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Enhanced functionality of T cell receptor-redirected T cells is defined by the transgene cassette

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An Erratum to this article was published on 29 May 2008

Abstract

The transfer of T cell receptor (TCR) genes allows to endow T cells with a new antigen specificity. For clinical applications of TCR-redirected T cells, efficient functional expression of the transgenic TCR is a key prerequisite. Here, we compared the influence of the transgene cassette on the expression and function of the murine TCR P14 (recognizing a LCMV gp33 epitope) and the human TCR WT-1 (recognizing an epitope of the tumor-associated antigen WT-1). We constructed different vectors, in which TCRα- and β-chain genes were either (a) linked by an internal ribosomal entry site (IRES), (b) combined by a 2A peptide, or (c) introduced into two individual retroviral constructs. While in a TCR-deficient T cell line TCR P14 was expressed equally well by all constructs, we found that IRES- but not 2A-employing TCR expression is hampered in a TCR-bearing cell line and in primary murine T cells where the transgenic TCR has to compete with endogenous TCR chains. Similarly, 2A-linked TCR WT-1 genes yielded highest expression and function as measured by tetramer binding and peptide-specific IFN-γ secretion. Differences in expression were independent of copy number integration as shown by real-time PCR. Thus, linking TCRα- and β-chain genes by a 2A peptide is superior to an IRES for TCR expression and T cell function.

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References

  1. Xue S, Gillmore R, Downs A, Tsallios A, Holler A, Gao L, Wong V, Morris E, Stauss HJ (2005) Exploiting T cell receptor genes for cancer immunotherapy. Clin Exp Immunol 139:167–172

    Article  PubMed  CAS  Google Scholar 

  2. Tey SK, Bollard CM, Heslop HE (2006) Adoptive T-cell transfer in cancer immunotherapy. Immunol Cell Biol 84:281–289

    Article  PubMed  CAS  Google Scholar 

  3. Engels B, Uckert W (2007) Redirecting T lymphocyte specificity by T cell receptor gene transfer—a new era for immunotherapy. Mol Aspects Med 28:115–142

    Article  PubMed  CAS  Google Scholar 

  4. Sommermeyer D, Neudorfer J, Weinhold M, Leisegang M, Engels B, Noessner E, Heemskerk MH, Charo J, Schendel DJ, Blankenstein T, Bernhard H, Uckert W (2006) Designer T cells by T cell receptor replacement. Eur J Immunol 36:3052–3059

    Article  PubMed  CAS  Google Scholar 

  5. Heemskerk MH, Hagedoorn RS, van der Hoorn MA, van der Veken LT, Hoogeboom M, Kester MG, Willemze R, Falkenburg JH (2007) Efficiency of T-cell receptor expression in dual-specific T cells is controlled by the intrinsic qualities of the TCR chains within the TCR-CD3 complex. Blood 109:235–243

    Article  PubMed  CAS  Google Scholar 

  6. Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, Royal RE, Topalian SL, Kammula US, Restifo NP, Zheng Z, Nahvi A, de Vries CR, Rogers-Freezer LJ, Mavroukakis SA, Rosenberg SA (2006) Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314:126–129

    Article  PubMed  CAS  Google Scholar 

  7. Cohen CJ, Zhao Y, Zheng Z, Rosenberg SA, Morgan RA (2006) Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability. Cancer Res 66:8878–8886

    Article  PubMed  CAS  Google Scholar 

  8. Voss RH, Kuball J, Engel R, Guillaume P, Romero P, Huber C, Theobald M (2006) Redirection of T cells by delivering a transgenic mouse-derived MDM2 tumor antigen-specific TCR and its humanized derivative is governed by the CD8 coreceptor and affects natural human TCR expression. Immunol Res 34:67–87

    Article  PubMed  CAS  Google Scholar 

  9. Kuball J, Dossett ML, Wolfl M, Ho WY, Voss RH, Fowler C, Greenberg PD (2007) Facilitating matched pairing and expression of TCR chains introduced into human T cells. Blood 109:2331–2338

    Article  PubMed  CAS  Google Scholar 

  10. Cohen CJ, Li YF, El-Gamil M, Robbins PF, Rosenberg SA, Morgan RA (2007) Enhanced antitumor activity of T cells engineered to express T-cell receptors with a second disulfide bond. Cancer Res 67:3898–3903

