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Publications (10 of 13) Show all publications
Malm, M., Frejd, F. Y., Ståhl, S. & Löfblom, J. (2016). Targeting HER3 using mono- and bispecific antibodies or alternative scaffolds. mAbs, 8(7), 1195-1209
Open this publication in new window or tab >>Targeting HER3 using mono- and bispecific antibodies or alternative scaffolds
2016 (English)In: mAbs, ISSN 1942-0862, E-ISSN 1942-0870, Vol. 8, no 7, p. 1195-1209Article in journal (Refereed) Published
Abstract [en]

The human epidermal growth factor receptor 3 (HER3) has in recent years been recognized as a key node in the complex signaling network of many different cancers. It is implicated in de novo and acquired resistance against therapies targeting other growth factor receptors, e.g., EGFR, HER2, and it is a major activator of the PI3K/Akt signaling pathway. Consequently, HER3 has attracted substantial attention, and is today a key target for drugs in clinical development. Sophisticated protein engineering approaches have enabled the generation of a range of different affinity proteins targeting this receptor, including antibodies and alternative scaffolds that are either mono- or bispecific. Here, we describe HER3 and its role as a key tumor target, and give a comprehensive review of HER3-targeted proteins currently in development, including discussions on the opportunities and challenges of targeting this receptor.

Place, publisher, year, edition, pages
Taylor & Francis, 2016
Keywords
Affibody molecules, alternative scaffolds, bispecific antibodies, ErbB3, HER3, monoclonal antibodies, protein therapeutics, tumor targeting
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-195263 (URN)10.1080/19420862.2016.1212147 (DOI)000385569400002 ()2-s2.0-84989339198 (Scopus ID)
Note

QC 20161115

Available from: 2016-11-15 Created: 2016-11-02 Last updated: 2017-11-29Bibliographically approved
Andersson, K. G., Rosestedt, M., Varasteh, Z., Malm, M., Sandström, M., Tolmachev, V., . . . Orlova, A. (2015). Comparative evaluation of 111In-labeled NOTA‑conjugated affibody molecules for visualization of HER3 expression in malignant tumors. Oncology Reports, 34(2), 1042-8
Open this publication in new window or tab >>Comparative evaluation of 111In-labeled NOTA‑conjugated affibody molecules for visualization of HER3 expression in malignant tumors
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2015 (English)In: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 34, no 2, p. 1042-8Article in journal (Refereed) Published
Abstract [en]

Expression of human epidermal growth factor receptor type 3 (HER3) in malignant tumors has been associated with resistance to a variety of anticancer therapies. Several anti-HER3 monoclonal antibodies are currently under pre-clinical and clinical development aiming to overcome HER3-mediated resistance. Radionuclide molecular imaging of HER3 expression may improve treatment by allowing the selection of suitable patients for HER3-targeted therapy. Affibody molecules are a class of small (7kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. In a recent study, we selected affibody molecules with affinity to HER3 at a low picomolar range. The aim of the present study was to develop an anti-HER3 affibody molecule suitable for labeling with radiometals. The HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA HER3-specific affibody molecules were labeled with indium‑111 (111In) and assessed invitro and invivo for imaging properties using single photon emission computed tomography (SPECT). Labeling of HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA with 111In provided stable conjugates. Invitro cell tests demonstrated specific binding of the two conjugates to HER3-expressing BT‑474 breast carcinoma cells. In mice bearing BT‑474 xenografts, the tumor uptake of the two conjugates was receptor‑specific. Direct invivo comparison of 111In-HEHEHE-Z08698-NOTA and 111In-HEHEHE-Z08699‑NOTA demonstrated that the two conjugates provided equal radioactivity uptake in tumors, although the tumor-to-blood ratio was improved for 111In-HEHEHE-Z08698-NOTA [12±3 vs. 8±1, 4h post injection (p.i.)] due to more efficient blood clearance. 111In-HEHEHE-Z08698-NOTA is a promising candidate for imaging of HER3-expression in malignant tumors using SPECT. Results of the present study indicate that this conjugate could be used for patient stratification for anti-HER3 therapy.

