HEHEHE-tagged affibody molecule may be purified by IMAC, is conveniently labeled with [99mTc(CO)3]+, and shows improved biodistribution with reduced hepatic radioactivity accumulation

Vladimir Tolmachev, Camilla Hofström, Jennie Malmberg, Sara Ahlgren, Seyed Jalal Hosseinimehr, Mattias Sandström, Lars Abrahmsén, Anna Orlova, Torbjörn Gräslund

Research output: Contribution to journalArticlepeer-review

59 Citations (Scopus)

Abstract

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 [99mTc(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 ZHER2:342 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 [99mTc(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 ZHER2:342-H6 and (HE)3-ZHER2:342, respectively, could be efficiently purified using IMAC and stably labeled with [99mTc(CO) 3]+ and were subsequently compared with the parental H6-ZHER2:342 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 [99mTc(CO)3]+-ZHER2:342- H6 compared to [99mTc(CO)3]+-H 6-ZHER2:342, and more than 10-fold lower with [ 99mTc(CO)3]+-(HE)3-Z HER2:342. These differences translated into appreciably superior tumor-to-liver ratio for [99mTc(CO)3]+-(HE) 3-ZHER2:342 compared to the alternative conjugates. This information might be useful for development of other scaffold-based molecular imaging probes.

Original languageEnglish
Pages (from-to)2013-2022
Number of pages10
JournalBioconjugate Chemistry
Volume21
Issue number11
DOIs
Publication statusPublished - 17 Nov 2010
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

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