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

Abstract

e have been developing a non-viral cell-specific gene delivery system named immunoporter, using a modified monoclonal antibody.1-6) The original immunoporter was a conjugate between a monoclonal antibody and a cationic peptide poly-L-lysine (pLys).1) The Fab fragment of antibody was also conjugated with pLys.3) These whole-antibody or Fab-fragment immunoporters readily bound to DNA and the resulting complex, named an immunogene, delivered a reporter gene or therapeutic gene(s) to target cells in vitro and in vivo.5) Most importantly, the Fab immunoporter carrying herpes simplex virus (HSV) thymidine kinase (TK) gene together with ganciclovir treatment was effective in suppression of the growth of epidermal growth factor (EGF) receptor-hyperproducing tumor cells in nude mice. 5) Thus, the immunoporter/immunogene approach was considered as a promising alternative to viral vectors for targeted gene delivery. However, the chemical conjugation of monoclonal antibody or Fab fragment is laborious and unsuitable for large-scale preparation, which would be required for future clinical use. To overcome this potential problem, we exploited recombinant single-chain antibody (scFv) technology.7) The scFv is regarded as the minimal structural component of an antibody required for antigen-binding activity, 7) and can be produced in high quantity by means of gene expression in Escherichia coli, 8) yeast 9) or mammalian cells. 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. Moreover, we present evidence that recombinant immunogenes made with humanized scFv can be potent gene transfer vehicles to target particular tumor cells. Materials and MethodsCell culture. Human squamous carcinoma A431 cells over-expressing EGF receptors were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum at 37°C and 5% CO 2 .B4G7 monoclonal antibody. B4G7 antibody is an IgG2b class murine monoclonal antibody and is specific to human EGF receptor. 20,21) Plasmid preparation. pSRK-GL3 encodes luciferase (Luc) gene and pSRD-TK encodes HSV-TK gene.11) These recombinant plasmids were purified with Endfree QIAGEN Plasmid Purification Kit (QIAGEN).Purification of recombinant immunoporters. 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. The yields of mBBD20 and hCaCD20 wer...

Blocking translocation of cell surface molecules from the ER to the cell surface by intracellular antibodies targeted to the ER

“…The properties of PTD-linked intrabody genes need to be examined in detail. In another promising approach, specific targeting of the therapeutic herpes simplex virus thymidine kinase gene was achieved with a so-called immunoporter consisting of a Fab fragment against EGF receptor conjugated with a cationic polylysine chain [106]. It was shown that the Fab immunore-porter carrying the herpes TK gene was effective in preventing the growth of EGF receptor-overexpressing tumour cells.…”