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Engineering of bispecific affinity proteins with nanomolar affinity for both ErbB2 and albumin
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), Proteomics.
KTH, School of Biotechnology (BIO), Proteomics.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The epidermal growth factor receptor 2, ErbB2, is a well-validated target for cancer therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-antigen specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, requiring frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ErbB2- targeting with antibody-like pharmacokinetic properties, we have engineered bispecific ErbB2-binding affinity proteins that are based on a small albumin-binding domain. Phage display technology was used for identification of a lead candidate as well as for affinity maturation using second-generation libraries. Affinity matured binders were shown to bind human ErbB2 with high affinity while still retaining the natural interaction with human serum albumin. Hence, two important properties that may be utilized for tumor targeting and in vivo half-life extension were combined in one molecule.

National Category
Biological Sciences
URN: urn:nbn:se:kth:diva-105516OAI: diva2:571302

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, MikaelGeorgieva, MariyaBjörnmalm, MattiasLofblom, JohnHober, Sophia
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