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

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 radionuclide 99m Tc. The use of a GGGC chelator at the C terminus has provided the lowest renal radioactivity retention of the previously investigated peptide-based chelators. Previously, it has also been 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 immobilized metal ion affinity chromatography (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.