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Engineering Bispecificity into a Single Albumin-Binding Domain
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0002-6104-6446
KTH, School of Biotechnology (BIO), Proteomics.
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0003-0605-8417
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0001-9423-0541
2011 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 6, no 10, e25791- p.Article in journal (Refereed) Published
Abstract [en]

Bispecific antibodies as well as non-immunoglobulin based bispecific affinity proteins are considered to have a very high potential in future biotherapeutic applications. In this study, we report on a novel approach for generation of extremely small bispecific proteins comprised of only a single structural domain. Binding to tumor necrosis factor-alpha (TNF-alpha) was engineered into an albumin-binding domain while still retaining the original affinity for albumin, resulting in a bispecific protein composed of merely 46 amino acids. By diversification of the non albumin-binding side of the three-helix bundle domain, followed by display of the resulting library on phage particles, bispecific single-domain proteins were isolated using selections with TNF-alpha as target. Moreover, based on the obtained sequences from the phage selection, a second-generation library was designed in order to further increase the affinity of the bispecific candidates. Staphylococcal surface display was employed for the affinity maturation, enabling efficient isolation of improved binders as well as multiparameter-based sortings with both TNF-alpha and albumin as targets in the same selection cycle. Isolated variants were sequenced and the binding to albumin and TNF-alpha was analyzed. This analysis revealed an affinity for TNF-alpha below 5 nM for the strongest binders. From the multiparameter sorting that simultaneously targeted TNF-alpha and albumin, several bispecific candidates were isolated with high affinity to both antigens, suggesting that cell display in combination with fluorescence activated cell sorting is a suitable technology for engineering of bispecificity. To our knowledge, the new binders represent the smallest engineered bispecific proteins reported so far. Possibilities and challenges as well as potential future applications of this novel strategy are discussed.

Place, publisher, year, edition, pages
2011. Vol. 6, no 10, e25791- p.
National Category
Biological Sciences
URN: urn:nbn:se:kth:diva-48516DOI: 10.1371/journal.pone.0025791ISI: 000295943000071ScopusID: 2-s2.0-80053444381OAI: diva2:462610
QC 20111207Available from: 2011-12-07 Created: 2011-11-21 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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