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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
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
Bass, T. (2017). Affibody molecules targeting HER3 for cancer therapy. (Doctoral dissertation). KTH Royal Institute of Technology
Open this publication in new window or tab >>Affibody molecules targeting HER3 for cancer therapy
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The development of targeted therapy has contributed tremendously to the treatment of patients with cancer. The use of highly specific affinity proteins to target cancer cells has become a standard in treatment strategies for several different cancers. In light of this, many cancer cell markers are investigated for their potential use in diagnostics and therapy. One such marker is the human epidermal growth factor receptor 3, HER3. It has been established as an important contributor to many cancer types. The function of HER3 is to relay cell growth signals from outside of the cell to the inside. Interfering with- and inhibit- ing the function of HER3 has emerged as an interesting strategy for cancer therapeutics. The studies presented in this thesis aim to target HER3 with small, engineered affinity domain proteins for therapeutic purposes. Monomeric affibody molecules have previously been engineered to bind and inhibit HER3 in vitro. Due to the relatively low expression of HER3, an increase in valency appears promising to strengthen the therapeutic potential. Affibody molecules targeting the receptor were thus linked to form bivalent and bispecific constructs and evaluated both in vitro and in vivo. In the first study of this thesis affibody molecules specific for HER3 and HER2 were fused to an albumin binding domain to form bivalent and bispecific construct. The constructs inhibited ligand-induced receptor phos- phorylation of both HER2 and HER3 more efficiently than monomeric affibody molecules. A second approach to enhance the potential of affibody molecules in tumor targeting is described in the second study, where monomeric HER3-binding affibody molecules were engineered to increase their affinity for HER3. The resulting variants showed a 20-fold in- creased affinity and higher capacity to inhibit cancer cell growth. Combining the findings of the first two studies, the third study describes the evaluation of a HER3-targeting bivalent affibody construct for potential application as a therapeutic. Here, the bivalent construct inhibited cancer cell growth in vitro and was found to slow down tumor growth in mice, while being well tolerated and showing no visible toxicity. The fourth study built upon these findings and compares a very similar bivalent construct to the clinically-investigated HER3-specific monoclonal antibody seribantumab. The affibody construct showed very comparable efficacy with the antibody in terms of decreasing tumor growth rate and ex- tending mouse survival. Collectively, these works describe for the first time the use of alternative affinity protein constructs with therapeutic potential targeting HER3.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2017. p. 87
Keywords
Affibody molecule, cancer therapy, epidermal growth factor receptors, ErbB3, HER3, protein engineering
National Category
Other Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-204593 (URN)
Public defence
2017-05-12, FR4, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20170330

Available from: 2017-03-30 Created: 2017-03-29 Last updated: 2017-03-30Bibliographically 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
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
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 ()24678002 (PubMedID)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: 2020-03-09Bibliographically 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 ()23675426 (PubMedID)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: 2020-03-09Bibliographically approved
Gantelius, J., Bass, T., Sjöberg, R., Nilsson, P. & Andersson-Svahn, H. (2011). A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays. International Journal of Molecular Sciences, 12(11), 7748-7759
Open this publication in new window or tab >>A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays
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2011 (English)In: International Journal of Molecular Sciences, ISSN 1661-6596, Vol. 12, no 11, p. 7748-7759Article in journal (Refereed) Published
Abstract [en]

Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (<30 ng/mL) determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings.

Keywords
point of care, protein microarray, lateral flow
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-55264 (URN)10.3390/ijms12117748 (DOI)000297696100032 ()22174629 (PubMedID)2-s2.0-82255181715 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note
QC 20120102Available from: 2012-01-02 Created: 2012-01-02 Last updated: 2020-03-09Bibliographically approved
Gantelius, J., Bass, T., Gundberg, A., Sundberg, M., Sjöberg, R., Nilsson, P. & Andersson-Svahn, H. (2011). A ten-minute high density lateral flow protein microarray assay. In: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011: (MicroTAS 2011). Paper presented at 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 2 October 2011 through 6 October 2011, Seattle, WA (pp. 1176-1178).
Open this publication in new window or tab >>A ten-minute high density lateral flow protein microarray assay
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2011 (English)In: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011: (MicroTAS 2011), 2011, p. 1176-1178Conference paper, Published paper (Refereed)
Abstract [en]

Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (<50ng/ml) determination of antigen specific antibodies in less than ten minutes total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy (AUC=99.4%) was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies.

Keywords
Lateral flow, Point of care, Protein microarray, Biofluids, Cell phone camera, Human tissues, Laboratory environment, Nanobeads, Antibodies, Antigens, Bioassay, Biochips, Tools
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-148732 (URN)2-s2.0-84883789603 (Scopus ID)9781618395955 (ISBN)
Conference
15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 2 October 2011 through 6 October 2011, Seattle, WA
Note

QC 20140826

Available from: 2014-08-26 Created: 2014-08-11 Last updated: 2014-08-26Bibliographically approved
Bass, T.Evaluating the therapeutic potential of a dimeric HER3-binding affibody construct in comparison with a monoclonal antibody, seribantumab..
Open this publication in new window or tab >>Evaluating the therapeutic potential of a dimeric HER3-binding affibody construct in comparison with a monoclonal antibody, seribantumab.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

A number of monoclonal antibodies targeting HER3 are currently under clinical investigation as potential cancer therapeutics. We have earlier generated high affinity (low picomolar) affibody molecules targeting HER3. These are small, 58 amino acid, non-immunoglobulin based scaffold proteins that have proved suitable for tumor targeting applications, previously primarily for molecular imaging purposes. Our high affinity HER3-binding affibody molecule has demonstrated to have anti-proliferative capacity on HER3-positive tumor cells. When formatted as a bivalent construct, in which the two affibody moieties are flanking a small albumin-binding domain (ABD), we have recently demonstrated that tumor growth could be delayed in mice for HER3-positive xenografts. In this study, we have modified the construct further and reduced the size. In a comparative study, we evaluated safety, the capacity to delay tumor growth in mice with BxPC-3 xenografts, and mouse survival. Our novel construct was compared to the HER3-specific monoclonal antibody seribantumab (MM-121), presently in clinical development. They were found to be equally potent in their therapeutic effects and in their safety profile. We conclude that this format of bivalent HER3-binding affibody molecules seems promising for further evaluation as candidate therapeutics for treatment of HER3-overexpressing tumors.

Keywords
Affibody, Cancer, ErbB3, HER3, Protein Engineering
National Category
Other Medical Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-204613 (URN)
Note

QC 20170330

Available from: 2017-03-29 Created: 2017-03-29 Last updated: 2017-03-30Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-1598-8894

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