schulga, A., Konovalova, E., Güler, R., Mitran, B., Garousi, J., . . . Tolmachev, V. (2019). Comparison of tumor-targeting properties of directly and indirectly radioiodinated designed ankyrin repeat protein (DARPin) G3 variants for molecular imaging of HER2. International Journal of Oncology, 54(4), 1209-1220
Open this publication in new window or tab >>Comparison of tumor-targeting properties of directly and indirectly radioiodinated designed ankyrin repeat protein (DARPin) G3 variants for molecular imaging of HER2
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2019 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 54, no 4, p. 1209-1220Article in journal (Refereed) Published
Abstract [en]

Evaluation of human epidermal growth factor receptor 2 (HER2) expression levels in breast and gastroesophageal cancer is used for the stratification of patients for HER2-targeting therapies. The use of radionuclide molecular imaging may facilitate such evaluation in a non-invasive way. Designed ankyrin repeat proteins (DARPins) are engineered scaffold proteins with high potential as probes for radionuclide molecular imaging. DARPin G3 binds with high affinity to HER2 and may be used to visualize this important therapeutic target. Studies on other engineered scaffold proteins have demonstrated that selection of the optimal labeling approach improves the sensitivity and specificity of radionuclide imaging. The present study compared two methods of labeling G3, direct and indirect radioiodination, to select an approach providing the best imaging contrast. G3-H6 was labeled with iodine-124, iodine-125 and iodine-131 using a direct method. A novel construct bearing a C-terminal cysteine, G3-GGGC, was site-specifically labeled using [125I]I-iodo-[(4-hydroxyphenyl)ethyl]maleimide (HPEM). The two radiolabeled G3 variants preserved binding specificity and high affinity to HER2-expressing cells. The specificity of tumor targeting in vivo was demonstrated. Biodistribution comparison of [131I]I-G3-H6 and [125I]I-HPEM-G3-GGGC in mice, bearing HER2-expressing SKOV3 xenografts, demonstrated an appreciable contribution of hepatobiliary excretion to the clearance of [125I]I-HPEM-G3-GGGC and a decreased tumor uptake compared to [131I]I-G3-H6. The direct label provided higher tumor-to-blood and tumor-to-organ ratios compared with the indirect label at 4 h post-injection. The feasibility of high contrast PET/CT imaging of HER2 expression in SKOV3 xenografts in mice using [124I]I-G3-H6 was demonstrated. In conclusion, direct radioiodination is the preferable approach for labeling DARPin G3 with iodine-123 and iodine-124 for clinical single photon emission computed tomography and positron emission tomography imaging.

Place, publisher, year, edition, pages
SPANDIDOS PUBL LTD, 2019
Keywords
DARPin, HER2, imaging, radionuclide, iodine, radioiodination
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:kth:diva-248065 (URN)10.3892/ijo.2019.4712 (DOI)000461097600006 ()2-s2.0-85062653493 (Scopus ID)
Note

QC 20190429

Available from: 2019-04-29 Created: 2019-04-29 Last updated: 2019-04-29Bibliographically approved
Meister, S., Hendrikse, N. & Löfblom, J. (2019). Directed evolution of the 3C protease from coxsackievirus using a novel fluorescence-assisted intracellular method. Biological chemistry (Print), 400(3), 405-415
Open this publication in new window or tab >>Directed evolution of the 3C protease from coxsackievirus using a novel fluorescence-assisted intracellular method
2019 (English)In: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 400, no 3, p. 405-415Article in journal (Refereed) Published
Abstract [en]

