Cancer Immunology, Immunotherapy

, Volume 58, Issue 6, pp 977–987 | Cite as

Lentiviral vectors encoding human MUC1-specific, MHC-unrestricted single-chain TCR and a fusion suicide gene: potential for universal and safe cancer immunotherapy

  • Xiaochuan Chen
  • Wentao Gao
  • Andrea Gambotto
  • Olivera J. Finn
Original Article


MUC1 tumor antigen is a target for immunotherapy of most human adenocarcinomas and some hematological malignancies. Expression of a MUC1-specific, MHC-unrestricted single-chain T cell receptor (scTCR) on cells of both innate and adaptive immune system through reconstitution of lethally irradiated mice by retroviral vector-transduced bone marrow cells, had been shown to effectively control the growth of MUC1+ tumors independent of their MHC type, suggesting that this receptor is a good candidate for broadly applicable gene therapy/immunotherapy. However, the translational application of this immuno-gene therapy modality was discouraged by the progressive transgene silencing in reconstituted T and B cells, as well as the potential of tumorogenesis intrinsic to oncoretroviral vectors. To overcome these problems and facilitate the future clinical use of this receptor, we have constructed a panel of novel self-inactivating lentiviral vectors (LVs) which harbor two independent internal promoters, one driving expression of the scTCR gene and the other of a fusion suicide gene, the HSV-TK–EGFP fusion gene, allowing the transduced cells to be destroyable by the pro-drug ganciclovir. Despite the large size of insert, these vectors were efficiently packaged into high titer virus that transferred the expression of transgene in both T cell lines and primary T cells. Sustained expression was maintained in a T cell line for over 4 months in vitro, suggesting its efficient resistance to transgene silencing. Both scTCR and HSV-TK–EGFP genes were functional in the transduced cells, as evidenced by their specific recognition of MUC1+ tumors and efficient eradication by ganciclovir.


MUC1 T cell receptor (TCR) Lentiviral vector Cancer immunotherapy 


  1. 1.
    Vlad AM, Kettel JC, Alajez NM, Carlos CA, Finn OJ (2004) MUC1 immunobiology: from discovery to clinical applications. Adv Immunol 82:249–293PubMedCrossRefGoogle Scholar
  2. 2.
    Girling A, Bartkova J, Burchell J, Gendler S, Gillett C, Taylor-Papadimitriou J (1989) A core protein epitope of the polymorphic epithelial mucin detected by the monoclonal antibody SM-3 is selectively exposed in a range of primary carcinomas. Int J Cancer 43(6):1072–1076PubMedCrossRefGoogle Scholar
  3. 3.
    Gendler S, Taylor-Papadimitriou J, Duhig T, Rothbard J, Burchell JA (1988) A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats. J Biol Chem 263(26):12820–12823PubMedGoogle Scholar
  4. 4.
    Siddiqui J, Abe M, Hayes D, Shani E, Yunis E, Kufe D (1988) Isolation and sequencing of a cDNA coding for the human DF3 breast carcinoma-associated antigen. Proc Natl Acad Sci USA 85(7):2320–2323PubMedCrossRefGoogle Scholar
  5. 5.
    Brossart P, Schneider A, Dill P, Schammann T, Grunebach F, Wirths S, Kanz L, Buhring HJ, Brugger W (2001) The epithelial tumor antigen MUC1 is expressed in hematological malignancies and is recognized by MUC1-specific cytotoxic T-lymphocytes. Cancer Res 61(18):6846–6850PubMedGoogle Scholar
  6. 6.
    Teruya-Feldstein J, Donnelly GB, Goy A, Hegde A, Nanjangud G, Qin J, Thaler H, Gilles F, Dyomin VG, Lloyd KO, Zelenetz AD, Houldsworth J, Chaganti RS (2003) MUC-1 mucin protein expression in B-cell lymphomas. Appl Immunohistochem Mol Morphol 11(1):28–32PubMedCrossRefGoogle Scholar
  7. 7.
    Duperry C, Klein B, Durie BG M, Zhang X, Fourdan M, Poncelet R, Favier F, Vincent C, Brochier J, Lenoir G, Bataille R (1989) Phenotype analysis of human myeloma cell lines. Blood 73(2):566–572Google Scholar
  8. 8.
