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Article

^99mTc-maEEE-Z_HER2:342, an Affibody Molecule-Based Tracer for the Detection of HER2 Expression in Malignant Tumors

Thuy Tran^†, Torun Engfeldt^‡, Anna Orlova^†^§, Mattias Sandström, Joachim Feldwisch^†^§, Lars Abrahmsén^§, Anders Wennborg^§, Vladimir Tolmachev^†^§ and Amelie Eriksson Karlström*^‡

Division of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Sweden, School of Biotechnology, Division of Molecular Biotechnology, Royal Institute of Technology, Stockholm, Sweden, Affibody AB, Bromma, Sweden, Medical Radiation Physics, Uppsala University Hospital, Uppsala University, Uppsala, Sweden, and Department of Medical Sciences, Uppsala University, Uppsala, Sweden

Bioconjugate Chem., 2007, 18 (6), pp 1956–1964

DOI: 10.1021/bc7002617

Publication Date (Web): October 19, 2007

†

Division of Biomedical Radiation Sciences, Uppsala University.

‡

Royal Institute of Technology.

Affibody AB.

Medical Radiation Physics, Uppsala University.

Department of Medical Sciences, Uppsala University.

* Corresponding author. Amelie Eriksson Karlström, School of Biotechnology, Division of Molecular Biotechnology, Royal Institute of Technology, AlbaNova University Center, Roslagsvägen 30B, S-106 91 Stockholm, Sweden. Tel: +46-8-5537 8333 . Fax: +46-8-5537 8481. E-mail: amelie@biotech.kth.se.

Abstract

Detection of HER2-overexpression in tumors and metastases is important for the selection of patients who will benefit from trastuzumab treatment. Earlier investigations showed successful imaging of HER2-positive tumors in patients using indium- or gallium-labeled Affibody molecules. The goal of this study was to evaluate the use of ^99mTc-labeled Affibody molecules for the detection of HER2 expression. The Affibody molecule Z_HER2:342 with the chelator sequences mercaptoacetyl-Gly-Glu-Gly (maGEG) and mercaptoacetyl-Glu-Glu-Glu (maEEE) was synthesized by peptide synthesis and labeled with technetium-99m. Binding specificity, cellular retention, and in vitro stability were investigated. The biodistribution of ^99mTc-maGEG-Z_HER2:342 and ^99mTc-maEEE-Z_HER2:342 was compared with ^99mTc-maGGG-Z_HER2:342 in normal mice, and the tumor targeting properties of ^99mTc-maEEE-Z_HER2:342 were determined in SKOV-3 xenografted nude mice. The results showed that the Affibody molecules were efficiently labeled with technetium-99m. The labeled conjugates were highly stable in vitro with preserved HER2-binding capacity. The use of glutamic acid in the chelator sequences for ^99mTc-labeling of Z_HER2:342 reduced the hepatobiliary excretion 3-fold with a single Gly-to-Glu substitution and 10-fold with three Gly-to-Glu substitutions. ^99mTc-maEEE-Z_HER2:342 showed a receptor-specific tumor uptake of 7.9 ± 1.0 %IA/g and a tumor-to-blood ratio of 38 at 4 h pi. Gamma-camera imaging with ^99mTc-maEEE-Z_HER2:342 could detect HER2-expressing tumors in xenografts already at 1 h pi. It was concluded that peptide synthesis for the coupling of chelator sequences to Affibody molecules for ^99mTc labeling is an efficient way to modify the in vivo kinetics. Increased hydrophilicity, combined with improved stability of the mercaptoacetyl-triglutamyl chelator, resulted in favorable biodistribution, making ^99mTc-maEEE-Z_HER2:342 a promising tracer for clinical imaging of HER2 overexpression in tumors.