Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Stable antibody expression at therapeutic levels using the 2A peptide

Nature Biotechnology volume 23, pages 584–590 (2005)Cite this article

Abstract

Therapeutic monoclonal antibodies (mAbs) are currently being developed for the treatment of cancer and other diseases. Despite clinical success, widespread application of mAb therapies may be limited by manufacturing capabilities. In this paper, we describe a mAb delivery system that allows continuous production of a full-length antibody at high-concentrations in vivo after gene transfer. The mAb is expressed from a single open reading frame by linking the heavy and light chains with a 2A self-processing peptide derived from the foot-and-mouth disease virus. Using this expression system, we generated a recombinant adeno-associated virus vector encoding the VEGFR2-neutralizing mAb DC101 (rAAV8-DC101). A single dose of rAAV8-DC101 resulted in long-term expression of >1,000 μg/ml of DC101 in mice, demonstrating significant anti-tumor efficacy. This report describes the first feasible gene therapy approach for stable delivery of mAbs at therapeutic levels, which may serve as an attractive alternative to direct injection of mAbs.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Full-length mAb expression cassette using the FMDV 2A sequence.
Figure 2: In vitro expression of DC101 mAb using the 2A self-processing sequence–containing expression plasmids.
Figure 3: Biological activity of DC101 mAbs generated from 2A-containing expression cassettes.
Figure 4: Expression of DC101 mAb in vivo by rAAV8 vector-mediated gene transfer.
Figure 5: Anti-tumor efficacy after rAAV8-mediated gene transfer of DC101.

Similar content being viewed by others

References

  1. Hudson, P.J. & Souriau, C. Engineered antibodies. Nat. Med. 9, 129–134 (2003).

    Article  CAS  PubMed  Google Scholar 

  2. Green, L.L. Antibody engineering via genetic engineering of the mouse: XenoMouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies. J. Immunol. Methods 231, 11–23 (1999).

    Article  CAS  PubMed  Google Scholar 

  3. Maloney, D.G. et al. IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma. Blood 90, 2188–2195 (1997).

    CAS  PubMed  Google Scholar 

  4. Grimm, D. & Kay, M.A. From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy. Curr. Gene Ther. 3, 281–304 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Mizuguchi, H., Xu, Z., Ishii-Watabe, A., Uchida, E. & Hayakawa, T. IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Mol. Ther. 1, 376–382 (2000).

    Article  CAS  PubMed  Google Scholar 

  6. Ryan, M.D. & Drew, J. Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO J. 13, 928–933 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Donnelly, M.L., Gani, D., Flint, M., Monaghan, S. & Ryan, M.D. The cleavage activities of aphthovirus and cardiovirus 2A proteins. J. Gen. Virol. 78, 13–21 (1997).

    Article  CAS  PubMed  Google Scholar 

  8. Donnelly, M.L. et al. Analysis of the aphthovirus 2A/2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'. J. Gen. Virol. 82, 1013–1025 (2001).

    Article  CAS  PubMed  Google Scholar 

  9. Szymczak, A.L. et al. Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nat. Biotechnol. 22, 589–594 (2004).

    Article  CAS  PubMed  Google Scholar 

  10. de Felipe, P., Hughes, L.E., Ryan, M.D. & Brown, J.D. Co-translational, intraribosomal cleavage of polypeptides by the foot-and-mouth disease virus 2A peptide. J. Biol. Chem. 278, 11441–11448 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. de Felipe, P. & Ryan, M.D. Targeting of proteins derived from self-processing polyproteins containing multiple signal sequences. Traffic 5, 616–626 (2004).

    Article  CAS  PubMed  Google Scholar 

  12. Prewett, M. et al. Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors. Cancer Res. 59, 5209–5218 (1999).

    CAS  PubMed  Google Scholar 

  13. Niwa, H., Yamamura, K. & Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199 (1991).

    Article  CAS  PubMed  Google Scholar 

  14. Gao, G.P. et al. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc. Natl. Acad. Sci. USA 99, 11854–11859 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bakker, J.M., Bleeker, W.K. & Parren, W.H.I. Therapeutic antibody gene transfer: an active approach to passive immunity. Mol. Ther. 10, 411–416 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Davis, T.A. et al. Rituximab anti-CD20 monoclonal antibody therapy in non-Hodgkin's lymphoma: safety and efficacy of re-treatment. J. Clin. Oncol. 18, 3135–3143 (2000).

    Article  CAS  PubMed  Google Scholar 

  17. Lin, Y.S. et al. Preclinical pharmacokinetics, interspecies scaling, and tissue distribution of a humanized monoclonal antibody against vascular endothelial growth factor. J. Pharmacol. Exp. Ther. 288, 371–378 (1999).

