Abstract
Adoptive T-cell transfer for cancer immunotherapy requires genetic modification of T cells with recombinant T-cell receptors (TCRs). Amphotropic retroviral vectors (RVs) used for TCR transduction for this purpose are considered safe in principle. Despite this, TCR-coding and packaging vectors could theoretically recombine to produce replication competent vectors (RCVs), and transduced T-cell preparations must be proven free of RCV. To eliminate the need for RCV testing, we transduced human T cells with ecotropic RVs so potential RCV would be non-infectious for human cells. We show that transfection of synthetic messenger RNA encoding murine cationic amino-acid transporter 1 (mCAT-1), the receptor for murine retroviruses, enables efficient transient ecotropic transduction of human T cells. mCAT-1-dependent transduction was more efficient than amphotropic transduction performed in parallel, and preferentially targeted naive T cells. Moreover, we demonstrate that ecotropic TCR transduction results in antigen-specific restimulation of primary human T cells. Thus, ecotropic RVs represent a versatile, safe and potent tool to prepare T cells for the adoptive transfer.
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References
Gill S, Kalos M . T cell-based gene therapy of cancer. Transl Res 2012; 161: 365–379.
Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 2006; 314: 126–129.
Johnson LA, Morgan RA, Dudley ME, Cassard L, Yang JC, Hughes MS et al. Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood 2009; 114: 535–546.
Robbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME et al. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol 2011; 29: 917–924.
Wilson CA, Cichutek K . The US and EU regulatory perspectives on the clinical use of hematopoietic stem/progenitor cells genetically modified ex vivo by retroviral vectors. Methods Mol Biol 2009; 506: 477–488.
Albritton LM, Tseng L, Scadden D, Cunningham JM . Putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell 1989; 57: 659–666.
Bertran J, Miller JL, Yang Y, Fenimore-Justman A, Rueda F, Vanin EF et al. Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors. J Virol 1996; 70: 6759–6766.
Koch P, Siemen H, Biegler A, Itskovitz-Eldor J, Brustle O . Transduction of human embryonic stem cells by ecotropic retroviral vectors. Nucleic Acids Res 2006; 34: 12.
Scholz A and Beato M. Scholz A and Beato M. Transient transfection of ecotropic retrovirus receptor permits stable gene transfer into non-rodent cells with murine retroviral vectors. Nucleic Acids Res 1996; 24: 979–980.
Barrilleaux B, Knoepfler P . Transduction of human cells with polymer-complexed ecotropic lentivirus for enhanced biosafety. J Vis Exp 2011; 53: 2822.
Van Tendeloo VF, Ponsaerts P, Lardon F, Nijs G, Lenjou M, Van BC et al. Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood 2001; 98: 49–56.
Van Tendeloo VF, Willems R, Ponsaerts P, Lenjou M, Nijs G, Vanhove M et al. High-level transgene expression in primary human T lymphocytes and adult bone marrow CD34+ cells via electroporation-mediated gene delivery. Gene Ther 2000; 7: 1431–1437.
Nakazawa Y, Huye LE, Dotti G, Foster AE, Vera JF, Manuri PR et al. Optimization of the PiggyBac transposon system for the sustained genetic modification of human T lymphocytes. J Immunother 2009; 32: 826–836.
Zhao Y, Zheng Z, Cohen CJ, Gattinoni L, Palmer DC, Restifo NP et al. High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation. Mol Ther 2006; 13: 151–159.
Zhao Y, Moon E, Carpenito C, Paulos CM, Liu X, Brennan AL et al. Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor. Cancer Res 2010; 70: 9053–9061.
Birkholz K, Hombach A, Krug C, Reuter S, Kershaw M, Kampgen E et al. Transfer of mRNA encoding recombinant immunoreceptors reprograms CD4+ and CD8+ T cells for use in the adoptive immunotherapy of cancer. Gene Ther 2009; 16: 596–604.
Holtkamp S, Kreiter S, Selmi A, Simon P, Koslowski M, Huber C et al. Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood 2006; 108: 4009–4017.
Kuhn AN, Diken M, Kreiter S, Selmi A, Kowalska J, Jemielity J et al. Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo. Gene Ther 2010; 17: 961–971.
Kuhn AN, Diken M, Kreiter S, Vallazza B, Tureci O, Sahin U . Determinants of intracellular RNA pharmacokinetics: implications for RNA-based immunotherapeutics. RNA Biol 2011; 8: 35–43.
Orlic D, Girard LJ, Jordan CT, Anderson SM, Cline AP, Bodine DM . The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction. Proc Natl Acad Sci USA 1996; 93: 11097–11102.
Sabatino DE, Do BQ, Pyle LC, Seidel NE, Girard LJ, Spratt SK et al. Amphotropic or gibbon ape leukemia virus retrovirus binding and transduction correlates with the level of receptor mRNA in human hematopoietic cell lines. Blood Cells Mol Dis 1997; 23: 422–433.
Katari UL, Keirnan JM, Worth AC, Hodges SE, Leen AM, Fisher WE et al. Engineered T cells for pancreatic cancer treatment. HPB (Oxford) 2011; 13: 643–650.
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.
Thompson JF, Hayes LS, Lloyd DB . Modulation of firefly luciferase stability and impact on studies of gene regulation. Gene 1991; 103: 171–177.
van der Loo JC, Swaney WP, Grassman E, Terwilliger A, Higashimoto T, Schambach A et al. Scale-up and manufacturing of clinical-grade self-inactivating gamma-retroviral vectors by transient transfection. Gene Ther 2012; 19: 246–254.
Beissert T, Hundertmark A, Kaburova V, Travaglini L, Mian AA, Nervi C et al. Targeting of the N-terminal coiled coil oligomerization interface by a helix-2 peptide inhibits unmutated and imatinib-resistant BCR/ABL. Int J Cancer 2008; 122: 2744–2752.
Grignani F, Kinsella T, Mencarelli A, Valtieri M, Riganelli D, Grignani F et al. High-efficiency gene transfer and selection of human hematopoietic progenitor cells with a hybrid EBV/retroviral vector expressing the green fluorescence protein. Cancer Res 1998; 58: 14–19.
Kreiter S, Konrad T, Sester M, Huber C, Tureci O, Sahin U . Simultaneous ex vivo quantification of antigen-specific CD4+ and CD8+ T cell responses using in vitro transcribed RNA. Cancer Immunol Immunother 2007; 56: 1577–1587.
Acknowledgements
We thank Ines Eiser, Aileen Laubenheimer, Tina Hempel and Kathleen Hobohm for excellent technical assistance, Sebastian Kreiter for proofreading the manuscript and Stephen Reece for native-speaker review.
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ÖT and US are inventors on patent applications, which cover optimization of RNA-based therapeutics. The remaining authors declare no conflict of interest.
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Koste, L., Beissert, T., Hoff, H. et al. T-cell receptor transfer into human T cells with ecotropic retroviral vectors. Gene Ther 21, 533–538 (2014). https://doi.org/10.1038/gt.2014.25
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DOI: https://doi.org/10.1038/gt.2014.25