    Takahashi T, Makiguchi Y, Hinoda Y, Kakiuchi H, Nakagawa N, Imai K, Yachi A (1994) Expression of MUC1 on myeloma cells and induction of HLA-unrestricted CTL against MUC1 from a multiple myeloma patient. J Immunol 153(5):2102–2109PubMedGoogle Scholar
  9. 9.
    Barnd DL, Lan MS, Metzgar RS, Finn OJ (1989) Specific, major histocompatibility complex-unrestricted recognition of tumor-associated mucins by human cytotoxic T cells. Proc Natl Acad Sci USA 86(18):7159–7163PubMedCrossRefGoogle Scholar
  10. 10.
    Jerome KR, Domenech N, Finn OJ (1993) Tumor-specific cytotoxic T cell clones from patients with breast and pancreatic adenocarcinoma recognize EBV-immortalized B cells transfected with polymorphic epithelial mucin complementary DNA. J Immunol 151(3):1654–1662PubMedGoogle Scholar
  11. 11.
    Magarian-Blander J, Ciborowski P, Hsia S, Watkins SC, Finn OJ (1998) Intercellular and intracellular events following the MHC-unrestricted TCR recognition of a tumor-specific peptide epitope on the epithelial antigen MUC1. J Immunol 160(7):3111–3120PubMedGoogle Scholar
  12. 12.
    Alajez NM, Schmielau J, Alter MD, Cascio M, Finn OJ (2005) Therapeutic potential of a tumor-specific, MHC-unrestricted T-cell receptor expressed on effector cells of the innate and the adaptive immune system through bone marrow transduction and immune reconstitution. Blood 105(12):4583–4589PubMedCrossRefGoogle Scholar
  13. 13.
    Pannell D, Ellis J (2001) Silencing of gene expression: implications for design of retrovirus vectors. Rev Med Virol 11:205–217PubMedCrossRefGoogle Scholar
  14. 14.
    Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P, Lim A, Osborne CS, Pawliuk R, Morillon E, Sorensen R, Forster A, Fraser P, Cohen JI, t Basile G, Alexander I, Wintergerst U, Frebourg T, Aurias A, Stoppa-Lyonnet D, Romana S, Radford-Weiss I, Gross F, Valensi F, Delabesse E, Macintyre E, Sigaux F, Soulier J, Leiva LE, Wissler M, Prinz C, Rabbitts TH, Le Deist F, Fischer A, Cavazzana-Calvo M (2003) LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302(5644):415–419PubMedCrossRefGoogle Scholar
  15. 15.
    Kohn DB, Sadelain M, Glorioso JC (2003) Occurrence of leukaemia following gene therapy of X-linked SCID. Nat Rev Cancer 3(7):477–488PubMedCrossRefGoogle Scholar
  16. 16.
    Nienhuis AW, Dunbar CE, Sorrentino BP (2006) Genotoxicity of retroviral integration in hematopoietic cells. Mol Ther 13(6):1031–1049PubMedCrossRefGoogle Scholar
  17. 17.
    Schroder AR, Shinn P, Chen H, Berry C, Ecker JR, Bushman F (2002) HIV-1 integration in the human genome favors active genes and local hotspots. Cell 110(4):521–529PubMedCrossRefGoogle Scholar
  18. 18.
    Cattoglio C, Facchini G, Sartori D, Antonelli A, Miccio A, Cassani B, Schmidt M, von Kalle C, Howe S, Thrasher AJ, Aiuti A, Ferrari G, Recchia A, Mavilio F (2007) Hot spots of retroviral integration in human CD34+ hematopoietic cells. Blood 110(6):1770–1778PubMedCrossRefGoogle Scholar
  19. 19.
    Mohamedali A, Moreau-Gaudry F, Richard E, Xia P, Nolta J, Malik P (2004) Self-inactivating lentiviral vectors resist proviral methylation but do not confer position-independent expression in hematopoietic stem cells. Mol Ther 10(2):249–259PubMedCrossRefGoogle Scholar
  20. 20.
