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Engineering strategies for ABD-derived affinity proteins for therapeutic and diagnostic applications
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0003-4008-5275
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.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 9
Keyword [en]
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
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-186279ISBN: 978-91-7595-983-2 (print)OAI: oai:DiVA.org:kth-186279DiVA: diva2:926726
Public defence
2016-06-03, Q2, Osquldas väg 10, Q-huset, våningsplan 2, KTH Campus, Stockholm, 15:21 (English)
Opponent
Supervisors
Note

QC 20160510

Available from: 2016-05-10 Created: 2016-05-09 Last updated: 2016-05-16Bibliographically approved
List of papers
1. Development and characterization of small bispecific albumin-binding domains with high affinity for ErbB3
Open this publication in new window or tab >>Development and characterization of small bispecific albumin-binding domains with high affinity for ErbB3
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.

Keyword
Albumin-binding domain, ABD, Phage display, ErbB3, HSA, Bispecific
National Category
Biochemistry and Molecular Biology Cell Biology
Identifiers
urn:nbn:se:kth:diva-131707 (URN)10.1007/s00018-013-1370-9 (DOI)000324774000015 ()2-s2.0-84884908517 (Scopus ID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

QC 20131018

Available from: 2013-10-18 Created: 2013-10-17 Last updated: 2017-12-06Bibliographically approved
2. Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin
Open this publication in new window or tab >>Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin
Show others...
2014 (English)In: PLoS ONE, ISSN 1932-6203, E-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.

Keyword
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
Identifiers
urn:nbn:se:kth:diva-149967 (URN)10.1371/journal.pone.0103094 (DOI)000339812700014 ()2-s2.0-84905457095 (Scopus ID)
Funder
Swedish Research CouncilThe Royal Swedish Academy of Sciences
Note

QC 20140904

Available from: 2014-09-04 Created: 2014-08-29 Last updated: 2017-12-05Bibliographically approved
3. ADAPT, a Novel Scaffold Protein-Based Probe for Radionuclide Imaging of Molecular Targets That Are Expressed in Disseminated Cancers
Open this publication in new window or tab >>ADAPT, a Novel Scaffold Protein-Based Probe for Radionuclide Imaging of Molecular Targets That Are Expressed in Disseminated Cancers
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2015 (English)In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 75, no 20, 4364-4371 p.Article in journal (Refereed) Published
Abstract [en]

Small engineered scaffold proteins have attracted attention as probes for radionuclide-based molecular imaging. One class of these imaging probes, termed ABD-Derived Affinity Proteins (ADAPT), has been created using the albumin-binding domain (ABD) of streptococcal protein G as a stable protein scaffold. In this study, we report the development of a clinical lead probe termed ADAPT6 that binds HER2, an oncoprotein overexpressed in many breast cancers that serves as a theranostic biomarker for several approved targeting therapies. Surface-exposed amino acids of ABD were randomized to create a combinatorial library enabling selection of high-affinity binders to various proteins. Furthermore, ABD was engineered to enable rapid purification, to eradicate its binding to albumin, and to enable rapid blood clearance. Incorporation of a unique cysteine allowed site-specific conjugation to a maleimido derivative of a DOTA chelator, enabling radionuclide labeling, In-111 for SPECT imaging and Ga-68 for PET imaging. Pharmacologic studies in mice demonstrated that the fully engineered molecule In-111/Ga-68-DOTA(HE) 3-ADAPT6 was specifically bound and taken up by HER2-expressing tumors, with a high tumor-to-normal tissue ratio in xenograft models of human cancer. Unbound tracer underwent rapid renal clearance followed by high renal reabsorption. HER2-expressing xenografts were visualized by gamma-camera or PET at 1 hour after infusion. PET experiments demonstrated feasibility for discrimination of xenografts with high or low HER2 expression. Our results offer a preclinical proof of concept for the use of ADAPT probes for noninvasive in vivo imaging.

Place, publisher, year, edition, pages
American Association for Cancer Research Inc., 2015
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:kth:diva-180154 (URN)10.1158/0008-5472.CAN-14-3497 (DOI)000365601900013 ()26297736 (PubMedID)2-s2.0-84945567447 (Scopus ID)
Note

QC 20160113

Available from: 2016-01-13 Created: 2016-01-07 Last updated: 2017-11-30Bibliographically approved
4. Investigating affinity-maturation strategies and reproducibility of fluorescence-activated cell sorting using a recombinant ADAPT library displayed on staphylococci
Open this publication in new window or tab >>Investigating affinity-maturation strategies and reproducibility of fluorescence-activated cell sorting using a recombinant ADAPT library displayed on staphylococci
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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
National Category
Engineering and Technology Natural Sciences
Research subject
Järnvägsgruppen - Effektiva tågsystem för persontrafik
Identifiers
urn:nbn:se:kth:diva-184208 (URN)10.1093/protein/gzw006 (DOI)000376351600004 ()26984961 (PubMedID)2-s2.0-84965029316 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20160404

Available from: 2016-03-30 Created: 2016-03-30 Last updated: 2017-11-30Bibliographically approved

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