Targeting of biotinylated compounds to its target tissue using a low-density lipoprotein receptor–avidin fusion protein

Abstract

The very high binding affinity of avidin to biotin is one of the highest to occur in nature. We constructed a fusion protein composed of avidin and the endocytotic LDL receptor in order to target biotinylated molecules to cells of the desired tissues. In addition to the native avidin, charge-mutated and nonglycosylated avidins were utilized as part of the fusion proteins, in order to modify its properties. All of the fusion protein versions retained the biotin-binding capacity. Although the specificity was not increased, however, fusion proteins composed of natural avidin and nonglycosylated avidin bound most efficiently to the biotinylated ligands. Fluorescence microscopy and atomic force microscopy studies revealed the expression of the fusion protein on cell membranes, and demonstrated specific and high-affinity binding of biotin to the low-density lipoprotein receptor (LDLR)-avidin fusion protein in vitro. Additionally, systemically administered biotinylated ligand targeted with high specificity the intracerebral tumors of rats that were expressing fusion protein after the virus-mediated gene transfer. These results suggest that local gene transfer of the fusion protein to target tissues may offer a novel tool for the delivery of biotinylated molecules in vitro and in vivo for therapeutic and imaging purposes.

Novel Avidin-like Protein from a Root Nodule Symbiotic Bacterium, Bradyrhizobium japonicum

“…Medical applications of avidin-biotin technology (10,11) include, for example, gene therapy (12,13), imaging (14), and targeted drug delivery (15,16). In traditional one-step radioimmunotherapy, a therapeutic radioactive material is directly linked to a tumor-specific antibody (17)(18)(19).…”

Avidin Fusion Protein Strategies in Targeted Drug and Gene Delivery

“…The Lodavin expression was detected with biotinylated peroxidase in tumors, whereas no binding was seen in control animals. Furthermore, systemically administered biotinylated transferrin showed specific accumulation in gliomas having distribution congruent to Lodavin expression [15].…”

“…In the Lodavin construct the cytoplasmic, transmembrane and a part of the EGF-homology domains were included in the fusion [15]. In contrast to the C-terminal location of avidin in Scavidin, in Lodavin the biotin binder was positioned N-terminally.…”

“…avidin chimeras i.e. Scavidin, [14] and Lodavin [15]. These two fusion proteins are expressed on cell membranes where they provide an artificial receptor for biotinylated drug and imaging molecules.…”

Small Animal Imaging in Development of New Generation Diagnostic and Therapeutic Agents

“…Using adenovirus mediated gene transfer transient expression of the fusion protein on cell membrane was achieved (Lehtolainen et al 2002, Lehtolainen et al 2003. When biotinylated molecule binds to the fusion receptor, it is internalized into the cell.…”