    Chinnasamy D, Chinnasamy N, Enriquez MJ, Otsu M, Morgan RA, Candotti F (2000) Lentiviral-mediated gene transfer into human lymphocytes: role of HIV-1 accessory proteins. Blood 96(4):1309–1316PubMedGoogle Scholar
  21. 21.
    Costello E, Munoz M, Buetti E, Meylan PR, Diggelmann H, Thali M (2000) Gene transfer into stimulated and unstimulated T lymphocytes by HIV-1-derived lentiviral vectors. Gene Ther 7(7):596–604PubMedCrossRefGoogle Scholar
  22. 22.
    Verhoeyen E, Dardalhon V, Ducrey-Rundquist O, Trono D, Taylor N, Cosset FL (2003) IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes. Blood 101(6):2167–2174PubMedCrossRefGoogle Scholar
  23. 23.
    Zhou X, Cui Y, Huang X, Yu Z, Thomas AM, Ye Z, Pardoll DM, Jaffee EM, Cheng L (2003) Lentivirus-mediated gene transfer and expression in established human tumor antigen-specific cytotoxic T cells and primary unstimulated T cells. Hum Gene Ther 14(11):1089–1105PubMedCrossRefGoogle Scholar
  24. 24.
    Levine BL, Humeau LM, Boyer J, MacGregor RR, Rebello T, Lu X, Binder GK, Slepushkin V, Lemiale F, Mascola JR, Bushman FD, Dropulic B, June CH (2006) Gene transfer in humans using a conditionally replicating lentiviral vector. Proc Natl Acad Sci USA 103(46):17372–17377PubMedCrossRefGoogle Scholar
  25. 25.
    Pasanen T, Hakkarainen T, Timonen P, Parkkinen J, Tenhunen A, Loimas S, Wahlfors J (2003) TK-GFP fusion gene virus vectors as tools for studying the features of HSV-TK/ganciclovir cancer gene therapy in vivo. Int J Mol Med 12(4):525–531PubMedGoogle Scholar
  26. 26.
    Pellinen R, Hakkarainen T, Wahlfors T, Tulimäki K, Ketola A, Tenhunen A, Salonen T, Wahlfors J (2004) Cancer cells as targets for lentivirus-mediated gene transfer and gene therapy. Int J Oncol 25(6):1753–1762PubMedGoogle Scholar
  27. 27.
    Li X, Mukai T, Young D, Frankel S, Law P, Wong-Staal F (1998) Transduction of CD34 + cells by a vesicular stomach virus protein G (VSV-G) pseudotyped HIV-1 vector. Stable gene expression in progeny cells, including dendritic cells. J Hum Virol 1(5):346–352PubMedGoogle Scholar
  28. 28.
    Ramezani Ali, Hawley Teresa S, Hawley Robert G (2003) Performance- and safety-enhanced lentiviral vectors containing the human interferon-β scaffold attachment region and the chicken β-globin insulator. Blood 101(12):4717–4724PubMedCrossRefGoogle Scholar
  29. 29.
    Gao Z, Golob J, Tanavde VM, Civin CI, Hawley RG, Cheng L (2001) High levels of transgene expression following transduction of long-term NOD/SCID-repopulating human cells with a modified lentiviral vector. Stem Cells 19(3):247–259PubMedCrossRefGoogle Scholar
  30. 30.
    Cui Y, Golob J, Kelleher E, Ye Z, Pardoll D, Cheng L (2002) Targeting transgene expression to antigen-presenting cells derived from lentivirus-transduced engrafting human hematopoietic stem/progenitor cells. Blood 99(2):399–408PubMedCrossRefGoogle Scholar
  31. 31.
    Hasegawa Y, Emi N, Shimokata K, Abe A, Kawabe T, Hasegawa T, Kirioka T, Saito H (1993) Gene transfer of herpes simplex virus type I thymidine kinase gene as a drug sensitivity gene into human lung cancer cell lines using retroviral vectors. Am J Respir Cell Mol Biol 8(6):655–661PubMedGoogle Scholar
  32. 32.
    Iwakuma T, Cui Y, Chang LJ (1999) Self-inactivating lentiviral vectors with U3 and U5 modifications. Virology 261(1):120–132PubMedCrossRefGoogle Scholar
  33. 33.