Place, publisher, year, edition, pages
Spandidos Publications, 2015
Keywords
Affibody molecules, HER3, Indium-111, Molecular imaging, NOTA
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-170938 (URN)10.3892/or.2015.4046 (DOI)000357965600060 ()26059265 (PubMedID)2-s2.0-84931352417 (Scopus ID)
Funder
Swedish Cancer SocietySwedish Research Council
Note

QC 20150714

Available from: 2015-07-14 Created: 2015-07-13 Last updated: 2017-12-04Bibliographically approved
Malm, M., Bass, T., Gudmundsdotter, L., Lord, M., Frejd, F. Y., Ståhl, S. & Löfblom, J. (2014). Engineering of a bispecific affibody molecule towards HER2 and HER3 by addition of an albumin-binding domain allows for affinity purification and in vivo half-life extension. Biotechnology Journal, 9(9), 1215-1222
Open this publication in new window or tab >>Engineering of a bispecific affibody molecule towards HER2 and HER3 by addition of an albumin-binding domain allows for affinity purification and in vivo half-life extension
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2014 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 9, no 9, p. 1215-1222Article in journal (Refereed) Published
Abstract [en]

Emerging strategies in cancer biotherapy include the generation and application of bispecific antibodies, targeting two tumor-associated antigens for improved tumor selectivity and potency. Here, an alternative format for bispecific molecules was designed and investigated, in which two Affibody molecules were linked by an albumin-binding domain (ABD). Affibody molecules are small (6 kDa) affinity proteins and this new format allows for engineering of molecules with similar function as full-length bispecific antibodies, but in a dramatically smaller size (around eight-fold smaller). The ABD was intended to function both as a tag for affinity purification as well as for in vivo half-life extension in future preclinical and clinical investigations. Affinity-purified bispecific Affibody molecules, targeting HER2 and HER3, showed simultaneous binding to the three target proteins (HER2, HER3, and albumin) when investigated in biosensor assays. Moreover, simultaneous interactions with the receptors and albumin were demonstrated using flow cytometry on cancer cells. The bispecific Affibody molecules were also able to block ligand-induced phosphorylation of the HER receptors, indicating an anti-proliferative effect. We believe that this compact and flexible format has great potential for developing new potent bispecific affinity proteins in the future, as it combines the benefits of a small size (e.g. improved tissue penetration and reduced cost of goods) with a long circulatory half-life.

Keywords
Affibody molecules, Albumin-binding domain, Bispecific, Half-life extension, HER3
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-153852 (URN)10.1002/biot.201400009 (DOI)000341694200013 ()2-s2.0-84906948848 (Scopus ID)
Funder
Swedish Foundation for Strategic Research Swedish Research Council, 2012-9975Swedish Cancer Society, CAN 2013/586Vinnova
Note

QC 20141013

Available from: 2014-10-13 Created: 2014-10-09 Last updated: 2017-12-05Bibliographically approved
Orlova, A., Malm, M., Rosestedt, M., Varasteh, Z., Andersson, K., Selvaraju, R. K., . . . Löfblom, J. (2014). Imaging of HER3-expressing xenografts in mice using a Tc-99m(CO)(3)-HEHEHE-Z(HER3:08699) affibody molecule. European Journal of Nuclear Medicine and Molecular Imaging, 41(7), 1450-1459
Open this publication in new window or tab >>Imaging of HER3-expressing xenografts in mice using a Tc-99m(CO)(3)-HEHEHE-Z(HER3:08699) affibody molecule
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2014 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, no 7, p. 1450-1459Article in journal (Refereed) Published
Abstract [en]