Proteases are crucial for regulating biological processes in organisms through hydrolysis of peptide bonds. Recombinant proteases have moreover become important tools in biotechnological, and biomedical research and as therapeutics. We have developed a label-free high-throughput method for quantitative assessment of proteolytic activity in Escherichia coli. The screening method is based on co-expression of a protease of interest and a reporter complex. This reporter consists of an aggregation-prone peptide fused to a fluorescent protein via a linker that contains the corresponding substrate sequence. Cleavage of the substrate rescues the fluorescent protein from aggregation, resulting in increased fluorescence that correlates to proteolytic activity, which can be monitored using flow cytometry. In one round of flow-cytometric cell sorting, we isolated an efficiently cleaved tobacco etch virus (TEV) substrate from a 1:100 000 background of non-cleavable sequences, with around 6000-fold enrichment. We then engineered the 3C protease from coxsackievirus B3 (CVB3 3C(pro)) towards improved proteolytic activity on the substrate LEVLFQ down arrow GP. We isolated highly proteolytic active variants from a randomly mutated CVB3 3C(pro) library with up to 4-fold increase in activity. The method enables simultaneous measurement of proteolytic activity and protease expression levels and can therefore be applied for protease substrate profiling, as well as directed evolution of proteases.

Place, publisher, year, edition, pages
WALTER DE GRUYTER GMBH, 2019
Keywords
Coxsackievirus B3, FACS, GFP-fusion, intracellular assay, protease engineering, protease substrate profiling
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-245129 (URN)10.1515/hsz-2018-0362 (DOI)000458628400014 ()30521472 (PubMedID)2-s2.0-85058219114 (Scopus ID)
Note

QC 20190313

Available from: 2019-03-13 Created: 2019-03-13 Last updated: 2019-03-18Bibliographically approved
Dahlsson Leitao, C., Rinne, S. S., Mitran, B., Vorobyeva, A., Andersson, K. G., Tolmachev, V., . . . Orlova, A. (2019). Molecular Design of HER3-Targeting Affibody Molecules: Influence of Chelator and Presence of HEHEHE-Tag on Biodistribution of 68 Ga-Labeled Tracers. International Journal of Molecular Sciences, 20(5)
Open this publication in new window or tab >>Molecular Design of HER3-Targeting Affibody Molecules: Influence of Chelator and Presence of HEHEHE-Tag on Biodistribution of 68 Ga-Labeled Tracers
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2019 (English)In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, no 5Article in journal (Refereed) Published
Abstract [en]

Affibody-based imaging of HER3 is a promising approach for patient stratification. We investigated the influence of a hydrophilic HEHEHE-tag ((HE)₃-tag) and two different gallium-68/chelator-complexes on the biodistribution of Z08698 with the aim to improve the tracer for PET imaging. Affibody molecules (HE)₃-Z08698-X and Z08698-X (X = NOTA, NODAGA) were produced and labeled with gallium-68. Binding specificity and cellular processing were studied in HER3-expressing human cancer cell lines BxPC-3 and DU145. Biodistribution was studied 3 h p.i. in Balb/c nu/nu mice bearing BxPC-3 xenografts. Mice were imaged 3 h p.i. using microPET/CT. Conjugates were stably labeled with gallium-68 and bound specifically to HER3 in vitro and in vivo. Association to cells was rapid but internalization was slow. Uptake in tissues, including tumors, was lower for (HE)₃-Z08698-X than for non-tagged variants. The neutral [68Ga]Ga-NODAGA complex reduced the hepatic uptake of Z08698 compared to positively charged [68Ga]Ga-NOTA-conjugated variants. The influence of the chelator was more pronounced in variants without (HE)3-tag. In conclusion, hydrophilic (HE)₃-tag and neutral charge of the [68Ga]Ga-NODAGA complex promoted blood clearance and lowered hepatic uptake of Z08698. [68Ga]Ga-(HE)₃-Z08698-NODAGA was considered most promising, providing the lowest blood and hepatic uptake and the best imaging contrast among the tested variants.

Place, publisher, year, edition, pages
NLM (Medline), 2019
Keywords
affibody, gallium-68, HER3, molecular imaging, NODAGA, NOTA, PET
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-246490 (URN)10.3390/ijms20051080 (DOI)000462542300079 ()30832342 (PubMedID)2-s2.0-85062394960 (Scopus ID)
Note

QC 20190326

Available from: 2019-03-26 Created: 2019-03-26 Last updated: 2019-04-23Bibliographically approved
Vorobyeva, A., Schulga, A., Konovalova, E., Güler, R., Löfblom, J., Sandstrom, M., . . . Deyev, S. M. (2019). Optimal composition and position of histidine-containing tags improves biodistribution of Tc-99m-labeled DARP in G3. Scientific Reports, 9, Article ID 9405.
Open this publication in new window or tab >>Optimal composition and position of histidine-containing tags improves biodistribution of Tc-99m-labeled DARP in G3
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 9405Article in journal (Refereed) Published
Abstract [en]

