Immunotherapy

Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors

Abstract

Immunotherapy with T cell modified with gamma-retroviral or lentiviral (LV) vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in clinical trials. However, the potential for insertional mutagenesis and genotoxicity of viral vectors is a safety concern, and their cost and regulatory demands a roadblock for rapid and broad clinical translation. Here, we demonstrate that CAR T cells can be engineered through non-viral Sleeping Beauty (SB) transposition of CAR genes from minimalistic DNA vectors called minicircles (MCs). We analyzed genomic distribution of SB and LV integrations and show that a significantly higher proportion of MC-derived CAR transposons compared with LV integrants had occurred outside of highly expressed and cancer-related genes into genomic safe harbor loci that are not expected to cause mutagenesis or genotoxicity. CD19-CAR T cells engineered with our enhanced SB approach conferred potent reactivity in vitro and eradicated lymphoma in a xenograft model in vivo. Intriguingly, electroporation of SB MCs is substantially more effective and less toxic compared with conventional plasmids, and enables cost-effective rapid preparation of therapeutic CAR T-cell doses. This approach sets a new standard in advanced cellular and gene therapy and will accelerate and increase the availability of CAR T-cell therapy to treat hematologic malignancies.

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References

  1. 1

    Davila ML, Riviere I, Wang X, Bartido S, Park J, Curran K et al. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med 2014; 6: 224–225.

  2. 2

    Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 2014; 371: 1507–1517.

  3. 3

    Kochenderfer JN, Dudley ME, Kassim SH, Somerville RP, Carpenter RO, Stetler-Stevenson M et al. Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Oncol 2015; 33: 540–549.

  4. 4

    Field AC, Vink C, Gabriel R, Al-Subki R, Schmidt M, Goulden N et al. Comparison of lentiviral and sleeping beauty mediated alphabeta T cell receptor gene transfer. PloS One 2013; 8: e68201.

  5. 5

    Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003; 302: 415–419.

  6. 6

    Wang GP, Levine BL, Binder GK, Berry CC, Malani N, McGarrity G et al. Analysis of lentiviral vector integration in HIV+ study subjects receiving autologous infusions of gene modified CD4+ T cells. Mol Ther 2009; 17: 844–850.

  7. 7

    Izsvak Z, Hackett PB, Cooper LJ, Ivics Z . Translating Sleeping Beauty transposition into cellular therapies: victories and challenges. BioEssays 2010; 32: 756–767.

  8. 8

    Swierczek M, Izsvak Z, Ivics Z . The Sleeping Beauty transposon system for clinical applications. Exp Opin Biol Ther 2012; 12: 139–153.

  9. 9

    Ivics Z, Hackett PB, Plasterk RH, Izsvak Z . Molecular reconstruction of sleeping beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 1997; 91: 501–510.

  10. 10

    Mates L, Chuah MK, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A et al. Molecular evolution of a novel hyperactive sleeping beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet 2009; 41: 753–761.

  11. 11

    Peng PD, Cohen CJ, Yang S, Hsu C, Jones S, Zhao Y et al. Efficient nonviral sleeping beauty transposon-based TCR gene transfer to peripheral blood lymphocytes confers antigen-specific antitumor reactivity. Gene Ther 2009; 16: 1042–1049.

  12. 12

    Huang X, Guo H, Kang J, Choi S, Zhou TC, Tammana S et al. Sleeping Beauty transposon-mediated engineering of human primary T cells for therapy of CD19+ lymphoid malignancies. Mol Ther 2008; 16: 580–589.

  13. 13

    Jin Z, Maiti S, Huls H, Singh H, Olivares S, Mates L et al. The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor. Gene Ther 2011; 18: 849–856.

  14. 14

    Singh H, Manuri PR, Olivares S, Dara N, Dawson MJ, Huls H et al. Redirecting specificity of T-cell populations for CD19 using the Sleeping Beauty system. Cancer Res 2008; 68: 2961–2971.

  15. 15

    Singh H, Figliola MJ, Dawson MJ, Olivares S, Zhang L, Yang G et al. Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using Sleeping Beauty system and artificial antigen presenting cells. PloS One 2013; 8: e64138.

  16. 16

    Singh H, Huls H, Kebriaei P, Cooper LJ . A new approach to gene therapy using Sleeping Beauty to genetically modify clinical-grade T cells to target CD19. Immunol Rev 2014; 257: 181–190.

  17. 17

    June CH, Riddell SR, Schumacher TN . Adoptive cellular therapy: a race to the finish line. Sci Transl Med 2015; 7: 280ps287.

