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Investigating affinity-maturation strategies and reproducibility of fluorescence-activated cell sorting using a recombinant ADAPT library displayed on staphylococci
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0003-4008-5275
KTH, School of Biotechnology (BIO), Protein Technology. Centre for Cellular and Biomolecular Research, Donnelly Centre, University of Toronto, Toronto, ON, Canada.ORCID iD: 0000-0002-6104-6446
KTH, School of Biotechnology (BIO), Protein Technology. Department of Chemical and Biomolecular Engineering, University of Melbourne, Parkville, VIC, Australia.
KTH, School of Biotechnology (BIO), Protein Technology.
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2016 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 29, no 5, 187-195 p.Article in journal (Refereed) Published
Abstract [en]

During the past decades, advances in protein engineering have resulted in the development of various in vitro selection techniques (e.g. phage display) to facilitate discovery of new and improved proteins. The methods are based on linkage between genotype and phenotype and are often performed in successive rounds of selection. Since the resulting output depends on the selection pressures used and the applied strategy, parameters in each round must be carefully considered. In addition, studies have reported biases that can cause enrichment of unwanted clones and/or low correlation between abundance in output and affinity. We have recently developed a selection method based on display of protein libraries on Staphylococcus carnosus and isolation of affinity proteins by fluorescence-activated cell sorting. Here, we compared duplicate selections for affinity maturation using equilibrium binding at different target concentrations and kinetic off-rate selection. The results showed that kinetic selection is efficient for isolation of high-affinity binders and that equilibrium selection at subnanomolar concentrations should be avoided. Furthermore, the reproducibility of the selection was high and a clear correlation was observed between enrichment and affinity. This work reports on the reproducibility of bacterial display in combination with FACS and provides insights into selection design to help guide the development of new affinity proteins.

Place, publisher, year, edition, pages
Oxford University Press, 2016. Vol. 29, no 5, 187-195 p.
National Category
Engineering and Technology Natural Sciences
Research subject
Järnvägsgruppen - Effektiva tågsystem för persontrafik
URN: urn:nbn:se:kth:diva-184208DOI: 10.1093/protein/gzw006ISI: 000376351600004PubMedID: 26984961ScopusID: 2-s2.0-84965029316OAI: diva2:915592
Swedish Research Council

QC 20160404

Available from: 2016-03-30 Created: 2016-03-30 Last updated: 2016-06-10Bibliographically 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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Åstrand, MikaelNilvebrant, JohanBjörnmalm, MattiasLindbo, SarahHober, SophiaLöfblom, John
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