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Nonviral RNA Transfection to Transiently Modify T Cells with Chimeric Antigen Receptors for Adoptive Therapy

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Synthetic Messenger RNA and Cell Metabolism Modulation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 969))

Abstract

Redirecting T cells with a chimeric antigen receptor (CAR) of predefined specificity showed remarkable efficacy in the adoptive therapy trials of malignant diseases. The CAR consists of a single chain fragment of variable region (scFv) antibody targeting domain covalently linked to the CD3ζ signalling domain of the T cell receptor complex to mediate T cell activation upon antigen engagement. By using an antibody-derived targeting domain a CAR can potentially redirect T cells towards any target expressed on the cell surface as long as a binding domain is available. Antibody-mediated targeting moreover circumvents MHC restriction of the targeted antigen, thereby broadening the potential of applicability of adoptive T cell therapy. While T cells were so far genetically modified by viral transduction, transient modification with a CAR by RNA transfection gained increasing interest during the last years. This chapter focuses on methods to modify human T cells from peripheral blood with a CAR by electroporation of in vitro transcribed RNA and to test modified T cells for function for use in adoptive immunotherapy.

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References

  1. Gross G, Waks T, Eshhar Z (1989) Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci USA 86:10024–10028

    Article  PubMed  CAS  Google Scholar 

  2. Hawkins RE, Gilham DE, Debets R, Eshhar Z, Taylor N, Abken H, Schumacher TN (2010) Development of adoptive cell therapy for cancer: a clinical perspective. Hum Gene Ther 21:665–672

    Article  PubMed  CAS  Google Scholar 

  3. Kohn DB, Dotti G, Brentjens R, Savoldo B, Jensen M, Cooper LJ, June CH, Rosenberg S, Sadelain M, Heslop HE (2011) CARs on track in the clinic. Mol Ther 19(3):432–438

    Article  PubMed  CAS  Google Scholar 

  4. Brenner MK, Heslop HE (2010) Adoptive T cell therapy of cancer. Curr Opin Immunol 22:251–257

    Article  PubMed  CAS  Google Scholar 

  5. Bridgeman JS, Hawkins RE, Hombach AA, Abken H, Gilham DE (2010) Building better chimeric antigen receptors for adoptive T cell therapy. Curr Gene Ther 10:77–90

    Article  PubMed  CAS  Google Scholar 

  6. Morgan RA, Dudley ME, Rosenberg SA (2010) Adoptive cell therapy: genetic modification to redirect effector cell specificity. Cancer J 16:336–341

    Article  PubMed  CAS  Google Scholar 

  7. Kershaw MH, Teng MW, Smyth MJ, Darcy PK (2005) Supernatural T cells: genetic modification of T cells for cancer therapy. Nat Rev Immunol 5(12):928–940

    Article  PubMed  CAS  Google Scholar 

  8. Hombach A, Heuser C, Sircar R, Tillmann T, Diehl V, Kruis W, Pohl C, Abken H (1997) T cell targeting of TAG72+ tumor cells by a chimeric receptor with antibody-like specificity for a carbohydrate epitope. Gastroenterology 113(4):1163–1170

    Article  PubMed  CAS  Google Scholar 

  9. Lamers CH, Sleijfer S, Vulto AG, Kruit WH, Kliffen M, Debets R, Gratama JW, Stoter G, Oosterwijk E (2006) Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. J Clin Oncol 24(13):e20–e22

    Article  PubMed  Google Scholar 

  10. Ferrand C, Robinet E, Contassot E, Certoux JM, Lim A, Herve P, Tiberghien P (2000) Retrovirus-mediated gene transfer in primary T lymphocytes: influence of the transduction/selection process and of ex vivo expansion on the T cell receptor beta chain hypervariable region repertoire. Hum Gene Ther 11:1151–1164

    Article  PubMed  CAS  Google Scholar 

  11. Sauce D, Bodinier M, Garin M, Petracca B, Tonnelier N, Duperrier A, Melo JV, Apperley JF, Ferrand C, Herve P, Lang F, Tiberghien P, Robinet E (2002) Retrovirus-mediated gene transfer in primary T lymphocytes impairs their anti-Epstein-Barr virus potential through both culture-dependent and selection process-dependent mechanisms. Blood 99:1165–1173

    Article  PubMed  CAS  Google Scholar 

  12. Sauce D, Tonnelier N, Duperrier A, Petracca B, de Carvalho Bittencourt M, Saadi M, Saas P, Ferrand C, Herve P, Tiberghien P, Robinet E (2002) Influence of ex vivo expansion and retrovirus-mediated gene transfer on primary T lymphocyte phenotype and functions. J Hematother Stem Cell Res 11:929–940

    Article  PubMed  CAS  Google Scholar 

  13. Schaft N, Dorrie J, Muller I, Beck V, Baumann S, Schunder T et al (2006) A new way to generate cytolytic tumor-specific T cells: electroporation of RNA coding for a T cell receptor into T lymphocytes. Cancer Immunol Immunother 55:1132–1141

    Article  PubMed  CAS  Google Scholar 

  14. Zhao Y, Zheng Z, Cohen CJ, Gattinoni L, Palmer DC, Restifo NP et al (2006) High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation. Mol Ther 13:151–159