  18. 18

    Ramos CA, Savoldo B, Dotti G . CD19-CAR trials. Cancer J 2014; 20: 112–118.

  19. 19

    Mayrhofer P, Schleef M, Jechlinger W . Use of minicircle plasmids for gene therapy. Methods Mol Biol 2009; 542: 87–104.

  20. 20

    Chen ZY, He CY, Ehrhardt A, Kay MA, Minicircle DNA . vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo. Mol Ther 2003; 8: 495–500.

  21. 21

    Kay MA, He CY, Chen ZY . A robust system for production of minicircle DNA vectors. Nat Biotechnol 2010; 28: 1287–1289.

  22. 22

    Mayrhofer P, Blaesen M, Schleef M, Jechlinger W . Minicircle-DNA production by site specific recombination and protein-DNA interaction chromatography. J Gene Med 2008; 10: 1253–1269.

  23. 23

    Chabot S, Orio J, Schmeer M, Schleef M, Golzio M, Teissie J, Minicircle DNA . Electrotransfer for efficient tissue-targeted gene delivery. Gene Ther 2013; 20: 62–68.

  24. 24

    Kobelt D, Schleef M, Schmeer M, Aumann J, Schlag PM, Walther W . Performance of high quality minicircle DNA for in vitro and in vivo gene transfer. Mol Biotechnol 2013; 53: 80–89.

  25. 25

    Sharma N, Cai Y, Bak RO, Jakobsen MR, Schroder LD, Mikkelsen JG . Efficient sleeping beauty DNA transposition from DNA minicircles. Mol Ther Nucleic Acids 2013; 2: e74.

  26. 26

    Cui Z, Geurts AM, Liu G, Kaufman CD, Hackett PB . Structure-function analysis of the inverted terminal repeats of the sleeping beauty transposon. J Mol Biol 2002; 318: 1221–1235.

  27. 27

    Hudecek M, Sommermeyer D, Kosasih PL, Silva-Benedict A, Liu L, Rader C et al. The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol Res 2015; 3: 125–135.

  28. 28

    Wang X, Chang WC, Wong CW, Colcher D, Sherman M, Ostberg JR et al. A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood 2011; 118: 1255–1263.

  29. 29

    Hudecek M, Lupo-Stanghellini MT, Kosasih PL, Sommermeyer D, Jensen MC, Rader C et al. Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T cells. Clin Cancer Res 2013; 19: 3153–3164.

  30. 30

    Hudecek M, Schmitt TM, Baskar S, Lupo-Stanghellini MT, Nishida T, Yamamoto TN et al. The B-cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor. Blood 2010; 116: 4532–4541.

  31. 31

    Brown CE, Wright CL, Naranjo A, Vishwanath RP, Chang WC, Olivares S et al. Biophotonic cytotoxicity assay for high-throughput screening of cytolytic killing. J Immunol Methods 2005; 297: 39–52.

  32. 32

    Sommermeyer D, Hudecek M, Kosasih PL, Gogishvili T, Maloney DG, Turtle CJ et al. Chimeric antigen receptor-modified T cells derived from defined CD8 and CD4 subsets confer superior antitumor reactivity in vivo. Leukemia 2015; 30: 492–500.

  33. 33

    Frigault MJ, Lee J, Basil MC, Carpenito C, Motohashi S, Scholler J et al. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells. Cancer Immunol Res 2015; 3: 356–367.

  34. 34

    Zayed H, Izsvak Z, Walisko O, Ivics Z . Development of hyperactive sleeping beauty transposon vectors by mutational analysis. Mol Ther 2004; 9: 292–304.

  35. 35

    Vigdal TJ, Kaufman CD, Izsvak Z, Voytas DF, Ivics Z . Common physical properties of DNA affecting target site selection of sleeping beauty and other Tc1/mariner transposable elements. J Mol Biol 2002; 323: 441–452.

  36. 36

    Schones DE, Cui K, Cuddapah S, Roh TY, Barski A, Wang Z et al. Dynamic regulation of nucleosome positioning in the human genome. Cell 2008; 132: 887–898.

  37. 37

    Papapetrou EP, Lee G, Malani N, Setty M, Riviere I, Tirunagari LM et al. Genomic safe harbors permit high beta-globin transgene expression in thalassemia induced pluripotent stem cells. Nat Biotechnol 2011; 29: 73–78.

  38. 38

    Sadelain M, Papapetrou EP, Bushman FD . Safe harbours for the integration of new DNA in the human genome. Nat Revi Cancer 2012; 12: 51–58.

