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Comparative evaluation of tumor targeting using the anti-HER2 ADAPT scaffold protein labeled at the C-terminus with indium-111 or technetium-99m
KTH, School of Biotechnology (BIO), Protein Technology.
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 14780Article in journal (Refereed) Published
Abstract [en]

ABD-Derived Affinity Proteins (ADAPTs) is a novel class of engineered scaffold proteins derived from an albumin-binding domain of protein G. The use of ADAPT6 derivatives as targeting moiety have provided excellent preclinical radionuclide imaging of human epidermal growth factor 2 (HER2) tumor xenografts. Previous studies have demonstrated that selection of nuclide and chelator for its conjugation has an appreciable effect on imaging properties of scaffold proteins. In this study we performed a comparative evaluation of the anti-HER2 ADAPT having an aspartate-glutamate-alanine-valine-aspartate-alanine-asparagine-serine (DEAVDANS) N-terminal sequence and labeled at C-terminus with (99)mTc using a cysteine-containing peptide based chelator, glycine-serine-serine-cysteine (GSSC), and a similar variant labeled with In-111 using a maleimido derivative of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. Both (99)mTc-DEAVDANS-ADAPT6-GSSC and In-111-DEAVDANS-ADAPT6-GSSC-DOTA accumulated specifically in HER2-expressing SKOV3 xenografts. The tumor uptake of both variants did not differ significantly and average values were in the range of 19-21% ID/g. However, there was an appreciable variation in uptake of conjugates in normal tissues that resulted in a notable difference in the tumor-to-organ ratios. The In-111-DOTA label provided 2-6 fold higher tumor-to-organ ratios than (99)mTc-GSSC and is therefore the preferable label for ADAPTs.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP , 2017. Vol. 7, article id 14780
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:kth:diva-217934DOI: 10.1038/s41598-017-15366-wISI: 000414569900003Scopus ID: 2-s2.0-85033458225OAI: oai:DiVA.org:kth-217934DiVA, id: diva2:1158857
Note

QC 20171121

Available from: 2017-11-21 Created: 2017-11-21 Last updated: 2018-03-15Bibliographically approved
In thesis
1. 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
Keywords
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)
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Supervisors
Note

QC 20180315

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

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