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Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin
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.
KTH, School of Biotechnology (BIO), Protein Technology.
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2014 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 9, no 8, e103094- p.Article in journal (Refereed) Published
Abstract [en]

The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and 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-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein.

Place, publisher, year, edition, pages
2014. Vol. 9, no 8, e103094- p.
Keyword [en]
albumin, albumin binding domain derived affinity protein, epidermal growth factor receptor 2, epitope, peptides and proteins, trastuzumab, unclassified drug, amino acid sequence, amino acid substitution, antigen binding, article, binding affinity, binding site, controlled study, DNA sequence, fluorescence activated cell sorting, nonhuman, protein binding, protein domain, protein engineering, protein expression, protein protein interaction, protein purification, protein targeting
National Category
Biological Sciences
URN: urn:nbn:se:kth:diva-149967DOI: 10.1371/journal.pone.0103094ISI: 000339812700014ScopusID: 2-s2.0-84905457095OAI: diva2:743573
Swedish Research CouncilThe Royal Swedish Academy of Sciences

QC 20140904

Available from: 2014-09-04 Created: 2014-08-29 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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Nilvebrant, JohanÅstrand, MikaelGeorgieva-Kotseva, MariaBjörnmalm, MattiasLöfblom, JohnHober, Sophia
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