    Article  PubMed  CAS  Google Scholar 

  11. Scholten KB, Kramer D, Kueter EW, Graf M, Schoedl T, Meijer CJ, Schreurs MW, Hooijberg E (2006) Codon modification of T cell receptors allows enhanced functional expression in transgenic human T cells. Clin Immunol 119:135–145

    Article  PubMed  CAS  Google Scholar 

  12. Xue SA, Gao L, Hart D, Gillmore R, Qasim W, Thrasher A, Apperley J, Engels B, Uckert W, Morris E, Stauss H (2005) Elimination of human leukemia cells in NOD/SCID mice by WT1-TCR gene-transduced human T cells. Blood 106:3062–3067

    Article  PubMed  CAS  Google Scholar 

  13. Clay TM, Custer MC, Sachs J, Hwu P, Rosenberg SA, Nishimura MI (1999) Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity. J Immunol 163:507–513

    PubMed  CAS  Google Scholar 

  14. Emerman M, Temin HM (1984) Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell 39:449–467

    Article  PubMed  CAS  Google Scholar 

  15. Cheng L, Du C, Murray D, Tong X, Zhang YA, Chen BP, Hawley RG (1997) A GFP reporter system to assess gene transfer and expression in human hematopoietic progenitor cells. Gene Ther 4:1013–1022

    Article  PubMed  CAS  Google Scholar 

  16. Engels B, Noessner E, Frankenberger B, Blankenstein T, Schendel DJ, Uckert W (2005) Redirecting human T lymphocytes toward renal cell carcinoma specificity by retroviral transfer of T cell receptor genes. Hum Gene Ther 16:799–810

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  18. Holst J, Vignali KM, Burton AR, Vignali DA (2006) Rapid analysis of T-cell selection in vivo using T cell-receptor retrogenic mice. Nat Methods 3:191–197

    Article  PubMed  CAS  Google Scholar 

  19. de Felipe P, Martin V, Cortes ML, Ryan M, Izquierdo M (1999) Use of the 2A sequence from foot-and-mouth disease virus in the generation of retroviral vectors for gene therapy. Gene Ther 6:198–208

    Article  PubMed  Google Scholar 

  20. Klump H, Schiedlmeier B, Vogt B, Ryan M, Ostertag W, Baum C (2001) Retroviral vector-mediated expression of HoxB4 in hematopoietic cells using a novel coexpression strategy. Gene Ther 8:811–817

    Article  PubMed  CAS  Google Scholar 

  21. Szymczak AL, Workman CJ, Wang Y, Vignali KM, Dilioglou S, Vanin EF, Vignali DA (2004) Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nat Biotechnol 22:589–594

    Article  PubMed  CAS  Google Scholar 

  22. Pircher H, Burki K, Lang R, Hengartner H, Zinkernagel RM (1989) Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen. Nature 342:559–561

    Article  PubMed  CAS  Google Scholar 

  23. Engels B, Cam H, Schuler T, Indraccolo S, Gladow M, Baum C, Blankenstein T, Uckert W (2003) Retroviral vectors for high-level transgene expression in T lymphocytes. Hum Gene Ther 14:1155–1168

    Article  PubMed  CAS  Google Scholar 

  24. Morita S, Kojima T, Kitamura T (2000) Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther 7:1063–1066

    Article  PubMed  CAS  Google Scholar 

  25. Letourneur F, Malissen B (1989) Derivation of a T cell hybridoma variant deprived of functional T cell receptor alpha and beta chain transcripts reveals a nonfunctional alpha-mRNA of BW5147 origin. Eur J Immunol 19:2269–2274

    Article  PubMed  CAS  Google Scholar 

  26. Karttunen J, Sanderson S, Shastri N (1992) Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens. Proc Natl Acad Sci U S A 89:6020–6024

    Article  PubMed  CAS  Google Scholar 

  27. Uckert W, Becker C, Gladow M, Klein D, Kammertoens T, Pedersen L, Blankenstein T (2000) Efficient gene transfer into primary human CD8+ T lymphocytes by MuLV-10A1 retrovirus pseudotype. Hum Gene Ther 11:1005–1014