Human epidermal growth factor receptor type 3 (HER3) is a transmembrane receptor tyrosine kinase belonging to the HER (ErbB) receptor family. Membranous expression of HER3 is associated with trastuzumab resistance in breast cancer and the transition to androgen independence in prostate cancer. Imaging of HER3 expression in malignant tumors may provide important diagnostic information that can influence patient management. Affibody molecules with low picomolar affinity to HER3 were recently selected. The aim of this study was to investigate the feasibility of HER3 imaging using radiolabeled Affibody molecules. A HER3-binding Affibody molecule, Z(08699), with a HEHEHE-tag on N-terminus was labeled with Tc-99m(CO)(3) using an IsoLink kit. In vitro and in vivo binding specificity and the cellular processing of the labeled binder were evaluated. Biodistribution of Tc-99m(CO)(3)-HEHEHE-Z(08699) was studied over time in mice bearing HER3-expressing xenografts. HEHEHE-Z(08699) was labeled with Tc-99m(CO)(3) with an isolated yield of > 80 % and a purity of > 99 %. Binding of Tc-99m(CO)(3)-HEHEHE-Z(08699) was specific to BT474 and MCF7 (breast cancer), and LS174T (colon cancer) cells. Cellular processing showed rapid binding and relatively quick internalization of the receptor/Affibody molecule complex (70 % of cell-associated radioactivity was internalized after 24 h). The tumor targeting was receptor mediated and the excretion was predominantly renal. Receptor-mediated uptake was also found in the liver, lung, stomach, intestine, and salivary glands. At 4 h pi, tumor-to-blood ratios were 7 +/- 3 for BT474, and 6 +/- 2 for LS174T xenografts. LS174T tumors were visualized by microSPECT 4 h pi. The results of this study suggest the feasibility of HER3-imaging in malignant tumors using Affibody molecules.

Keywords
HER3, Affibody molecule, Technetium-99m, Molecular imaging, Molecular targeting
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-147939 (URN)10.1007/s00259-014-2733-7 (DOI)000337286200021 ()2-s2.0-84903722742 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20140714

Available from: 2014-07-14 Created: 2014-07-10 Last updated: 2017-12-05Bibliographically approved
Orlova, A., Malm, M., Lindberg, H., Varasteh, Z., Rosestedt, M., Tolmachev, V., . . . Löfblom, J. (2013). Feasibility of radionuclide imaging of HER3-expressing tumors using affibody molecules. Journal of labelled compounds & radiopharmaceuticals, 56, S11-S11
Open this publication in new window or tab >>Feasibility of radionuclide imaging of HER3-expressing tumors using affibody molecules
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2013 (English)In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 56, p. S11-S11Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Wiley-Blackwell, 2013
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-124297 (URN)000318694100012 ()
Note

QC 20130701

Available from: 2013-07-01 Created: 2013-06-28 Last updated: 2017-12-06Bibliographically approved
Orlova, A., Malm, M., Lindberg, H., Varasteh, Z., Selvaraju, R. K., Kronqvist, N., . . . Tolmachev, V. (2013). Feasibility of radionuclide imaging of HER3-expressing tumours using technetium-99m labeled affibody molecules. Paper presented at Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 19-23, 2013, Lyon, France. European Journal of Nuclear Medicine and Molecular Imaging, 40, S185-S186
Open this publication in new window or tab >>Feasibility of radionuclide imaging of HER3-expressing tumours using technetium-99m labeled affibody molecules
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2013 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, p. S185-S186Article in journal, Meeting abstract (Other academic) Published
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:kth:diva-134576 (URN)000325853400293 ()
Conference
Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 19-23, 2013, Lyon, France
Note

QC 20131127

Available from: 2013-11-27 Created: 2013-11-25 Last updated: 2017-12-06Bibliographically approved
Malm, M. (2013). Generation and characterization of Affibody molecules targeting HER3. (Doctoral dissertation). Stockholm: KTH Royal Institute of Technology
Open this publication in new window or tab >>Generation and characterization of Affibody molecules targeting HER3
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the field of oncology, the ability to target specific tumor cells using highly selective targeting molecules is an attractive and emerging concept. In this context, the epidermal growth factor receptor HER3 has proven central to the biology behind many different human cancers and inhibition of the signaling mediated by this receptor could provide antitumoral effects. Consequently, this receptor has emerged as a suitable target for imaging, functional blocking or delivery of toxic payloads. A promising targeting-molecule for such applications is the small non-immunoglobulin derived Affibody molecule. The work upon which this thesis is based, revolves around HER3 with the aim to generate and characterize Affibody molecules targeting this receptor.

