A lysine-free mutant of epidermal growth factor as targeting moiety of a targeted toxin

Abstract

AimsElevated levels of epidermal growth factor (EGF) receptor are observed on several human tumors, e.g. cervical carcinoma and mamma carcinomas. The natural ligand EGF is an alternative to established antibodies and tyrosine kinase inhibitors for targeting EGF receptor-overexpressing tumor cells for therapy. Conjugations of compounds to EGF lack the necessary homogeneity for an intended application, since several amino acids may react with the chemical linker. Main methodsWe designed an EGF variant (EGFRR) in which the two lysines were substituted with arginine (K28R and K48R). EGFRR was fused to the protein toxin saporin to obtain a model protein for detailed analyses on EGF receptor binding and on both the enzymatic activity of saporin and the cytotoxicity of the fusion protein. Key findingsThe mutation decreased the enzymatic activity of saporin 2.3-fold and the binding of EGFRR retained its specificity for EGF receptor while increasing the Kd 5.5-fold. In spite of these differences the cytotoxicity of the fusion protein was unchanged in comparison to a fusion protein with EGF both when applied alone and in combination with cytotoxicity augmenting saponin. SignificanceWe conclude that EGFRR retained its ability to bind with high specificity to EGF receptor and is thus suitable for a number of chemical linkage applications such as targeting drugs or dyes to EGF receptor-expressing cells.