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Use of a HEHEHE Purification Tag Instead of a Hexahistidine Tag Improves Biodistribution of Affibody Molecules Site-Specifically Labeled with Tc-99m, In-111 and I-125
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
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2011 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 54, no 11, 3817-3826 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules are a class of small (similar to 7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging in vivo. The attachment of a hexahistidine (His(6))-tag to the Affibody molecule allows facile purification by immobilized metal ion affinity chromatography (IMAC) but leads to high accumulation of radioactivity in the liver. Earlier, we have demonstrated that replacement of the His(6)- tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of Affibody molecules by IMAC, enables labeling with [Tc-99m(CO)(3)](+), and provides low hepatic accumulation of radioactivity. In this study, we compared the biodistribution of cysteine-containing Affibody molecules site-specifically labeled with In-111, Tc-99m, and I-125 at the C-terminus, having a His(6)-tag at the N- or C-terminus or a HEHEHE-tag at the N-terminus. We show that the use of a HEHEHE-tag provides appreciable reduction of hepatic radioactivity, especially for radiometal labels. We hope that this information can also be useful for development of other scaffold protein-based imaging agents.

Place, publisher, year, edition, pages
2011. Vol. 54, no 11, 3817-3826 p.
Keyword [en]
National Category
Other Basic Medicine
URN: urn:nbn:se:kth:diva-34657DOI: 10.1021/jm200065eISI: 000291082500010ScopusID: 2-s2.0-79958148943OAI: diva2:426890
Swedish Research Council
QC 20110627Available from: 2011-06-27 Created: 2011-06-13 Last updated: 2013-01-29Bibliographically approved
In thesis
1. Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting
Open this publication in new window or tab >>Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Affibody molecules are small (7 kDa) affinity proteins of non-immunoglobulin origin that have been generated to specifically interact with a large number of clinically important molecular targets.

In this thesis, Affibody molecules have been employed as tracers for radionuclide molecular imaging of HER2- and IGF-1R-expressing tumors, paper I-IV, and for surface knock-down of EGFR, paper V. In paper I, a tag with the amino acid sequence HEHEHE was fused to the N-terminus of a HER2-specific Affibody molecule, (ZHER2), and was shown to enable facile IMAC purification and efficient tri-carbonyl 99mTc-labeling. In vivo evaluation of radioactivity uptake in different organs showed an improved biodistribution, including a 10-fold lower radioactivity uptake in liver, compared to the same construct with a H6-tag. In paper II, it was further shown that an N-terminally placed HEHEHE-tag on ZHER2 provided lower unspecific uptake of radioactivity in liver compared to its H6-tagged counterpart even when radiolabeling was at the C-terminus using alternative chemistries to attach 99mTc, 111In or 125I. In paper III, the H6-tag’s composition and position was varied with regards to charge, hydrophobicity and its C- or N-terminal placement on ZHER2. Among the ten variants investigated, it was found that an N-terminal HEHEHE-tag provided the most favorable overall biodistribution profile and that introduction of hydrophobic and positively charged amino acids provoked liver uptake of radioactivity. In paper IV, the HEHEHE-tag was shown to enable IMAC purification and tri-carbonyl 99mTc-labeling of an IGF-1R-specific Affibody molecule and improved its overall biodistribution when compared to the same construct with a H6-tag. In paper V, the aim was to develop an intracellular receptor-entrapment system to reduce the surface levels of EGFR. An EGFR-specific Affibody molecule was expressed as a fusion to different mutants of an intracellular transport protein in SKOV-3 cells, resulting in a collection of cell lines with 50%, 60%, 80% and 96% reduced surface level of EGFR. Analysis of the proliferation rate of these cell lines showed that a modest reduction (15%) in proliferation occurs between 60% and 80% reduction of the surface level of EGFR.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2013. xi, 65 p.
Trita-BIO-Report, ISSN 1654-2312 ; 2013:2
Affibody molecules, affinity proteins, radionuclide molecular imaging, intracellular targeting, EGFR, HER2, IGF-1R
National Category
Biochemistry and Molecular Biology Medical Biotechnology
Research subject
SRA - Molecular Bioscience
urn:nbn:se:kth:diva-116884 (URN)978-91-7501-613-9 (ISBN)
Public defence
2013-02-20, FR4, AlbaNova University Center Roslagstullsbacken 21, Stockholm, 10:30 (English)
Swedish Research CouncilKnut and Alice Wallenberg Foundation

QC 20130129

Available from: 2013-01-29 Created: 2013-01-29 Last updated: 2013-01-29Bibliographically approved

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