    CAS  PubMed  Google Scholar 

  18. Armbruster, C. et al. A phase I trial with two human monoclonal antibodies (hMAb 2F5, 2G12) against HIV-1. AIDS 16, 227–233 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. Perez, N. et al. Regulatable systemic production of monoclonal antibodies by in vivo muscle electroporation. Genet. Vaccines Ther. 2, 2 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Noel, D. et al. In vitro and in vivo secretion of cloned antibodies by genetically modified myogenic cells. Hum. Gene Ther. 8, 1219–1229 (1997).

    Article  CAS  PubMed  Google Scholar 

  21. Noel, D., Pelegrin, M., Brockly, F., Lund, A.H. & Piechaczyk, M. Sustained systemic delivery of monoclonal antibodies by genetically modified skin fibroblasts. J. Invest. Dermatol. 115, 740–745 (2000).

    Article  CAS  PubMed  Google Scholar 

  22. Lewis, A.D., Chen, R., Montefiori, D.C., Johnson, P.R. & Clark, K.R. Generation of neutralizing activity against human immunodeficiency virus type 1 in serum by antibody gene transfer. J. Virol. 76, 8769–8775 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Noel, D. et al. High in vivo production of a model monoclonal antibody on adenoviral gene transfer. Hum. Gene Ther. 13, 1483–1493 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. Jooss, K. & Chirmule, N. Immunity to adenovirus and adeno-associated viral vectors: implications for gene therapy. Gene Ther. 10, 955–963 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Monahan, P.E., Jooss, K. & Sands, M.S. Safety of adeno-associated virus gene therapy vectors: a current evaluation. Expert Opin. Drug Saf. 1, 79–91 (2002).

    Article  CAS  PubMed  Google Scholar 

  26. Lu, Y. Recombinant adeno-associated virus as delivery vector for gene therapy–a review. Stem. Cells. Dev. 13, 133–145 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Flotte, T.R. et al. Phase I trial of intramuscular injection of a recombinant adeno-associated virus alpha 1-antitrypsin (rAAV2-CB-hAAT) gene vector to AAT-deficient adults. Hum. Gene Ther. 15, 93–128 (2004).

    Article  PubMed  Google Scholar 

  28. Moss, R.B. et al. Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis: a multicenter, double-blind, placebo-controlled trial. Chest 125, 509–521 (2004).

    Article  PubMed  Google Scholar 

  29. Manno, C.S. et al. AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B. Blood 101, 2963–2972 (2003).

    Article  CAS  PubMed  Google Scholar 

  30. Janson, C. et al. Clinical protocol. Gene therapy of Canavan disease: AAV-2 vector for neurosurgical delivery of aspartoacylase gene (ASPA) to the human brain. Hum. Gene Ther. 13, 1391–1412 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Luo, J. et al. J. Subthalamic GAD gene therapy in a Parkinson's disease rat model. Science 298, 425–429 (2002).

    Article  CAS  PubMed  Google Scholar 

  32. Ryan, M.D., King, A.M. & Thomas, G.P. Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence. J. Gen. Virol. 72, 2727–2732 (1991).

    Article  CAS  PubMed  Google Scholar 

  33. Jang, S.K., Pestova, T.V., Hellen, C.U., Witherell, G.W. & Wimmer, E. Cap-independent translation of picornavirus RNAs: structure and function of the internal ribosomal entry site. Enzyme 44, 292–309 (1990).

    Article  CAS  PubMed  Google Scholar 

  34. Xiao, X., Li, J. & Samulski, R.J. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J. Virol. 72, 2224–2232 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Liu, F., Song, Y. & Liu, D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther. 6, 1258–1266 (1999).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Mingxia Shi, Sandra Sanchez, Lei Xu, Gail Colbern and the animal service group of Cell Genesys for technical assistance, John Leszyk at the University of Massachusetts Medical School for carrying out mass spectrometry analysis and Peter Working for critical reading of the manuscript.

Author information

Authors and Affiliations

  1. Department of Preclinical Oncology and Immunology, Cell Genesys, Inc., 500 Forbes Blvd., S. San Francisco, 94080, California, USA

    Jianmin Fang, Jing-Jing Qian, Saili Yi, Thomas C Harding, Guang Huan Tu, Melinda VanRoey & Karin Jooss

Authors
  1. Jianmin Fang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jing-Jing Qian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Saili Yi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas C Harding
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Guang Huan Tu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Melinda VanRoey
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Karin Jooss
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jianmin Fang.

Ethics declarations

Competing interests

The authors are employed by Cell Genesys, Inc.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fang, J., Qian, JJ., Yi, S. et al. Stable antibody expression at therapeutic levels using the 2A peptide. Nat Biotechnol 23, 584–590 (2005). https://doi.org/10.1038/nbt1087

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1087

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing
pFad - Phonifier reborn

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.

Note: This service is not intended for secure transactions such as banking, social media, email, or purchasing. Use at your own risk. We assume no liability whatsoever for broken pages.


Alternative Proxies:

Alternative Proxy

pFad Proxy

pFad v3 Proxy

pFad v4 Proxy