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Development and characterization of small bispecific three-helical ErbB3/albumin-binding domains aimed at therapeutic applications
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0002-6104-6446
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0003-4008-5275
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0001-9423-0541
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0003-0605-8417
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Affinity proteins based on small scaffolds are currently emerging as alternatives to antibodies for therapy. Similarly to antibodies, they can be engineered to have high affinity for specific proteins. A potential problem with small proteins and peptides is their short in vivo circulation time, which might limit the therapeutic efficacy. To circumvent this issue, we have engineered bispecificity into an albumin-binding domain (ABD) derived from streptococcal protein G. The inherent albumin binding was preserved while the opposite side of the molecule was randomized to create a flexible surface for selection of high-affinity binders. Here we present novel ABD-variants selected by phage display with the ability to bind to the epidermal growth factor receptor 3 (ErbB3). Isolated candidates were thoroughly characterized regarding affinity and stability. Importantly, they were shown to still have affinity to albumin, hence demonstrating that the intended strategy to engineer bispecific single-domain proteins was successful. Moreover, competition assays revealed that the new binders could block the natural ligand Neuregulin from binding to ErbB3, indicating a potential anti-proliferative effect. These new binders thus represent promising candidates for further development into ErbB3-signaling inhibitors, where the albumin interaction could result in prolonged in vivo half-life.

Keyword [en]
Albumin-binding domain, ABD, phage display, ErbB3, human serum albumin, HSA, bispecific
National Category
Biological Sciences
URN: urn:nbn:se:kth:diva-105514OAI: diva2:571298

QS 2012

Available from: 2012-11-22 Created: 2012-11-22 Last updated: 2012-11-22Bibliographically approved
In thesis
1. An albumin-binding domain as a scaffold for bispecific affinity proteins
Open this publication in new window or tab >>An albumin-binding domain as a scaffold for bispecific affinity proteins
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein engineering and in vitro selection systems are powerful methods to generate binding proteins. In nature, antibodies are the primary affinity proteins and their usefulness has led to a widespread use both in basic and applied research. By means of combinatorial protein engineering and protein library technology, smaller antibody fragments or alternative non-immunoglobulin protein scaffolds can be engineered for various functions based on molecular recognition. In this thesis, a 46 amino acid small albumin-binding domain derived from streptococcal protein G was evaluated as a scaffold for the generation of affinity proteins. Using protein engineering, the albumin binding has been complemented with a new binding interface localized to the opposite surface of this three-helical bundle domain. By using in vitro selection from a combinatorial library, bispecific protein domains with ability to recognize several different target proteins were generated. In paper I, a bispecific albumin-binding domain was selected by phage display and utilized as a purification tag for highly efficient affinity purification of fusion proteins. The results in paper II show how protein engineering, in vitro display and multi-parameter fluorescence-activated cell sorting can be used to accomplish the challenging task of incorporating two high affinity binding-sites, for albumin and tumor necrosis factor-alpha, into this new bispecific protein scaffold. Moreover, the native ability of this domain to bind serum albumin provides a useful characteristic that can be used to extend the plasma half-lives of proteins fused to it or potentially of the domain itself. When combined with a second targeting ability, a new molecular format with potential use in therapeutic applications is provided. The engineered binding proteins generated against the epidermal growth factor receptors 2 and 3 in papers III and IV are aimed in this direction. Over-expression of these receptors is associated with the development and progression of various cancers, and both are well-validated targets for therapy. Small bispecific binding proteins based on the albumin-binding domain could potentially contribute to this field. The new alternative protein scaffold described in this thesis is one of the smallest structured affinity proteins reported. The bispecific nature, with an inherent ability of the same domain to bind to serum albumin, is unique for this scaffold. These non-immunoglobulin binding proteins may provide several advantages as compared to antibodies in several applications, particularly when a small size and an extended half-life are of key importance. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. x, 103 p.
Trita-BIO-Report, ISSN 1654-2312 ; 2012:21
albumin-binding domain, bispecific, albumin, affinity protein, phage display, staphylococcal display, orthogonal affinity purification, TNF-alpha, ErbB2, ErbB3
National Category
Biochemistry and Molecular Biology Medical Biotechnology
Research subject
SRA - Molecular Bioscience
urn:nbn:se:kth:diva-105425 (URN)978-91-7501-569-9 (ISBN)
Public defence
2012-12-14, FR4, AlbaNova University Center, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Swedish Research CouncilKnut and Alice Wallenberg Foundation

QC 20121122

Available from: 2012-11-22 Created: 2012-11-21 Last updated: 2012-11-22Bibliographically approved

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Nilvebrant, JohanÅstrand, MikaelLofblom, JohnHober, Sophia
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