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.

  • Original Article
  • Published:

Phoenix-ampho outperforms PG13 as retroviral packaging cells to transduce human T cells with tumor-specific receptors: implications for clinical immunogene therapy of cancer

Cancer Gene Therapy volume 13, pages 503–509 (2006)Cite this article

Abstract

We have designed a transgene that encodes a scFv(G250) chimeric receptor, which is specific for carboxyanhydrase IX (G250-ligand, G250L), a molecule overexpressed by renal cell cancer (RCC). Retroviral transduction of this transgene into primary human T lymphocytes confers these cells with specific functional responses towards G250L-positive RCC cells. In preparation of a clinical phase (I/II) study in RCC patients, we set up a protocol for gene transduction and expansion of primary human T cells. For this purpose, we directly compared two packaging cell lines, that is, the GALV-pseudotyped MLV producing cell line PG13, and the MLV-A-producing cell line Phi-NX-Ampho (a.k.a. Phoenix-A). We generated and characterized stable scFv(G250)-positive clones of both PG13 and Phoenix cells and optimized the retrovirus production conditions. Transductions of primary human T cells yielded 30–60% scFv(G250)+ T cells using PG13-derived retrovirus versus up to 90% scFv(G250)+ T cells using Phoenix-derived retrovirus. The median number of transgene integrations per scFv(G250)+ T cell differed only 1.5-fold as determined by real-time PCR (mean number of integrations per T cell 2.6 and 3.7 for PG13 and Phoenix-based transductions, respectively). In addition, T cells transduced with Phoenix-derived retrovirus showed, on a per cell basis, 10–30% higher levels of scFv(G250)-mediated TNFα production and cytolysis of G250L+ RCC cells than T cells transduced with PG13-derived retrovirus. The improved functional transduction efficiency together with a limited increase in the number of integrations per recipient cell, made us select Phoenix clone 58 for our clinical immunogene therapy study.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1
Figure 2

Similar content being viewed by others

References

  1. Lamers CH, Willemsen RA, Luider BA, Debets R, Bolhuis RL . Protocol for gene transduction and expansion of human T lymphocytes for clinical immunogene therapy of cancer. Cancer Gene Ther 2002; 9: 613–623.

    Article  CAS  Google Scholar 

  2. 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 Therapy 1998; 5: 1195–1203.

    Article  CAS  Google Scholar 

  3. Weijtens ME, Hart EH, Bolhuis RL . Functional balance between T cell chimeric receptor density and tumor associated antigen density: CTL mediated cytolysis and lymphokine production. Gene Therapy 2000; 7: 35–42.

    Article  CAS  Google Scholar 

  4. Le Doux JM, Morgan JR, Yarmush ML . Differential inhibition of retrovirus transduction by proteoglycans and free glycosaminoglycans. Biotechnol Prog 1999; 15: 397–406.

    Article  CAS  Google Scholar 

  5. Swift S, Lorens J, Achacoso P, Nolan GP . Radid production of retroviruses for efficient gene delivery to mammalian cells using 293T cell-basd systems. Current Protocols in Immunology 1999; 10.17: 14–29.

    Google Scholar 

  6. Kustikova OS, Wahlers A, Kuhlcke K, Stahle B, Zander AR, Baum C et al. Dose finding with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population. Blood 2003; 102: 3934–3937.

    Article  CAS  Google Scholar 

  7. Schaft N, Willemsen RA, de Vries J, Lankiewicz B, Essers BW, Gratama JW et al. Peptide fine specificity of anti-glycoprotein 100 CTL is preserved following transfer of engineered TCR alpha beta genes into primary human T lymphocytes. J Immunol 2003; 170: 2186–2194.

    Article  CAS  Google Scholar 

  8. Baum C, Von Kalle C, Staal FJ, Li Z, Fehse B, Schmidt M et al. Chance or necessity? Insertional mutagenesis in gene therapy and its consequences. Mol Ther 2004; 9: 5–13.

    Article  CAS  Google Scholar 

  9. Bonini C, Grez M, Traversari C, Ciceri F, Marktel S, Ferrari G et al. Safety of retroviral gene marking with a truncated NGF receptor. Nat Med 2003; 9: 367–369.

    Article  CAS  Google Scholar 

  10. Yee C, Savage PA, Lee PP, Davis MM, Greenberg PD . Isolation of high avidity melanoma-reactive CTL from heterogeneous populations using peptide-MHC tetramers. J Immunol 1999; 162: 2227–2234.

    CAS  Google Scholar 

  11. Lamers CHJ, Sleijfer S, Willemsen RA, Debets R, Kruit WHJ, Gratama JW et al. Adoptive immuno-gene therapy of cancer with single chain antibody [scFv(Ig)] gene modified T lymphocytes. J Biol Reg Homeost Agents 2004; 18: 134–140.

    CAS  Google Scholar 

Download references

Acknowledgements

The authors thank Corrien Groot-v. Ruyven, Sabine Langeveld and Barbara Luider-Vrieling for their technical assistance. This work has been supported by the Dutch Cancer Foundation (DDHK99-1865), the EU Grant QLK3-1999-01-262, and the Cancer Research Institute, New York (Clinical Investigation Grant ‘Immunogenetherapy of metastatic renal cell cancer patients’).

Author information

Authors and Affiliations

  1. Department of Medical Oncology, Unit of Clinical and Tumor Immunology, Erasmus MC – Daniel den Hoed Cancer Center, Rotterdam, The Netherlands

    C H J Lamers, R A Willemsen, P van Elzakker, B A van Krimpen, J W Gratama & R Debets

Authors
  1. C H J Lamers
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R A Willemsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. P van Elzakker
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. B A van Krimpen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J W Gratama
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. R Debets
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to C H J Lamers.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lamers, C., Willemsen, R., van Elzakker, P. et al. Phoenix-ampho outperforms PG13 as retroviral packaging cells to transduce human T cells with tumor-specific receptors: implications for clinical immunogene therapy of cancer. Cancer Gene Ther 13, 503–509 (2006). https://doi.org/10.1038/sj.cgt.7700916

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.cgt.7700916

Keywords

This article is cited by

Search

Advanced search

Quick links