Designed ankyrin repeat protein (DARPin) Ec1, a small scaffold protein (18 kDa), binds with high affinity the epithelial cell adhesion molecule (EpCAM) that is overexpressed in several carcinomas. To enhance the targeted delivery of cytotoxic drugs using Ec1, we investigated the potential of fusing Ec1 with an albumin-binding domain (ABD) to improve its circulation time and decrease renal uptake. Two fusion proteins were created, Ec1-ABD, with the ABD at the C-terminus, and ABD-Ec1, with the ABD at the N-terminus. Both variants were labeled with 111In. ABD-fused variants bound specifically to EpCAM-expressing cells with picomolar affinity. Adding human albumin reduced the affinity. This effect was more pronounced for Ec1-ABD; however, the affinity remained in the subnanomolar range. The position of the ABD did not influence the internalization rate of both variants by human cancer cells. In mouse models with human cancer xenografts, both variants demonstrated over 10-fold lower renal uptake compared to the Ec1. Tumor uptake of the ABD-fused variants was higher than the uptake of Ec1. ABD-Ec1 provided two-fold higher tumor uptake, indicating fusion with an ABD as a promising way to modulate the targeting properties of an Ec1-based construct. However, the effect of fusion depends on the order of the domains.
Keywords: albumin-binding domain (ABD); designed ankyrin repeat protein; epithelial cell adhesion molecule (EpCAM); fusion protein; nude mice; xenograft.