Radionuclide molecular imaging of HER2 expression in disseminated cancer enables stratification of patients for HER2-targeted therapies. DARP in G3, a small (14 kDa) engineered scaffold protein, is a promising probe for imaging of HER2. We hypothesized that position (C- or N-terminus) and composition (hexahistidine or (HE)(3)) of histidine-containing tags would influence the biodistribution of [Tc-99m]Tc(CO)(3)-labeled DARP in G3. To test the hypothesis, G3 variants containing tags at N-terminus (H-6-G3 and (HE)(3)-G3) or at C-terminus (G3-H-6 and G3-(HE)(3)) were labeled with [Tc-99m]Tc(CO)(3). Labeling yield, label stability, specificity and affinity of the binding to HER2, biodistribution and tumor targeting properties of these variants were compared side-by-side. There was no substantial influence of position and composition of the tags on binding of [Tc-99m]Tc(CO)(3)-labeled variants to HER2. The specificity of HER2 targeting in vivo was confirmed. The tumor uptake in BALB/c nu/nu mice bearing SKOV3 xenografts was similar for all variants. On the opposite, there was a strong influence of the tags on uptake in normal tissues. The tumor-to-liver ratio for [Tc-99m]Tc(CO)(3)-(HE)(3)-G3 was three-fold higher compared to the hexahistidine-tag containing variants. Overall, [Tc-99m]Tc(CO)(3)-(HE)(3)-G3 variant provided the highest tumor-to-lung, tumor-to-liver, tumor-to-bone and tumor-to-muscle ratios, which should improve sensitivity of HER2 imaging in these common metastatic sites.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2019
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:kth:diva-255436 (URN)10.1038/s41598-019-45795-8 (DOI)000473130000026 ()31253840 (PubMedID)2-s2.0-85068220749 (Scopus ID)
Note

QC 20190820

Available from: 2019-08-20 Created: 2019-08-20 Last updated: 2019-08-20Bibliographically approved
Rinne, S. S., Leitao, C. D., Mitran, B., Bass, T., Andersson, K. G., Tolmachev, V., . . . Orlova, A. (2019). Optimization of HER3 expression imaging using affibody molecules: Influence of chelator for labeling with indium-111. Scientific Reports, 9, Article ID 655.
Open this publication in new window or tab >>Optimization of HER3 expression imaging using affibody molecules: Influence of chelator for labeling with indium-111
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 655Article in journal (Refereed) Published
Abstract [en]

Radionuclide molecular imaging of human epidermal growth factor receptor 3 (HER3) expression using affibody molecules could be used for patient stratification for HER3-targeted cancer therapeutics. We hypothesized that the properties of HER3-targeting affibody molecules might be improved through modification of the radiometal-chelator complex. Macrocyclic chelators NOTA (1,4,7-triazacyclononane-N,N',N ''-triacetic acid), NODAGA (1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid), and DOTAGA (1,4,7,10-tetraazacyclododececane, 1-(glutaric acid)-4,7,10-triacetic acid) were conjugated to the C-terminus of anti-HER3 affibody molecule Z(08698) and conjugates were labeled with indium-111. All conjugates bound specifically and with picomolar affinity to HER3 in vitro. In mice bearing HER3-expressing xenografts, no significant difference in tumor uptake between the conjugates was observed. Presence of the negatively charged In-111-DOTAGA-complex resulted in the lowest hepatic uptake and the highest tumor-to-liver ratio. In conclusion, the choice of chelator influences the biodistribution of indium-111 labeled anti-HER3 affibody molecules. Hepatic uptake of anti-HER3 affibody molecules could be reduced by the increase of negative charge of the radiometal-chelator complex on the C-terminus without significantly influencing the tumor uptake.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2019
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-243945 (URN)10.1038/s41598-018-36827-w (DOI)000456554600094 ()30679757 (PubMedID)2-s2.0-85060519832 (Scopus ID)
Note