    Chang LJ, Urlacher V, Iwakuma T, Cui Y, Zucali J (1999) Efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system. Gene Ther 6(5):715–728PubMedCrossRefGoogle Scholar
  34. 34.
    Zufferey R, Donello JE, Trono D, Hope TJ (1999) Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 73(4):2886–2892PubMedGoogle Scholar
  35. 35.
    Higashimoto T, Urbinati F, Perumbeti A, Jiang G, Zarzuela A, Chang LJ, Kohn DB, Malik P (2007) The woodchuck hepatitis virus post-transcriptional regulatory element reduces readthrough transcription from retroviral vectors. Gene Ther 14(17):1298–1304PubMedCrossRefGoogle Scholar
  36. 36.
    Charneau P, Clavel F (1991) A single-stranded gap in human immunodeficiency virus unintegrated linear DNA defined by a central copy of the polypurine tract. J Virol 65(5):2415–2421PubMedGoogle Scholar
  37. 37.
    Charneau P, Alizon M, Clavel F (1992) A second origin of DNA plus-strand synthesis is required for optimal human immunodeficiency virus replication. J Virol 66(5):2814–2820PubMedGoogle Scholar
  38. 38.
    Sirven A, Pflumio F, Zennou V, Titeux M, Vainchenker W, Coulombel L, Dubart-Kupperschmitt A, Charneau P (2000) The human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of human hematopoietic stem cells. Blood 96(13):4103–4110PubMedGoogle Scholar
  39. 39.
    Alajez NM, Eghtesad S, Finn OJ (2006) Cloning and expression of human membrane-bound and soluble engineered T cell receptors for immunotherapy. J Biomed Biotechnol 2006(2):68091PubMedGoogle Scholar
  40. 40.
    Qi R, An H, Yu Y, Zhang M, Liu S, Xu H, Guo Z, Cheng T, Cao X (2003) Notch1 signaling inhibits growth of human hepatocellular carcinoma through induction of cell cycle arrest and apoptosis. Cancer Res 63(23):8323–8329PubMedGoogle Scholar
  41. 41.
    Tiscornia G, Singer O, Verma IM (2006) Production and purification of lentiviral vectors. Nat Protoc 1(1):241–245PubMedCrossRefGoogle Scholar
  42. 42.
    Beatty PL, Plevy SE, Sepulveda AR, Finn OJ (2007) Cutting edge: transgenic expression of human MUC1 in IL-10 −/− mice accelerates inflammatory bowel disease and progression to colon cancer. J Immunol 179(2):735–739PubMedGoogle Scholar
  43. 43.
    Yu X, Zhan X, D’Costa J, Tanavde VM, Ye Z, Peng T, Malehorn MT, Yang X, Civin CI, Cheng L (2003) Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells. Mol Ther 7(6):827–838PubMedCrossRefGoogle Scholar
  44. 44.
    Semple-Rowland SL, Eccles KS, Humberstone EJ (2007) Targeted expression of two proteins in neural retina using self-inactivating, insulated lentiviral vectors carrying two internal independent promoters. Mol Vis 13:2001–2011PubMedGoogle Scholar
  45. 45.
    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 1:376–382PubMedCrossRefGoogle Scholar
  46. 46.
    Zychlinski D, Schambach A, Modlich U, Maetzig T, Meyer J, Grassman E, Mishra A, Baum C (2008) Physiological Promoters Reduce the Genotoxic Risk of Integrating Gene Vectors. Mol Ther 16:718–725PubMedCrossRefGoogle Scholar
  47. 47.
    Kumar M, Keller B, Makalou N, Sutton RE (2001) Systematic determination of the packaging limit of lentiviral vectors. Hum Gene Ther 12(15):1893–1905PubMedCrossRefGoogle Scholar
  48. 48.
    al Yacoub N, Romanowska M, Haritonova N, Foerster J (2007) Optimized production and concentration of lentiviral vectors containing large inserts. J Gene Med 9(7):579–584PubMedCrossRefGoogle Scholar
  49. 49.
