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Influence of Histidine-Containing Tags on the Biodistribution of ADAPT Scaffold Proteins.
KTH, School of Biotechnology (BIO), Protein Technology.
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0003-4008-5275
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2016 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 27, no 3, p. 716-726Article in journal (Refereed) Published
Abstract [en]

Engineered scaffold proteins (ESP) are high-affinity binders that can be used as probes for radionuclide imaging. Histidine-containing tags enable both efficient purification of ESP and radiolabeling with (99m)Tc(CO)3. Earlier studies demonstrated that the use of a histidine-glutamate-histidine-glutamate-histidine-glutamate (HE)3-tag instead of the commonly used hexahistidine (H6)-tag reduces hepatic uptake of radiolabeled ESP and short peptides. Here, we investigated the influence of histidine-containing tags on the biodistribution of a novel type of ESP, ADAPTs. A series of anti-HER2 ADAPT probes having H6- or (HE)3-tags in the N-termini were prepared. The constructs, (HE)3-ADAPT6 and H6-ADAPT6, were labeled with two different nuclides, (99m)Tc or (111)In. The labeling with (99m)Tc(CO)3 utilized the histidine-containing tags, while (111)In was attached through a maleimido derivative of DOTA conjugated to the N-terminus. For (111)In-labeled ADAPTs, the use of (HE)3 provided a significantly (p < 0.05) lower hepatic uptake at 1 h after injection, but there was no significant difference in hepatic uptake of (111)In-(HE)3-ADAPT6 and H6-ADAPT6 at later time points. Interestingly, in the case of (99m)Tc, (99m)Tc(CO)3-H6-ADAPT6 provided significantly (p < 0.05) lower uptake in a number of normal tissues and was more suitable as an imaging probe. Thus, the influence of histidine-containing tags on the biodistribution of the novel ADAPT scaffold proteins was different compared to its influence on other ESPs studied so far. Apparently, the effect of a histidine-containing tag on the biodistribution is highly dependent on the scaffold composition of the ESP.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2016. Vol. 27, no 3, p. 716-726
National Category
Biological Sciences Chemical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-184209DOI: 10.1021/acs.bioconjchem.5b00677ISI: 000372478600026PubMedID: 26781756Scopus ID: 2-s2.0-84962209236OAI: oai:DiVA.org:kth-184209DiVA, id: diva2:915597
Funder
Swedish Cancer Society, CAN 2015/350Swedish Research Council, 2015-02353 621-2012-5088
Note

QC 20160405

Available from: 2016-03-30 Created: 2016-03-30 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
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)
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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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Åstrand, MikaelHober, Sophia

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