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Radionuclide Tumor Targeting Using ADAPT Scaffold Proteins: Aspects of Label Positioning and Residualizing Properties of the Label
KTH, School of Biotechnology (BIO), Protein Technology.
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2018 (English)In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 59, no 1, p. 93-99Article in journal (Refereed) Published
Abstract [en]

Visualization of cancer-associated alterations of molecular phenotype using radionuclide imaging is a noninvasive approach to stratifying patients for targeted therapies. The engineered albumin-binding domain-derived affinity protein (ADAPT) is a promising tracer for radionuclide molecular imaging because of its small size (6.5 kDa), which satisfies the precondition for efficient tumor penetration and rapid clearance. Previous studies demonstrated that the human epidermal growth factor receptor type 2 (HER2)-targeting ADAPT6 labeled with radiometals at the N terminus is able to image HER2 expression in xenografts a few hours after injection. The aim of this study was to evaluate whether the use of a non-residualizing label or placement of the labels at the C terminus would further improve the targeting properties of ADAPT6. Methods: Two constructs, Cys(2)-ADAPT6 and Cys(59)-ADAPT6, having the (HE)(3)DANS sequence at the N terminus were produced and site-specifically labeled using In-111-DOTA or I-125-iodo-((4-hydroxyphenyl) ethyl) maleimide (HPEM). The conjugates were compared in vitro and in vivo. HER2-targeting properties and biodistribution were evaluated in BALB/C nu/nu mice bearing ovarian carcinoma cell (SKOV-3) xenografts. Results: Specific HER2 binding and high affinity were preserved after labeling. Both Cys(2)-ADAPT6 and Cys59-ADAPT6 were internalized slowly by HER2-expressing cancer cells. Depending on the label position, uptake at 4 h after injection varied from 10% to 22% of the injected dose per gram of tumor tissue. Regardless of terminus position, the I-125-HPEM label provided more than 140-fold lower renal uptake than the In-111-DOTA label at 4 after injection. The tumor-to-organ ratios were, in contrast, higher for both of the (111)InDOTA- labeled ADAPT variants in other organs. Tumor-to-blood ratios for In-111-labeled Cys(2)-ADAPT6 and Cys(59)-ADAPT6 did not differ significantly (250-280), but In-111-DOTA-Cys(59)-ADAPT6 provided significantly higher tumor-to-lung, tumor-to-liver, tumor-to-spleen, and tumor-to-muscle ratios. Radioiodinated variants had similar tumor-to-organ ratios, but I-125-HPEM-Cys(59)-ADAPT6 had significantly higher tumor uptake and a higher tumor-to-kidney ratio. Conclusion: Residualizing properties of the label strongly influence the targeting properties of ADAPT6. The position of the radiolabel influences targeting as well, although to a lesser extent. Placement of a label at the C terminus yields the best biodistribution features for both radiometal and radiohalogen labels. Low renal retention of the radioiodine label creates a precondition for radionuclide therapy using I-131-labeled HPEM-Cys(59)-ADAPT6.

Place, publisher, year, edition, pages
Society of Nuclear Medicine , 2018. Vol. 59, no 1, p. 93-99
Keywords [en]
molecular biology, ADAPT, HER2, biodistribution, molecular imaging, terminal sequence
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
URN: urn:nbn:se:kth:diva-221362DOI: 10.2967/jnumed.117.197202ISI: 000419116200021PubMedID: 28864631Scopus ID: 2-s2.0-85040078184OAI: oai:DiVA.org:kth-221362DiVA, id: diva2:1174944
Funder
Swedish Cancer SocietySwedish Research Council
Note

QC 20180117

Available from: 2018-01-17 Created: 2018-01-17 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)
Opponent
Supervisors
Note

QC 20180315

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

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