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HEHEHE-Tagged Affibody Molecule May Be Purified by IMAC, Is Conveniently Labeled with [Tc-99m(CO)(3)](+), and Shows Improved Biodistribution with Reduced Hepatic Radioactivity Accumulation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. (BMS)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. (BMS)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. (BMS)
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2010 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 21, no 11, 2013-2022 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules are a class of small (ca. 7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging of therapeutic targets in vivo. A hexahistidine tag at the N-terminus streamlines development of new imaging probes by enabling facile purification using immobilized metal ion affinity chromatography (IMAC), as well as convenient [Tc-99m(CO)(3)](+)-labeling. However, previous studies in mice have demonstrated that Affibody molecules labeled by this method yield higher liver accumulation of radioactivity, compared to the same tracer lacking the hexahistidine tag and labeled by an alternative method. Two variants of the HER2-binding Affibody molecule Z(HER2:342) were made in an attempt to create a tagged tracer that could be purified by immobilized metal affinity chromatography, yet would not result in anomalous hepatic radioactivity accumulation following labeling with [Tc-99m(CO)(3)](+). In one construct, the hexahistidine tag was moved to the C-terminus. In the other construct, every second histidine residue in the hexahistidine tag was replaced by the more hydrophilic glutamate, resulting in a HEHEHE-tag. Both variants, denoted Z(HER2:342)-H-6 and (HE)(3)-Z(HER2:342), respectively, could be efficiently purified using IMAC and stably labeled with [Tc-99m(CO)(3)](+) and were subsequently compared with the parental H-6-Z(HER2:342) having an N-terminal hexahistidine tag. All three variants were demonstrated to specifically bind to HER2-expressing cells in vitro. The hepatic accumulation of radioactivity in a murine model was 2-fold lower with [Tc-99m(CO)(3)](+)-Z(HER2:342)-H-6 compared to [Tc-99m(CO)(3)](+)-H-6-Z(HER2:342), and more than 10-fold lower with [Tc-99m(CO)(3)](+)-(HE)(3)-Z(HER2:342). These differences translated into appreciably superior tumor-to-liver ratio for [Tc-99m(CO)(3)](+)-(HE)(3)-Z(HER2:342) compared to the alternative conjugates. This information might be useful for development of other scaffold-based molecular imaging probes.

Place, publisher, year, edition, pages
2010. Vol. 21, no 11, 2013-2022 p.
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Medical and Health Sciences
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URN: urn:nbn:se:uu:diva-139356DOI: 10.1021/bc1002357ISI: 000284203200010OAI: oai:DiVA.org:uu-139356DiVA: diva2:381052
Available from: 2010-12-23 Created: 2010-12-23 Last updated: 2017-12-11Bibliographically approved

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Tolmachev, VladimirOrlova, Anna

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