 

In the first study, HER3-specific Affibody molecules were generated by combinatorial protein engineering using a combined approach where first generation binders were isolated from a phage-displayed naive library, followed by affinity maturation of these binders using a focused staphylococcal surface-displayed library and flow-cytometric cell sorting. This engineering strategy enabled the successful isolation of HER3-specific Affibody molecules with subnanomolar affinities for the receptor and the ability to compete with the natural ligand heregulin (HRG) for binding to HER3. In the second study, the cellular effects of these Affibody molecules were characterization in vitro. The results demonstrated that the ability to inhibit HRG-binding to the receptor translated into inhibition of ligand-induced phosphorylation of HER3, HER2 as well as the downstream signaling molecules Akt and Erk. As a result, the HER3-specific Affibody molecules also inhibited HRG-induced cell growth of two different breast cancer cell lines in vitro. These promising results, suggested that the HER3-targeting Affibody molecules could have a therapeutic effect in tumors that are dependent on ligand-induced signaling of HER3. However, due to the relatively low expression level of HER3 on tumor cells, we explored two different engineering approaches of the HER3-specific Affibody molecules in order to potentially improve its tumor targeting ability. One approach was to construct bispecific Affibody molecules where a HER3- and a HER2-specific Affibody molecule were fused on each side of an albumin-binding domain (ABD). In the third study, one such bispecific construct was shown to have increased ability to inhibit ligand-induced phosphorylation of HER2 and retained ability to inhibit HRG-induced activation of HER3, as compared to the monomeric anti-HER3 Affibody. Another strategy was to further increase the affinity of the HER3-specific Affibody molecules towards the receptor through a semi-rational affinity maturation approach. In the fourth study, a staphylococcal displayed affinity maturation library was screened by FACS using an off-rate selection procedure. This approach resulted in the successful isolation of picomolar HER3-binders with improved potency of inhibiting HRG-induced cell growth as compared to a first generation binder. Moreover, in the fifth study, in vivo characterization of these HER3-specific Affibody molecules was performed in both normal and xenograft mice. The results suggested specific targeting of HER3 in vivo and provided the first evidence of successful tumor imaging using a HER3-specific Affibody. Taken together, the work included in this thesis describes (to our knowledge) the first non-immunoglobulin derived affinity protein targeting HER3, with promising features for both therapeutic and imaging applications.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2013. p. vii, 87
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:1
Keywords
Affibody molecules, epidermal growth factor receptors, HER3, ErbB3, combinatorial protein engineering, combinatorial library, staphylococcal surface display
National Category
Natural Sciences
Identifiers
urn:nbn:se:kth:diva-137972 (URN)978-91-7501-955-0 (ISBN)
Public defence
2014-01-24, FR 4, AlbaNova, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20131217

Available from: 2013-12-17 Created: 2013-12-17 Last updated: 2014-01-22Bibliographically approved
Malm, M., Kronqvist, N., Lindberg, H., Gudmundsdotter, L., Bass, T., Frejd, F. Y., . . . Löfblom, J. (2013). Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification. PLoS ONE, 8(5), e62791
Open this publication in new window or tab >>Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 5, p. e62791-Article in journal (Refereed) Published
Abstract [en]

The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 PM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.

Place, publisher, year, edition, pages
Public Library Science, USA, 2013
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-124294 (URN)10.1371/journal.pone.0062791 (DOI)000318852400011 ()2-s2.0-84877609907 (Scopus ID)
Funder
Swedish Research CouncilSwedish Cancer Society
Note

QC 20130701

Available from: 2013-07-01 Created: 2013-06-28 Last updated: 2017-12-06Bibliographically approved
Göstring, L., Malm, M., Höidén-Guthenberg, I., Frejd, F. Y., Ståhl, S., Löfblom, J. & Gedda, L. (2012). Cellular Effects of HER3-Specific Affibody Molecules. PLoS ONE, 7(6), e40023
Open this publication in new window or tab >>Cellular Effects of HER3-Specific Affibody Molecules
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 6, p. e40023-Article in journal (Refereed) Published
Abstract [en]

