Development of drug targeting based on recombinant expression of the chicken avidin gene

Abstract

The chemistry required for covalent biotinylation of drugs, radiopharmaceuticals and other ligands is highly developed, and a large number of biotinylated reagents can be readily synthesized. In order to investigate whether expression of avidin cDNA in mammalian cells might be useful as part of a drug targeting strategy, we transiently expressed the avidin gene in two human tumor cell lines (the cervical carcinoma cell line, HeLa, and the liver derived line, Hep G2). Avidin protein as detected by either immunohistochemistry or binding of streptavidin-biotin complexes was present and functional following transient expression. This result indicated that the mechanisms underlying avidin oligomerization which are necessary for proper protein folding are present within mammalian carcinoma cell lines. Next, we generated a producer cell line (derived from psi2) capable of releasing a recombinant retrovirus encoding chicken avidin, and a tumorigenic murine breast cancer cell line (16/C) with stable avidin expression. We show that these cell lines are suitable for conferring functional expression of avidin in vitro. These experiments establish a means by which avidin gene expression can be explored as a mechanism for targeted gene delivery of biotin-derivitized drugs in vitro, and have important implications for utilization of this strategy in vivo.

Recombinant NeutraLite Avidin: a non‐glycosylated, acidic mutant of chicken avidin that exhibits high affinity for biotin and low non‐specific binding properties

“…Over the past two decades, the avidin‐biotin system has been developed for general applications in the life sciences [2–4]. The technology has also been used recently in targeted drug imaging and as an agent for delivery systems in medicine [25–28]. However, the presence of sugar and the positive charge of avidin can be a hindrance to its use in these applications, due to non‐specific binding to extraneous material.…”

Engineering of chicken avidin: a progressive series of reduced charge mutants

“…Throughout the years, avidin has become a frequently used tool in numerous biotechnological applications, including different localization, diagnostic and separation technologies [1]. Recently avidin has also found its use in affinity‐based targeting of drugs and imaging agents with promising results [2–5]. All these applications are generally based on the high affinity ( K d ∼10 −15 M) [6]avidin has for biotin, a low‐molecular‐weight vitamin, which can be readily attached to biologically active binders and detectable probes.…”

Biochemical characterization and crystal structure of a recombinant hen avidin and its acidic mutant expressed in Escherichia coli

“…Avidin has been expressed in Escherichia coli where it is accumulated in inclusion bodies, with only a minor proportion of soluble and functional protein [3,11]. Correctly assembled and functional recombinant avidin has been obtained from transfected mammalian cell lines [12] and, more recently, in a baculovirus expression system [13]. Mutated avidins, however, have not been described so far.…”