Use of a HEHEHE purification tag instead of a hexahistidine tag improves biodistribution of affibody molecules site-specifically labeled with ^99mTc, ¹¹¹In, and ¹²⁵I

Affibody molecules are a class of small (̃7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging in vivo. The attachment of a hexahistidine (His6)-tag to the Affibody molecule allows facile purification by immobilized metal ion affinity chromatography (IMAC) but leads to high accumulation of radioactivity in the liver. Earlier, we have demonstrated that replacement of the His₆-tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of Affibody molecules by IMAC, enables labeling with [ ^99mTc(CO)₃]⁺, and provides low hepatic accumulation of radioactivity. In this study, we compared the biodistribution of cysteine-containing Affibody molecules site-specifically labeled with ¹¹¹In, ^99mTc, and ¹²⁵I at the C-terminus, having a His₆-tag at the N- or C-terminus or a HEHEHE-tag at the N-terminus. We show that the use of a HEHEHEtag provides appreciable reduction of hepatic radioactivity, especially for radiometal labels. We hope that this information can also be useful for development of other scaffold protein-based imaging agents.