Single chain Fv: a ligand in receptor-mediated gene delivery

Abstract

We have used an anti-human polymeric immunoglobulin receptor (pIgR) single chain Fv (scFv) to deliver reporter genes to epithelial cells in vitro. The scFv was constructed from a monoclonal antibody directed against pIgR and a cysteine residue was added at the carboxyl end to facilitate its conjugation to polylysine (polyK) via the heterobifunctional cross-linker SPDP. ScFv-cys was expressed in Drosophila S2 cells and purified to homogeneity using conventional column chromatography. ScFv-polyK, and polyK as control, were condensed with a DNA expression plasmid containing the luciferase reporter gene driven by the CMV promoter into unimolecular (with respect to DNA) complexes under high salt conditions. Target cells were MDCK cells transfected with human pIgR and repeatedly sorted for highlevel receptor expression, with untransfected MDCK cells as

Targeting of Synthetic Gene Delivery Systems

“…A number of technologies for the de novo selection of potential binding ligands such as, for example, antibodies, phage display, combinatorial peptide, or nucleotide (aptamer) libraries, have recently become widely available and recombinant technology has greatly accelerated the discovery process. (Currently, phage display [81] drives the move to smaller targeting moieties such as antibody fragments [82, 83] and peptides [84, 85]. ) Ligands derived from these technologies have a number of advantages, in particular with respect to pharmaceutical, regulatory, and production issues, and some have already been tested in the clinic.…”

“…Antibodies to the transferrin receptor [82], the anti-platelet endothelial cell adhesion molecule PECAM [86], the polymeric immunoglobulin receptor pIgR [83], anti-CD5 [87], and the ErbB-2 receptor [88] have been used for the targeting of liposomes [11, 89], polylysine polyplexes [90], PEI polyplexes [86], and DNA-peptide complexes [91]. These systems in general mediated an increased uptake or expression in vivo compared to the untargeted vector.…”

Single‐Chain Fv Fragments from Phage Display Libraries

“…For targeted DNA delivery, ligands, such as antibodies, have been chemically coupled to polycations. This includes coupling to polylysine (Gupta et al, 2001), polyethylenimine (PEI) (O'Neill et al, 2001), or cationic amphiphiles, such as cholesterol spermine (Mohr et al, 1999). In case of short cationic peptides, these sequences can be directly incorporated into recombinant antibodies by means of genetic engineering.…”

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

“…An alternative to antibodies, or chemically prepared antibody fragments, are single-chain antibodies prepared by biotechnological methods [70]. 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.…”

“…An alternative strategy to promote cell-specific delivery is to use antibodies [9,51] or recombinant versions of the antibodies, such as single-chain Fv fragments [70]. For example, an anti-JL1 antibody coupled to polylysine has been used to deliver DNA to leukaemia cells [71], anti-CD3 antibodies coupled to PEI were used to enhance gene delivery to the Jurkat T cell line [72], and an antibody against the cancer-specific epithelial glycoprotein-2 was used to deliver lipoplexes to tumour cells [73].…”