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An albumin-binding domain as a scaffold for bispecific affinity proteins
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0002-6104-6446
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.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2012:21
Keyword [en]
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
Identifiers
URN: urn:nbn:se:kth:diva-105425ISBN: 978-91-7501-569-9 (print)OAI: oai:DiVA.org:kth-105425DiVA: diva2:571046
Public defence
2012-12-14, FR4, AlbaNova University Center, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

QC 20121122

Available from: 2012-11-22 Created: 2012-11-21 Last updated: 2012-11-22Bibliographically approved
List of papers
1. Engineering Bispecificity into a Single Albumin-Binding Domain
Open this publication in new window or tab >>Engineering Bispecificity into a Single Albumin-Binding Domain
2011 (English)In: PLoS ONE, ISSN 1932-6203, E-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.

National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-48516 (URN)10.1371/journal.pone.0025791 (DOI)000295943000071 ()2-s2.0-80053444381 (Scopus ID)
Note
QC 20111207Available from: 2011-12-07 Created: 2011-11-21 Last updated: 2017-12-08Bibliographically approved
2. A small bispecific protein selected for orthogonal affinity purification
Open this publication in new window or tab >>A small bispecific protein selected for orthogonal affinity purification
Show others...
2010 (English)In: BIOTECHNOL J, ISSN 1860-6768, Vol. 5, no 6, 605-617 p.Article in journal (Refereed) Published
Abstract [en]

A novel protein domain with dual affinity has been created by randomization and selection. The small alkali-stabilized albumin-binding domain (ABD(star)), used as scaffold to construct the library, has affinity to human serum albumin (HSA) and is constituted of 46 amino acids of which 11 were randomized. To achieve a dual binder, the binding site of the inherent HSA affinity was untouched and the randomization was made on the opposite side of the molecule. Despite its small size and randomization of almost a quarter of its amino acids, a bifunctional molecule, ABDz1, with ability to bind to both HSA and the Z(2) domain/protein A was successfully selected using phage display. Moreover, the newly selected variant showed improved affinity for HSA compared to the parental molecule. This novel protein domain has been characterized regarding secondary structure and affinity to the two different ligands. The possibility for affinity purification on two different matrices has been investigated using the two ligands, the HSA matrix and the protein A-based, MabSelect SuRe matrix, and the new protein domain was purified to homogeneity. Furthermore, gene fusions between the new domain and three different target proteins with different characteristics were made. To take advantage of both affinities, a purification strategy referred to as orthogonal affinity purification using two different matrices was created. Successful purification of all three versions was efficiently carried out using this strategy.

Keyword
Albumin binding domain, Bispecific binder, Orthogonal purification, Phage display, Z domain
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-27283 (URN)10.1002/biot.201000041 (DOI)000278969600009 ()20518064 (PubMedID)
Note

QC 20101214

Available from: 2010-12-14 Created: 2010-12-09 Last updated: 2012-11-22Bibliographically approved
3. Engineering of bispecific affinity proteins with nanomolar affinity for both ErbB2 and albumin
Open this publication in new window or tab >>Engineering of bispecific affinity proteins with nanomolar affinity for both ErbB2 and albumin
Show others...
(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
Identifiers
urn:nbn:se:kth:diva-105516 (URN)
Note

QS 2012

Available from: 2012-11-22 Created: 2012-11-22 Last updated: 2012-11-22Bibliographically approved
4. Development and characterization of small bispecific three-helical ErbB3/albumin-binding domains aimed at therapeutic applications
Open this publication in new window or tab >>Development and characterization of small bispecific three-helical ErbB3/albumin-binding domains aimed at therapeutic applications
(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
Albumin-binding domain, ABD, phage display, ErbB3, human serum albumin, HSA, bispecific
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-105514 (URN)
Note

QS 2012

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

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