QC 20190305

Available from: 2019-03-05 Created: 2019-03-05 Last updated: 2019-03-05Bibliographically approved
Rinne, S., Mitran, B., Gentry, J., Vorobyeva, A., Leitao, C. D., Andersson, K. G., . . . Orlova, A. (2019). Optimizing the molecular design of Ga-68-labeled affibody molecules for in vivo PET imaging of HER3 expression. Journal of labelled compounds & radiopharmaceuticals, 62, S468-S470
Open this publication in new window or tab >>Optimizing the molecular design of Ga-68-labeled affibody molecules for in vivo PET imaging of HER3 expression
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2019 (English)In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 62, p. S468-S470Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
WILEY, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-254032 (URN)000468965200391 ()
Note

QC 20190814

Available from: 2019-08-14 Created: 2019-08-14 Last updated: 2019-08-14Bibliographically approved
Oroujeni, M., Garousi, J., Andersson, K. G., Löfblom, J., Mitran, B., Orlova, A. & Tolmachev, V. (2018). Comparative evaluation of anti-EFGR affibody molecules labelled with gallium-68 and zirconium-89 using desferrioxamine B as a chelator. 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, S674-S675
Open this publication in new window or tab >>Comparative evaluation of anti-EFGR affibody molecules labelled with gallium-68 and zirconium-89 using desferrioxamine B as a chelator
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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. S674-S675Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
SPRINGER, 2018
National Category
Medical Genetics
Identifiers
urn:nbn:se:kth:diva-239823 (URN)10.1007/s00259-018-4148-3 (DOI)000449266206125 ()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
Orlova, A., Bass, T., Rinne, S. S., Leitao, C. D., Rosestedt, M., Atterby, C., . . . Ståhl, S. (2018). Evaluation of the Therapeutic Potential of a HER3-Binding Affibody Construct TAM-HER3 in Comparison with a Monoclonal Antibody, Seribantumab. Molecular Pharmaceutics, 15(8), 3394-3403
Open this publication in new window or tab >>Evaluation of the Therapeutic Potential of a HER3-Binding Affibody Construct TAM-HER3 in Comparison with a Monoclonal Antibody, Seribantumab
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2018 (English)In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 15, no 8, p. 3394-3403Article in journal (Refereed) Published
Abstract [en]

Human epidermal growth factor receptor type 3 (HER3) is recognized to be involved in resistance to HER targeting therapies. A number of HER3-targeting monoclonal antibodies are under clinical investigation as potential cancer therapeutics. Smaller high-affinity scaffold proteins are attractive non-Fc containing alternatives to antibodies. A previous study indicated that anti-HER3 affibody molecules could delay the growth of xenografted HER3-positive tumors. Here, we designed a second-generation HER3-targeting construct (TAM-HER3), containing two HER3-specific affibody molecules bridged by an albumin-binding domain (ABD) for extension of blood circulation. Receptor blocking activity was demonstrated in vitro. In mice bearing BxPC-3 xenografts, the therapeutic efficacy of TAM-HER3 was compared to the HER3-specific monoclonal antibody seribantumab (MM-121). TAM-HER3 inhibited heregulin-induced phosphorylation in a panel of HER3-expressing cancer cells and was found to be equally as potent as seribantumab in terms of therapeutic efficacy in vivo and with a similar safety profile. Median survival times were 60 days for TAM-HER3, 54 days for seribantumab, and 41 days for the control group. No pathological changes were observed in cytopathological examination. The multimeric HER3-binding affibody molecule in fusion to ABD seems promising for further evaluation as candidate therapeutics for treatment of HER3-overexpressing tumors.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2018
Keywords
affibody molecule, HER3, targeting therapy, preclinical
National Category
Clinical Medicine
Identifiers
urn:nbn:se:kth:diva-233605 (URN)10.1021/acs.molpharmaceut.8b00393 (DOI)000441476800049 ()29995421 (PubMedID)2-s2.0-85049877449 (Scopus ID)
Note

QC 20180827

Available from: 2018-08-27 Created: 2018-08-27 Last updated: 2018-09-03Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0001-9423-0541

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