RESEARCH ARTICLE Evaluation of a HER2-targeting affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for 99m Tc-labelling at the C terminus Hanna Lindberg & Camilla Hofström & Mohamed Altai & Hadis Honorvar & Helena Wållberg & Anna Orlova & Stefan Ståhl & Torbjörn Gräslund & Vladimir Tolmachev Received: 10 October 2011 / Accepted: 21 December 2011 / Published online: 17 January 2012 # International Society of Oncology and BioMarkers (ISOBM) 2012 Abstract Affibody molecules are a class of small (ca.7 kDa) robust scaffold proteins with high potential as tracers for radionuclide molecular imaging in vivo. Incorporation of a cysteine-containing peptide-based chelator at the C terminus provides an opportunity for stable labelling with the radionu- clide 99m Tc. The use of a GGGC chelator at the C terminus has provided the lowest renal radioactivity retention of the previ- ously investigated peptide-based chelators. Previously, it has also been demonstrated that replacement of the His 6 -tag with the negatively charged histidine-glutamate-histidine-gluta- mate-histidine-glutamate (HEHEHE)-tag permits purification of affibody molecules by immobilized metal ion affinity chro- matography (IMAC) and provides low hepatic accumulation of radioactivity of conjugates site-specifically labelled at the C terminus using several different nuclides. We hypothesized that the combination of a HEHEHE-tag at the N terminus and a GGGC chelator at the C terminus of an affibody molecule would be a favourable format permitting IMAC purification and providing low uptake in excretory organs. To investigate this hypothesis, a (HE) 3 -Z HER2:342 -GGGC affibody molecule was generated. It could be efficiently purified by IMAC and stably labelled with 99m Tc. 99m Tc-(HE) 3 -Z HER2:342 -GGGC preserved specific binding to HER2-expressing cells. In NMRI mice, hepatic uptake of 99m Tc-(HE) 3 -Z HER2:342 -GGGC was lower than the uptake of the control affibody molecules, 99m Tc-Z HER2:2395 -VDC and 99m Tc-Z HER2:342 -GGGC. At 1 and 4 h after injection, the renal uptake of 99m Tc-(HE) 3 - Z HER2:342 -GGGC was 2–3-fold lower than uptake of 99m Tc- Z HER2:2395 -VDC, but it was substantially higher than uptake of 99m Tc-Z HER2:342 -GGGC. Further investigation indicated that a fraction of 99m Tc was chelated by the HEHEHE-tag which caused a higher accumulation of radioactivity in the kidneys. Thus, a combination of a HEHEHE-tag and the GGGC chelator in targeting scaffold proteins was found to be undesirable in the case of 99m Tc labelling due to a partial loss of site-specificity of nuclide chelation. Keywords Affibody molecules . Radionuclide molecular imaging . Technetium-99m . HEHEHE-tag . GGGC chelator . Biodistribution Introduction Selective systemic therapy is a promising strategy for treatment of disseminated cancers using drugs targeting malignancy- associated alterations on the tumour cells, using, e.g. mono- clonal antibodies. An example of successful application of this strategy is the targeting of human epidermal growth factor receptor type 2 (HER2)-expressing breast cancer. HER2 is a member of the transmembrane tyrosine kinase receptor family, which is overexpressed in 25–30% of breast and ovarian carcinomas [1, 2] and in 80% of urinary bladder carcinomas Hanna Lindberg and Camilla Hofström contributed equally to this study. H. Lindberg : C. Hofström : H. Wållberg : S. Ståhl : T. Gräslund Division of Molecular Biotechnology, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology, Stockholm, Sweden M. Altai : H. Honorvar : V. Tolmachev (*) Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, 751 85 Uppsala, Sweden e-mail: vladimir.tolmachev@bms.uu.se A. Orlova Department of Medicinal Chemistry, Preclinical PET Platform, Uppsala University, Uppsala, Sweden Tumor Biol. (2012) 33:641–651 DOI 10.1007/s13277-011-0305-z