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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 × 5), yielding 5 mg/l with a purity of over 95%. The scFv-D4S × 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.

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References

  1. Kaneko S et al. Adenovirus-mediated gene therapy of hepatocellular carcinoma using cancer-specific gene expression. Cancer Res 1995; 55: 5283–5287.

    CAS  PubMed  Google Scholar 

  2. Ido A et al. Gene therapy for hepatoma cells using a retrovirus vector carrying herpes simplex virus thymidine kinase gene under the control of human alpha-fetoprotein gene promoter. Cancer Res 1995; 55: 3105–3109.

    CAS  PubMed  Google Scholar 

  3. Romanczuk H et al. Modification of an adenoviral vector with biologically selected peptides: a novel strategy for gene delivery to cells of choice. Hum Gene Ther 1999; 10: 2615–2626.

    Article  CAS  PubMed  Google Scholar 

  4. Smith JS et al. Redirected infection of directly biotinylated recombinant adenovirus vectors through cell surface receptors and antigens. Proc Natl Acad Sci USA 1999; 96: 8855–8860.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Weijtens ME, Willemsen RA, Hart EH, Bolhuis RL . A retroviral vector system ‘STITCH’ in combination with an optimized single chain antibody chimeric receptor gene structure allows efficient gene transduction and expression in human T lymphocytes. Gene Ther 1998; 5: 1195–1203.

    Article  CAS  PubMed  Google Scholar 

  6. Jiang A et al. Cell-type-specific gene transfer into human cells with retroviral vectors that display single-chain antibodies. J Virol 1998; 72: 10148–10156.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Martin F A et al. Retroviral vector targeting to melanoma cells by single-chain antibody incorporation in envelope. Hum Gene Ther 1998; 9: 737–746.

    Article  PubMed  Google Scholar 

  8. Chen J, Gamou S, Takayanagi A, Shimizu N . A novel gene delivery system using EGF receptor-mediated endocytosis. FEBS Lett 1994; 338:167–169.

    Article  CAS  PubMed  Google Scholar 

  9. Shimizu N, Chen J, Gamou S, Takayanagi A . Immunogene approach toward cancer therapy using epidermal growth factor receptor-mediated gene delivery. Cancer Gene Ther 1996; 3: 113–120.

    CAS  PubMed  Google Scholar 

  10. Chen J et al. Receptor-mediated gene delivery using the Fab fragments of anti-epidermal growth factor receptor antibodies: improved immunogene approach. Cancer Gene Ther 1998; 5: 357–364.

    CAS  PubMed  Google Scholar 

  11. Ohtake Y et al. Ex vivo delivery of suicide genes into melanoma cells using epidermal growth factor receptor-specific Fab immunogene. Jpn J Cancer Res 1999; 90:460–468.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Chen J et al. Targeted in vivo delivery of therapeutic gene into experimental squamous cell carcinomas using anti-epidermal growth factor receptor antibody: immunogene approach. Hum Gene Ther 1998; 9: 2673–2681.

    Article  CAS  PubMed  Google Scholar 

  13. Hochman J, Inbar D, Givol D . An active antibody fragment (Fv) composed of the variable portions of heavy and light chains. Biochemistry 1973; 12: 1130–1135.

    Article  CAS  PubMed  Google Scholar 

  14. Ward ES et al. Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 1989; 341: 544–546.

    Article  CAS  PubMed  Google Scholar 

  15. Field H, Yarranton GT, Rees AR . Expression of mouse immunoglobulin light and heavy chain variable regions in Escherichia coli and reconstitution of antigen-binding activity. Protein Eng 1990; 3: 641–647.

    Article  CAS  PubMed  Google Scholar 

  16. Glockshuber R, Stadlmuller J, Pluckthun A . Mapping and modification of an antibody hapten binding site: a site-directed mutagenesis study of McPC603. Biochemistry 1991; 30: 3049–3054.

    Article  CAS  PubMed  Google Scholar 

  17. McManus S, Riechmann L . Use of 2D NMR, protein engineering, and molecular modeling to study the hapten-binding site of an antibody Fv fragment against 2-phenyloxazolone. Biochemistry 1991; 30: 5851–5857.

    Article  CAS  PubMed  Google Scholar 

  18. Takahashi H, Igarashi T, Shimada I, Arata Y . Preparation of the Fv fragment from a short-chain mouse IgG2a anti-dansyl monoclonal antibody and use of selectively deuterated Fv analogues for two-dimensional 1H NMR analyses of the antigen-antibody interactions. Biochemistry 1991; 30: 2840–2847.

    Article  CAS  PubMed  Google Scholar 

  19. Boussif O et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA 1995; 92: 7297–7301.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by a Fund for ‘Research for the Future’ Program from the Japan Society for the Promotion of Science.

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Suzuki, M., Takayanagi, A. & Shimizu, N. Recombinant single-chain antibodies with various oligopeptide tails for targeted gene delivery. Gene Ther 10, 781–788 (2003). https://doi.org/10.1038/sj.gt.3301952

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