Influence of Histidine-Containing Tags on the Biodistribution of ADAPT Scaffold Proteins

Engineered scaffold proteins (ESP) are high-affinity binders that can be used as probes for radionuclide imaging. Histidine-containing tags enable both efficient purification of ESP and radiolabeling with ^99mTc(CO)₃. Earlier studies demonstrated that the use of a histidine-glutamate-histidine-glutamate-histidine-glutamate (HE)₃-tag instead of the commonly used hexahistidine (H₆)-tag reduces hepatic uptake of radiolabeled ESP and short peptides. Here, we investigated the influence of histidine-containing tags on the biodistribution of a novel type of ESP, ADAPTs. A series of anti-HER2 ADAPT probes having H₆- or (HE)₃-tags in the N-termini were prepared. The constructs, (HE)₃-ADAPT6 and H₆-ADAPT6, were labeled with two different nuclides, ^99mTc or ¹¹¹In. The labeling with ^99mTc(CO)₃ utilized the histidine-containing tags, while ¹¹¹In was attached through a maleimido derivative of DOTA conjugated to the N-terminus. For ¹¹¹In-labeled ADAPTs, the use of (HE)₃ provided a significantly (p < 0.05) lower hepatic uptake at 1 h after injection, but there was no significant difference in hepatic uptake of ¹¹¹In-(HE)₃-ADAPT6 and H₆-ADAPT6 at later time points. Interestingly, in the case of ^99mTc, ^99mTc(CO)₃-H₆-ADAPT6 provided significantly (p < 0.05) lower uptake in a number of normal tissues and was more suitable as an imaging probe. Thus, the influence of histidine-containing tags on the biodistribution of the novel ADAPT scaffold proteins was different compared to its influence on other ESPs studied so far. Apparently, the effect of a histidine-containing tag on the biodistribution is highly dependent on the scaffold composition of the ESP.