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ADAPT, a Novel Scaffold Protein-Based Probe for Radionuclide Imaging of Molecular Targets That Are Expressed in Disseminated Cancers
KTH, School of Biotechnology (BIO), Protein Technology.
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
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2015 (English)In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 75, no 20, p. 4364-4371Article in journal (Refereed) Published
Resource type
Text
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. Vol. 75, no 20, p. 4364-4371
National Category
Cancer and Oncology
Identifiers
URN: urn:nbn:se:kth:diva-180154DOI: 10.1158/0008-5472.CAN-14-3497ISI: 000365601900013PubMedID: 26297736Scopus ID: 2-s2.0-84945567447OAI: oai:DiVA.org:kth-180154DiVA, id: diva2:893692
Note

QC 20160113

Available from: 2016-01-13 Created: 2016-01-07 Last updated: 2018-03-15Bibliographically 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. p. xii, 72
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 9
Keyword
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: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)
Opponent
Supervisors
Note

QC 20160510

Available from: 2016-05-10 Created: 2016-05-09 Last updated: 2016-05-16Bibliographically approved
2. Generation and engineering of ABD-derived affinity proteins for clinical applications
Open this publication in new window or tab >>Generation and engineering of ABD-derived affinity proteins for clinical applications
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins that specifically recognize and bind to other molecules or structures are important tools in industrial and medical applications. Binding proteins engineered from small stable scaffold proteins have been utilized for several purposes due to their favorable biophysical properties, tolerance to mutagenesis, efficient tissue penetration and ease of production. The 46 amino acid long albumin-binding domain (ABD) derived from the bacterial receptor Protein G is a promising scaffold that has been explored in this thesis. The scaffold was subjected to combinatorial protein engineering for generation of ABD-derived binding proteins with novel specificities. Furthermore, the medical potential of engineered ABD- derived affinity proteins (ADAPTs) was evaluated in a series of pre-clinical studies.

In the first studies, ADAPTs suitability as tracers for radionuclide molecular imaging was evaluated. Factors influencing biodistribution and tumor targeting properties were assessed in mice models bearing HER2 positive xenografts. All tested ADAPT constructs demonstrated high and specific targeting of HER2-expressing tumor cells as well as fast clearance from circulation. The results also showed that the size and character of the N- terminus affected the biodistribution profile of ADAPTs. Moreover, the targeting properties of ADAPTs proved to be highly influenced by the residualizing properties of the attached radionuclide label. Taken together, the results provided the first evidence that tumor imaging can be performed using ADAPTs and the favorable pharmacokinetic profiles in the studied mice models suggest that the scaffold is a promising candidate for clinical applications.

In the last study, a platform for generation of stable ABD-derived affinity proteins with novel binding specificities was established using a multi-step approach combining directed evolution and rational protein design. A broad combinatorial protein library with 20 randomized positions in ABD was designed and binders against three distinct targets were selected using phage display. Characterization of the selected binders provided information regarding optimal positions to randomize in a final library. In addition, the isolated binders were subjected to mutagenesis in certain surface exposed positions and mutations that provided increased stability were introduced into the original scaffold. Finally, a more focused combinatorial protein library consisting of 11 randomized positions was designed and constructed. The library was validated by selections against the same set of targets as for the first, broad library. The isolation of highly stable affinity ligands confirms that the library can be used for generation of diverse and stable affinity molecules.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2018. p. 84
Series
TRITA-CBH-FOU ; 2018:7
Keyword
ABD, ADAPT, affinity proteins, protein engineering, radionuclide molecular imaging, HER2
National Category
Pharmaceutical Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-224256 (URN)978-91-7729-715-4 (ISBN)
Public defence
2018-04-13, FR4 Oskar Kleins Auditorium, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20180315

Available from: 2018-03-15 Created: 2018-03-15 Last updated: 2018-03-15Bibliographically approved

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Nilvebrant, JohanÅstrand, MikaelHober, Sophia

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