Recent studies have led to the recognition of the epidermal growth factor receptor HER3 as a key player in cancer, and consequently this receptor has gained increased interest as a target for cancer therapy. We have previously generated several Affibody molecules with subnanomolar affinity for the HER3 receptor. Here, we investigate the effects of two of these HER3-specific Affibody molecules, Z05416 and Z05417, on different HER3-overexpressing cancer cell lines. Using flow cytometry and confocal microscopy, the Affibody molecules were shown to bind to HER3 on three different cell lines. Furthermore, the receptor binding of the natural ligand heregulin (HRG) was blocked by addition of Affibody molecules. In addition, both molecules suppressed HRG-induced HER3 and HER2 phosphorylation in MCF-7 cells, as well as HER3 phosphorylation in constantly HER2-activated SKBR-3 cells. Importantly, Western blot analysis also revealed that HRG-induced downstream signalling through the Ras-MAPK pathway as well as the PI3K-Akt pathway was blocked by the Affibody molecules. Finally, in an in vitro proliferation assay, the two Affibody molecules demonstrated complete inhibition of HRG-induced cancer cell growth. Taken together, our findings demonstrate that Z05416 and Z05417 exert an anti-proliferative effect on two breast cancer cell lines by inhibiting HRG-induced phosphorylation of HER3, suggesting that the Affibody molecules are promising candidates for future HER3-targeted cancer therapy.

Keywords
Epidermal-Growth-Factor, Breast-Cancer Cells, Oncogene Product, Antitumor Action, Egf Receptor, Kinase, Ligand, Erbb2, Therapy, Family
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-99416 (URN)10.1371/journal.pone.0040023 (DOI)000305892100176 ()2-s2.0-84863110374 (Scopus ID)
Funder
Swedish Research Council, 2009-5758
Note

QC 20120731

Available from: 2012-07-31 Created: 2012-07-30 Last updated: 2017-12-07Bibliographically approved
Kronqvist, N., Malm, M., Göstring, L., Gunneriusson, E., Nilsson, M., Höidén-Guthenberg, I., . . . Löfblom, J. (2011). Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules. Protein Engineering Design & Selection, 24(4), 385-396
Open this publication in new window or tab >>Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules
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2011 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 24, no 4, p. 385-396Article in journal (Refereed) Published
Abstract [en]

Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers. ErbB3 has recently also been implicated in resistance to ErbB2-targeting therapies. Here we report the generation of high-affinity ErbB3-specific Affibody molecules intended for future molecular imaging and biotherapeutic applications. Using a high-complexity phage-displayed Affibody library, a number of ErbB3 binders were isolated and specific cell-binding activity was demonstrated in immunofluorescence microscopic studies. Subsequently, a second-generation library was constructed based on sequences of the candidates from the phage display selection. By exploiting the sensitive affinity discrimination capacity of a novel bacterial surface display technology, the affinity of candidate Affibody molecules was further increased down to subnanomolar affinity. In summary, the demonstrated specific targeting of native ErbB3 receptor on human cancer cell lines as well as competition with the heregulin/ErbB3 interaction indicates that these novel biological agents may become useful tools for diagnostic and therapeutic targeting of ErbB3-expressing cancers. Our studies also highlight the powerful approach of combining the advantages of different display technologies for generation of functional high-affinity protein-based binders. Potential future applications, such as radionuclide-based diagnosis and treatment of human cancers are discussed.

Keywords
Affibody, cell surface display, combinatorial protein engineering, HER3, Staphylococcus carnosus
National Category
Condensed Matter Physics
Identifiers
urn:nbn:se:kth:diva-14208 (URN)10.1093/protein/gzq118 (DOI)000288269600005 ()
Funder
Swedish Research Council, 2009-5758
Note

QC 20100726 Uppdaterad från manuskript till artikel i tidskrift (20110408)

Available from: 2010-07-26 Created: 2010-07-26 Last updated: 2017-12-12Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-1763-9073

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