    Article  PubMed  CAS  Google Scholar 

  15. Birkholz K, Hombach A, Krug C, Reuter S, Kershaw M, Kämpgen E, Schuler G, Abken H, Schaft N, Dörrie J (2009) Transfer of mRNA encoding recombinant immunoreceptors reprograms CD4+ and CD8+ T cells for use in the adoptive immunotherapy of cancer. Gene Ther 16(5):596–604

    Article  PubMed  CAS  Google Scholar 

  16. Rabinovich PM, Komarovskaya ME, Wrzesinski SH, Alderman JL, Budak-Alpdogan T, Karpikov A, Guo H, Flavell RA, Cheung NK, Weissman SM, Bahceci E (2009) Chimeric receptor mRNA transfection as a tool to generate antineoplastic lymphocytes. Hum Gene Ther 20(1):51–61

    Article  PubMed  CAS  Google Scholar 

  17. Rabinovich PM, Komarovskaya ME, Ye ZJ, Imai C, Campana D, Bahceci E, Weissman SM (2006) Synthetic messenger RNA as a tool for gene therapy. Hum Gene Ther 17(10):1027–1035

    Article  PubMed  CAS  Google Scholar 

  18. Almåsbak H, Rian E, Hoel HJ, Pulè M, Wälchli S, Kvalheim G, Gaudernack G, Rasmussen AM (2011) Transiently redirected T cells for adoptive transfer. Cytotherapy 13(5):629–640

    Article  PubMed  Google Scholar 

  19. Zhao Y, Moon E, Carpenito C, Paulos CM, Liu X, Brennan AL, Chew A, Carroll RG, Scholler J, Levine BL, Albelda SM, June CH (2010) Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor. Cancer Res 70(22):9053–9061

    Article  PubMed  CAS  Google Scholar 

  20. Choi Y, Yuen C, Maiti SN, Olivares S, Gibbons H, Huls H, Raphael R, Killian TC, Stark DJ, Lee DA, Torikai H, Monticello D, Kelly SS, Kebriaei P, Champlin RE, Biswal SL, Cooper LJ (2010) A high throughput microelectroporation device to introduce a chimeric antigen receptor to redirect the specificity of human T cells. Biomed Microdevices 12(5):855–863

    Article  PubMed  CAS  Google Scholar 

  21. Almåsbak H, Lundby M, Rasmussen AM (2010) Non-MHC-dependent redirected T cells against tumor cells. Methods Mol Biol 629:453–493

    PubMed  Google Scholar 

  22. Boczkowski D, Nair SK, Nam JH, Lyerly HK, Gilboa E (2000) Induction of tumor immunity and cytotoxic T lymphocyte responses using dendritic cells transfected with messenger RNA amplified from tumor cells. Cancer Res 60:1028–1034

    PubMed  CAS  Google Scholar 

  23. Jost LM, Kirkwood JM, Whiteside TL (1992) Improved short- and long-term XTT-based colorimetric cellular cytotoxicity assay for melanoma and other tumor cells. J Immunol Methods 147(2):153–165

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  25. Hollyman D, Stefanski J, Przybylowski M, Bartido S, Borquez-Ojeda O, Taylor C, Yeh R, Capacio V, Olszewska M, Hosey J, Sadelain M, Brentjens RJ, Riviere I (2009) Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy. J Immunother 32:169–180

    Article  PubMed  CAS  Google Scholar 

  26. Pouw N, Treffers-Westerlaken E, Kraan J, Wittink F, ten Hagen T, Verweij J, Debets R (2010) Combination of IL-21 and IL-15 enhances tumour-specific cytotoxicity and cytokine production of TCR-transduced primary T cells. Cancer Immunol Immunother 59:921–931

    Article  PubMed  CAS  Google Scholar 

  27. Suhoski MM, Golovina TN, Aqui NA, Tai VC, Varela-Rohena A, Milone MC, Carroll RG, Riley JL, June CH (2007) Engineering artificial antigen-presenting cells to express a diverse array of co-stimulatory molecules. Mol Ther 15:981–988

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department I of Internal Medicine, Tumor Genetics, and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany

    Tobias Riet, Astrid Holzinger & Hinrich Abken

  2. Department of Dermatology, University Hospital Erlangen, Erlangen, Germany

    Jan Dörrie, Niels Schaft & Gerold Schuler

Authors
  1. Tobias Riet
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  2. Astrid Holzinger
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  3. Jan Dörrie
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  4. Niels Schaft
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  5. Gerold Schuler
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  6. Hinrich Abken
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Corresponding author

Correspondence to Hinrich Abken .

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Editors and Affiliations

  1. The Anlyan Center, Department of Genetics, Yale School of Medicine, Cedar Street 300, New Haven, 06520-8005, Connecticut, USA

    Peter M. Rabinovich

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Riet, T., Holzinger, A., Dörrie, J., Schaft, N., Schuler, G., Abken, H. (2013). Nonviral RNA Transfection to Transiently Modify T Cells with Chimeric Antigen Receptors for Adoptive Therapy. In: Rabinovich, P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology, vol 969. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-260-5_12

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  • DOI: https://doi.org/10.1007/978-1-62703-260-5_12

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-259-9

  • Online ISBN: 978-1-62703-260-5

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