    Article  PubMed  CAS  Google Scholar 

  28. Soneoka Y, Cannon PM, Ramsdale EE, Griffiths JC, Romano G, Kingsman SM, Kingsman AJ (1995) A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res 23:628–633

    Article  PubMed  CAS  Google Scholar 

  29. Reuss S, Biese P, Cosset FL, Takeuchi Y, Uckert W (2007) Suspension packaging cell lines for the simplified generation of T-cell receptor encoding retrovirus vector particles. Gene Ther 14:595–603

    Article  PubMed  CAS  Google Scholar 

  30. Baum C, Dullmann J, Li Z, Fehse B, Meyer J, Williams DA, von Kalle C (2003) Side effects of retroviral gene transfer into hematopoietic stem cells. Blood 101:2099–2114

    Article  PubMed  CAS  Google Scholar 

  31. Inoue K, Ogawa H, Sonoda Y, Kimura T, Sakabe H, Oka Y, Miyake S, Tamaki H, Oji Y, Yamagami T, Tatekawa T, Soma T, Kishimoto T, Sugiyama H (1997) Aberrant overexpression of the Wilms tumor gene (WT1) in human leukemia. Blood 89:1405–1412

    PubMed  CAS  Google Scholar 

  32. Oji Y, Ogawa H, Tamaki H, Oka Y, Tsuboi A, Kim EH, Soma T, Tatekawa T, Kawakami M, Asada M, Kishimoto T, Sugiyama H (1999) Expression of the Wilms' tumor gene WT1 in solid tumors and its involvement in tumor cell growth. Jpn J Cancer Res 90:194–204

    PubMed  CAS  Google Scholar 

  33. Rosenberg SA (2001) Progress in human tumour immunology and immunotherapy. Nature 411:380–384

    Article  PubMed  CAS  Google Scholar 

  34. Garcia KC, Degano M, Stanfield RL, Brunmark A, Jackson MR, Peterson PA, Teyton L, Wilson IA (1996) An alphabeta T cell receptor structure at 2.5 A and its orientation in the TCR–MHC complex. Science 274:209–219

    Article  PubMed  CAS  Google Scholar 

  35. Rudolph MG, Stanfield RL, Wilson IA (2006) How TCRs bind MHCs, peptides, and coreceptors. Annu Rev Immunol 24:419–466

    Article  PubMed  CAS  Google Scholar 

  36. Weinhold M, Sommermeyer D, Uckert W, Blankenstein T (2007) Dual T cell receptor expressing CD8+ T cells with tumor- and self-specificity can inhibit tumor growth without causing severe autoimmunity. J Immunol 179:5534–5542

    PubMed  CAS  Google Scholar 

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Acknowledgments

We thank S. Shalapour for helpful advice on how to design and analyze real-time PCR assays and K. Hummel and J. Hauchwitz for excellent technical assistance. This study was supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich TR36 and SPP1230) and the Leukaemia Research Fund.

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Authors and Affiliations

  1. Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, 13092, Berlin, Germany

    Matthias Leisegang, Elisa Kieback, Daniel Sommermeyer, Simone Reuß & Wolfgang Uckert

  2. Department of Pathology and Committee on Immunology, University of Chicago, Chicago, Illinois, USA

    Boris Engels

  3. Institute of Biology, Humboldt University, Berlin, Germany

    Peter Meyerhuber & Wolfgang Uckert

  4. Department of Immunology and Molecular Pathology, Royal Free Hospital, University College London, London, UK

    Shao-An Xue & Hans Stauss

Authors
  1. Matthias Leisegang
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  2. Boris Engels
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  3. Peter Meyerhuber
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  4. Elisa Kieback
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  5. Daniel Sommermeyer
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  9. Wolfgang Uckert
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Correspondence to Wolfgang Uckert.

Additional information

An erratum to this article can be found at http://dx.doi.org/10.1007/s00109-008-0361-z

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Leisegang, M., Engels, B., Meyerhuber, P. et al. Enhanced functionality of T cell receptor-redirected T cells is defined by the transgene cassette. J Mol Med 86, 573–583 (2008). https://doi.org/10.1007/s00109-008-0317-3

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  • DOI: https://doi.org/10.1007/s00109-008-0317-3

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