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Development and characterization of small bispecific albumin-binding domains with high affinity for ErbB3
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0002-6104-6446
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0003-4008-5275
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0001-9423-0541
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0003-0605-8417
2013 (English)In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 70, no 20, 3973-3985 p.Article in journal (Refereed) Published
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 for selection of high-affinity binders. Here we present novel ABD variants with the ability to bind to the epidermal growth factor receptor 3 (ErbB3). Isolated candidates were shown to have an extraordinary thermal stability and affinity for ErbB3 in the nanomolar range. Importantly, they were also shown to retain their affinity to albumin, hence demonstrating that the intended strategy to engineer bispecific single-domain proteins against a tumor-associated receptor was successful. Moreover, competition assays revealed that the new binders could block the natural ligand Neuregulin-1 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.

Place, publisher, year, edition, pages
2013. Vol. 70, no 20, 3973-3985 p.
Keyword [en]
Albumin-binding domain, ABD, Phage display, ErbB3, HSA, Bispecific
National Category
Biochemistry and Molecular Biology Cell Biology
URN: urn:nbn:se:kth:diva-131707DOI: 10.1007/s00018-013-1370-9ISI: 000324774000015ScopusID: 2-s2.0-84884908517OAI: diva2:657220
Swedish Research CouncilKnut and Alice Wallenberg Foundation

QC 20131018

Available from: 2013-10-18 Created: 2013-10-17 Last updated: 2016-05-10Bibliographically approved
In thesis
1. Engineering strategies for ABD-derived affinity proteins for therapeutic and diagnostic applications
Open this publication in new window or tab >>Engineering strategies for ABD-derived affinity proteins for therapeutic and diagnostic applications
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Small stable protein domains are attractive scaffolds for engineering affinity proteins due to their high tolerance to mutagenesis without loosing structural integrity. The albuminbinding domain is a 5 kDa three-helix bundle derived from the bacterial receptor Protein G with low-nanomolar affinity to albumin. In this thesis, the albumin-binding domain is explored as a scaffold for engineering novel affinity proteins with the possible benefit of combining a prolonged serum half-life with specific targeting in a single small scaffold protein. Previously, a library was created by randomizing surface-exposed residues in order to engineer affinity to a new target antigen in addition to the inherent albumin affinity. Here, phage display selections were separately performed against the tumor antigens ERBB2 and ERBB3. The ERBB3 selection resulted in a panel of candidates that were found to have varying affinities to ERBB3 in the nanomolar range, while still retaining a high affinity to albumin. Further characterization concluded that the clones also competed for binding to ERBB3 with the natural activating ligand Heregulin. The selections against ERBB2 resulted in sub-nanomolar affinities to ERBB2 where the binding site was found to overlap with the antibody Trastuzumab. The binding sites on ABD to albumin and either target were found in both selections to be mutually exclusive, as increased concentrations of albumin reduced the level of binding to ERBB2 or ERBB3. An affinity-matured ERBB2 binder, denoted ADAPT6, which lacked affinity to albumin was evaluated as a radionuclide-labeled imaging tracer for diagnosing ERBB2-positive tumors. Biodistribution studies in mice showed a high renal uptake consistent with affinity proteins in the same size range and the injected ADAPT quickly localized to the implanted tumor. High contrast images could be generated and ERBB2-expressing tissue could be distinguished from normal tissue with high contrast, demonstrating the feasibility of the scaffold for use as diagnostic tool. In a fourth study, affinity maturation strategies using staphylococcal cell-surface display were evaluated by comparing two replicate selections and varying the stringency. A sub-nanomolar target concentration was concluded to be inappropriate for equilibrium selection as the resulting output was highly variable between replicates. In contrast, equilibrium sorting at higher concentrations followed by kinetic-focused off-rate selection resulted in high output overlap between attempts and a clear correlation between affinity and enrichment.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. xii, 72 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 9
ABD, ADAPT, Protein G, ERBB2, ERBB3, Directed evolution, phage display, staphylococcal display, bispecific
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
urn:nbn:se:kth:diva-186279 (URN)978-91-7595-983-2 (ISBN)
Public defence
2016-06-03, Q2, Osquldas väg 10, Q-huset, våningsplan 2, KTH Campus, Stockholm, 15:21 (English)

QC 20160510

Available from: 2016-05-10 Created: 2016-05-09 Last updated: 2016-05-16Bibliographically approved

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