Molecular Design of HER3-Targeting Affibody Molecules: Influence of Chelator and Presence of HEHEHE-Tag on Biodistribution of 68Ga-Labeled Tracers

Affibody-based imaging of HER3 is a promising approach for patient stratification. We investigated the influence of a hydrophilic HEHEHE-tag ((HE)(3)-tag) and two different gallium-68/chelator-complexes on the biodistribution of Z(08698) with the aim to improve the tracer for PET imaging. Affibody molecules (HE)(3)-Z(08698)-X and Z(08698)-X (X = NOTA, NODAGA) were produced and labeled with gallium-68. Binding specificity and cellular processing were studied in HER3-expressing human cancer cell lines BxPC-3 and DU145. Biodistribution was studied 3 h p.i. in Balb/c nu/nu mice bearing BxPC-3 xenografts. Mice were imaged 3 h p.i. using microPET/CT. Conjugates were stably labeled with gallium-68 and bound specifically to HER3 in vitro and in vivo. Association to cells was rapid but internalization was slow. Uptake in tissues, including tumors, was lower for (HE)(3)-Z(08698)-X than for non-tagged variants. The neutral [Ga-68]Ga-NODAGA complex reduced the hepatic uptake of Z(08698) compared to positively charged [Ga-68]Ga-NOTA-conjugated variants. The influence of the chelator was more pronounced in variants without (HE)(3-)tag. In conclusion, hydrophilic (HE)(3)-tag and neutral charge of the [Ga-68]Ga-NODAGA complex promoted blood clearance and lowered hepatic uptake of Z(08698). [Ga-68]Ga-(HE)(3)-Z(08698)-NODAGA was considered most promising, providing the lowest blood and hepatic uptake and the best imaging contrast among the tested variants.