  39. 39

    Papapetrou EP, Schambach A . Gene insertion into genomic safe harbors for human gene therapy. Mol Ther 2016; 24: 678–684.

  40. 40

    Izsvak Z, Ivics Z, Plasterk RH . Sleeping Beauty, a wide host-range transposon vector for genetic transformation in vertebrates. J Mol Biol 2000; 302: 93–102.

  41. 41

    Lukacs GL, Haggie P, Seksek O, Lechardeur D, Freedman N, Verkman AS, Size-dependent DNA . Mobility in cytoplasm and nucleus. J Biol Chem 2000; 275: 1625–1629.

  42. 42

    Turtle CJ, Hanafi LA, Berger C, Gooley TA, Cherian S, Hudecek M et al. CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. J Clin Invest 2016; 126: 2123–2138.

  43. 43

    Wilber A, Frandsen JL, Geurts JL, Largaespada DA, Hackett PB, McIvor RS . RNA as a source of transposase for Sleeping Beauty-mediated gene insertion and expression in somatic cells and tissues. Mol Ther 2006; 13: 625–630.

  44. 44

    Yant SR, Wu X, Huang Y, Garrison B, Burgess SM, Kay MA . High-resolution genome-wide mapping of transposon integration in mammals. Mol Cell Biol 2005; 25: 2085–2094.

  45. 45

    de Jong J, Akhtar W, Badhai J, Rust AG, Rad R, Hilkens J et al. Chromatin landscapes of retroviral and transposon integration profiles. PLoS Genet 2014; 10: e1004250.

  46. 46

    Grabundzija I, Irgang M, Mates L, Belay E, Matrai J, Gogol-Doring A et al. Comparative analysis of transposable element vector systems in human cells. Mol Therapy 2010; 18: 1200–1209.

  47. 47

    Huang X, Guo H, Tammana S, Jung YC, Mellgren E, Bassi P et al. Gene transfer efficiency and genome-wide integration profiling of Sleeping Beauty, Tol2, and piggyBac transposons in human primary T cells. Mol Ther 2010; 18: 1803–1813.

  48. 48

    Maldarelli F, Wu X, Su L, Simonetti FR, Shao W, Hill S et al. HIV latency. Specific HIV integration sites are linked to clonal expansion and persistence of infected cells. Science 2014; 345: 179–183.

  49. 49

    Voigt K, Gogol-Doring A, Miskey C, Chen W, Cathomen T, Izsvak Z et al. Retargeting sleeping beauty transposon insertions by engineered zinc finger DNA-binding domains. Mol Ther 2012; 20: 1852–1862.

  50. 50

    Ivics Z, Katzer A, Stuwe EE, Fiedler D, Knespel S, Izsvak Z . Targeted Sleeping Beauty transposition in human cells. Mol Ther 2007; 15: 1137–1144.

  51. 51

    Riddell SR, Sommermeyer D, Berger C, Liu LS, Balakrishnan A, Salter A et al. Adoptive therapy with chimeric antigen receptor-modified T cells of defined subset composition. Cancer J 2014; 20: 141–144.

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Acknowledgements

We thank Silke Frenz and Elke Spirk for expertise in performing the mouse experiments, and Christa Kruesemann for cloning of MCs. We thank Nirav Malani and Frederic Bushman for kindly providing a raw data set for LV insertions in T cells. Razieh Monjezi was supported by a grant of the German Excellence Initiative to the Graduate School of Life Sciences, University of Würzburg. Michael Hudecek is a member of the Young Scholar Program (Junges Kolleg) of the Bavarian Academy of Sciences. This work was supported by grants from the German Cancer Aid (Deutsche Krebshilfe e.V., Max Eder Program 110313, M.H.) and the IZKF Würzburg (Interdisziplinäres Zentrum für Klinische Forschung, Projekt D-244, M.H.). Csaba Miskey and Zoltán Ivics were supported by the Center for Cell and Gene Therapy of the LOEWE (Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz) program in Hessen, Germany.

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Correspondence to M Hudecek.

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MH and ZI are co-inventors on a patent application related to this work that has been filed by the University of Würzburg and the Paul-Ehrlich Institute. M Schleef and M Schmeer are co-inventors on patent applications related to MC-based transposon- and SB-vectors and are employed at PlasmidFactory GmbH & Co. KG, Bielefeld, Germany.

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Monjezi, R., Miskey, C., Gogishvili, T. et al. Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors. Leukemia 31, 186–194 (2017). https://doi.org/10.1038/leu.2016.180

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