Recombinant single-chain antibodies with various oligopeptide tails for targeted gene delivery

Abstract

The single-chain antibody (scFv) made by recombinant DNA technology is one of the most useful tools for basic research and clinical applications. To develop a novel targeted gene delivery method, we engineered the scFv gene for the antibody against human epidermal growth factor (EGF) receptor by connecting with DNA sequences for various oligopeptides with negative or positive charges. The resulting recombinant genes encoding artificial scFv with negative or positive tails were expressed in Escherichia coli and yeast Pichia pastris. In E. coli, all the scFv with negatively charged tails were expressed but mainly as an insoluble form, whereas those with positively charged tails were barely expressed. In yeast P. pastris, all the scFv with negatively charged tails were efficiently expressed and secreted into the culture medium. Addition of high salt into the yeast culture increased their secretion. Purification procedure was established for the scFv with the longest negatively charged tail (D4S Â 5), yielding 5 mg/l with a purity of over 95%. The scFv-D4S Â 5 was designated as a recombinant immunoporter, which was then mixed with plasmid DNA and polyethylenimine (PEI). The resulting DNA/PEI/immunoporter complex (designated recombinant immunogene) exhibited efficient gene delivery to EGF receptor overexpressing A431 tumor cells.

Gene delivery to vascular endothelium using chemical vectors: implications for cardiovascular gene therapy

“…Those can be made either by cloning the genes for the heavy chain and the light chain variable domains of an antibody with a sequence encoding a flexible linker, or they can be identified by phage display. One such system used antiepidermal growth factor receptor antibodies conjugated to PEI to deliver DNA to epidermal growth factor receptor-expressing tumour cells [76]. Another approach used a fusion of an anti-erbB single chain antibodies and protamine to enhance delivery to erbB-positive cells [77].…”

Targeted gene delivery using humanized single‐chain antibody with negatively charged oligopeptide tail

“…The murine type recombinant immunoporter (mBBD20) and humanized immunoporter (hCaCD20) were purified as previously described. 11) In brief, the immunoporters secreted by yeast Pichia pastris into the medium were purified by successive use of anion-exchange chromatography on "STREAMLINE" Q-XL (Amersham Pharmacia Biotech), immobilized metal ion-affinity chromatography and gel filtration. Finally, the immunoporters were dialyzed against 600 mM NaCl.…”

“…11) One of the negatively charged oligopeptide tails was composed of five repeats of a unit consisting of four aspartates and one serine (D4S×5, D20) (Fig. 1A).…”

“…10) Recently, we were able to construct several modified scFv genes for use as immunoporter/immunogene systems. 11) One of these recombinant immunoporters consisting of scFv and a negatively charged oligopeptide tail was capable of delivering genes to A431 tumor cells through the EGF receptor. 11) In this paper, we studied various conditions necessary for the formation of proper recombinant immunogenes.…”

“…11) One of these recombinant immunoporters consisting of scFv and a negatively charged oligopeptide tail was capable of delivering genes to A431 tumor cells through the EGF receptor. 11) In this paper, we studied various conditions necessary for the formation of proper recombinant immunogenes. Moreover, we present evidence that recombinant immunogenes made with humanized scFv can be potent gene transfer vehicles to target particular tumor cells.…”