    Logan AC, Haas DL, Kafri T, Kohn DB (2004) Integrated self-inactivating lentiviral vectors produce full-length genomic transcripts competent for encapsidation and integration. J Virol 78(16):8421–8436PubMedCrossRefGoogle Scholar
  50. 50.
    Hanawa H, Persons DA, Nienhuis AW (2005) Mobilization and Mechanism of Transcription of Integrated Self-Inactivating Lentiviral Vectors. J Virol 79:8410–8421PubMedCrossRefGoogle Scholar
  51. 51.
    Lucke S, Grunwald T, Uberla K (2005) Reduced Mobilization of Rev-Responsive Element-Deficient Lentiviral Vectors. J Virol 79:9359–9362PubMedCrossRefGoogle Scholar
  52. 52.
    Acres B, Limacher JM (2005) MUC1 as a target antigen for cancer immunotherapy. Expert Rev Vaccines 4(4):493–502PubMedCrossRefGoogle Scholar
  53. 53.
    Engelmann K, Shen H, Finn OJ (2008) MCF7 side population cells with characteristics of cancer stem/progenitor cells express the tumor antigen MUC1. Cancer Res 68(7):2419–2426PubMedCrossRefGoogle Scholar
  54. 54.
    Zhao Y, Zheng Z, Robbins PF, Khong HT, Rosenberg SA, Morgan RA (2005) Primary human lymphocytes transduced with NY-ESO-1 antigen-specific TCR genes recognize and kill diverse human tumor cell lines. J Immunol 174(7):4415–4423PubMedGoogle Scholar
  55. 55.
    Tsuji T, Yasukawa M, Matsuzaki J, Ohkuri T, Chamoto K, Wakita D, Azuma T, Niiya H, Miyoshi H, Kuzushima K, Oka Y, Sugiyama H, Ikeda H, Nishimura T (2005) Generation of tumor-specific, HLA class I-restricted human Th1 and Tc1 cells by cell engineering with tumor peptide-specific T-cell receptor genes. Blood 106(2):470–476PubMedCrossRefGoogle Scholar
  56. 56.
    Micucci F, Zingoni A, Piccoli M, Frati L, Santoni A, Galandrini R (2006) High-efficient lentiviral vector-mediated gene transfer into primary human NK cells. Exp Hematol 34(10):1344–1352PubMedCrossRefGoogle Scholar
  57. 57.
    Budak-Alpdogan T, Banerjee D, Bertino JR (2005) Hematopoietic stem cell gene therapy with drug resistance genes: an update. Cancer Gene Ther 12(11):849–863PubMedCrossRefGoogle Scholar
  58. 58.
    Yang L, Baltimore D (2005) Long-term in vivo provision of antigen-specific T cell immunity by programming hematopoietic stem cells. Proc Natl Acad Sci USA 102(12):4518–4523PubMedCrossRefGoogle Scholar
  59. 59.
    Dudley ME, Rosenberg SA (2003) Adoptive-cell-transfer therapy for the treatment of patients with cancer. Nat Rev Cancer 3(9):666–675PubMedCrossRefGoogle Scholar
  60. 60.
    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(7):799–810PubMedCrossRefGoogle Scholar
  61. 61.
    de Witte MA, Jorritsma A, Kaiser A, van den Boom MD, Dokter M, Bendle GM, Haanen JB, Schumacher TN (2008) Requirements for Effective Antitumor Responses of TCR Transduced T Cells. J Immunol 181(7):5128–5136PubMedGoogle Scholar
  62. 62.
    Bubeník J (2004) MHC class I down-regulation: tumour escape from immune surveillance? Int J Oncol 25(2):487–491PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Xiaochuan Chen
    • 1
    • 2
  • Wentao Gao
    • 3
  • Andrea Gambotto
    • 4
  • Olivera J. Finn
    • 1
  1. 1.Department of ImmunologyUniversity of Pittsburgh School of MedicinePittsburghUSA
  2. 2.Garden State Cancer CenterCenter for Molecular Medicine and ImmunologyBellevilleUSA
  3. 3.Department of PathologyUniversity of Pittsburgh School of MedicinePittsburghUSA
  4. 4.Center for Biotechnology and Institute of Molecular MedicineUniversity of Pittsburgh School of MedicinePittsburghUSA

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