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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
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0003-1763-9073
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0003-1598-8894
KTH, School of Biotechnology (BIO), Protein Technology.
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2014 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 9, no 9, 1215-1222 p.Article 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.

Place, publisher, year, edition, pages
2014. Vol. 9, no 9, 1215-1222 p.
Keyword [en]
Affibody molecules, Albumin-binding domain, Bispecific, Half-life extension, HER3
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-153852DOI: 10.1002/biot.201400009ISI: 000341694200013Scopus ID: 2-s2.0-84906948848OAI: oai:DiVA.org:kth-153852DiVA: diva2:754866
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
In thesis
1. Affibody molecules targeting HER3 for cancer therapy
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. 87 p.
Keyword
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)
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Note

QC 20170330

Available from: 2017-03-30 Created: 2017-03-29 Last updated: 2017-03-30Bibliographically approved

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Malm, MagdalenaBass, TarekStåhl, StefanLöfblom, John

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