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The immunogenicity of virus-derived 2A sequences in immunocompetent individuals


Genetic engineering of T cells for adoptive immunotherapy in cancer patients has shown significant promise. To ensure optimal antitumor activity and safety, the simultaneous expression of multiple genes is frequently required, and short viral-derived 2A sequences are increasingly preferred for this purpose. Concerns exist, however, that these virus-derived sequences may induce unwanted immune responses, and thus diminish persistence of the gene-modified cells after adoptive transfer. Whereas such responses were absent in immunocompromised recipients, potential immunogenicity in immunocompetent individuals remains a concern. We now address whether ex vivo T cell responses can be elicited against the most widely used 2A sequences (2A-Thosea asigna virus (TAV) or 2A-equine rhinitis virus (ERAV), specifically) in immunocompetent individuals. We used a potent ex vivo culture system previously validated to induce T cell responses even against weakly immunogenic antigens. Of the sixteen donors tested, only five released very low levels of interferon-γ in response to 2A-TAV peptide mixtures (single peptide specificity in three donors, adjacent self-antigen peptide specificity in one donor and nonspecific reactivity in one donor). None of them produced cytotoxic activity or responded to 2A-ERAV. These results suggest that exposure to viral-derived 2A sequences is unlikely to produce unwanted T cell responses in immunocompetent individuals and further supports their continued use for studies of human gene therapy.

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  1. Restifo NP, Dudley ME, Rosenberg SA . Adoptive immunotherapy for cancer: harnessing the T cell response. Nat Rev Immunol 2012; 12: 269–281.

    Article  CAS  Google Scholar 

  2. Hoyos V, Savoldo B, Dotti G . Genetic modification of human T lymphocytes for the treatment of hematological malignancies. Haematologica 2012; 97: 1622–1631.

    Article  CAS  Google Scholar 

  3. Kerkar SP, Restifo NP . Cellular constituents of immune escape within the tumor microenvironment. Cancer Res 2012; 72: 3125–3130.

    Article  CAS  Google Scholar 

  4. Craddock JA, Lu A, Bear A, Pule M, Brenner MK, Rooney CM et al. Enhanced tumor trafficking of GD2 chimeric antigen receptor T cells by expression of the chemokine receptor CCR2b. J Immunother 2010; 33: 780–788.

    Article  CAS  Google Scholar 

  5. Di Stasi A, De Angelis B, Rooney CM, Zhang L, Mahendravada A, Foster AE et al. T lymphocytes coexpressing CCR4 and a chimeric antigen receptor targeting CD30 have improved homing and antitumor activity in a Hodgkin tumor model. Blood 2009; 113: 6392–6402.

    Article  CAS  Google Scholar 

  6. Quintarelli C, Vera JF, Savoldo B, Giordano Attianese GM, Pule M, Foster AE et al. Co-expression of cytokine and suicide genes to enhance the activity and safety of tumor-specific cytotoxic T lymphocytes. Blood 2007; 110: 2793–2802.

    Article  CAS  Google Scholar 

  7. Hoyos V, Savoldo B, Quintarelli C, Mahendravada A, Zhang M, Vera J et al. Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety. Leukemia 2010; 24: 1160–1170.

    Article  CAS  Google Scholar 

  8. Zhang L, Kerkar SP, Yu Z, Zheng Z, Yang S, Restifo NP et al. Improving adoptive T cell therapy by targeting and controlling IL-12 expression to the tumor environment. Mol Ther 2011; 19: 751–759.

    Article  CAS  Google Scholar 

  9. Liu D, Song L, Wei J, Courtney AN, Gao X, Marinova E et al. IL-15 protects NKT cells from inhibition by tumor-associated macrophages and enhances antimetastatic activity. J Clin Invest 2012; 122: 2221–2233.

    Article  CAS  Google Scholar 

  10. Milone MC, Fish JD, Carpenito C, Carroll RG, Binder GK, Teachey D et al. Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol Ther 2009; 17: 1453–1464.

    Article  CAS  Google Scholar 

  11. Savoldo B, Ramos CA, Liu E, Mims MP, Keating MJ, Carrum G et al. CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients. J Clin Invest 2011; 121: 1822–1826.

    Article  CAS  Google Scholar 

  12. Ciceri F, Bonini C, Stanghellini MT, Bondanza A, Traversari C, Salomoni M et al. Infusion of suicide-gene-engineered donor lymphocytes after family haploidentical haemopoietic stem-cell transplantation for leukaemia (the TK007 trial): a non-randomised phase I-II study. Lancet Oncol 2009; 10: 489–500.

    Article  Google Scholar 

  13. Di Stasi A, Tey SK, Dotti G, Fujita Y, Kennedy-Nasser A, Martinez C et al. Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med 2011; 365: 1673–1683.

    Article  CAS  Google Scholar 

  14. Szymczak-Workman AL, Vignali KM, Vignali DA . Design and construction of 2A peptide-linked multicistronic vectors. Cold Spring Harb Protoc 2012; 2012: 199–204.

    PubMed  Google Scholar 

  15. de Felipe P . Skipping the co-expression problem: the new 2A ‘CHYSEL’ technology. Genet Vaccines Ther 2004; 2: 13.

    Article  Google Scholar 

  16. Szymczak AL, Workman CJ, Wang Y, Vignali KM, Dilioglou S, Vanin EF et al. Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nat Biotechnol 2004; 22: 589–594.

    Article  CAS  Google Scholar 

  17. 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.

    Article  CAS  Google Scholar 

  18. Quintarelli C, Dotti G, De Angelis B, Hoyos V, Mims M, Luciano L et al. Cytotoxic T lymphocytes directed to the preferentially expressed antigen of melanoma (PRAME) target chronic myeloid leukemia. Blood 2008; 112: 1876–1885.

    Article  CAS  Google Scholar 

  19. Hanley PJ, Cruz CR, Savoldo B, Leen AM, Stanojevic M, Khalil M et al. Functionally active virus-specific T cells that target CMV, adenovirus, and EBV can be expanded from naive T-cell populations in cord blood and will target a range of viral epitopes. Blood 2009; 114: 1958–1967.

    Article  CAS  Google Scholar 

  20. Quintarelli C, Dotti G, Hasan ST, De Angelis B, Hoyos V, Errichiello S et al. High-avidity cytotoxic T lymphocytes specific for a new PRAME-derived peptide can target leukemic and leukemic-precursor cells. Blood 2011; 117: 3353–3362.

    Article  CAS  Google Scholar 

  21. Tey SK, Dotti G, Rooney CM, Heslop HE, Brenner MK . Inducible caspase 9 suicide gene to improve the safety of allodepleted T cells after haploidentical stem cell transplantation. Biol Blood Marrow Transplant 2007; 13: 913–924.

    Article  CAS  Google Scholar 

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CA is supported by Oncosuisse (BIL KFS 02506-08-2009), NIH-NHLBI T32 HL092332 (Trainee) and an American Society for Blood and Marrow Transplantation (ASBMT)/Celgene New Investigator Award 2012; HEH is supported by a Dan L Duncan Chair, P50 CA126752, PO1 CA94237 and a SCOR from the Leukemia and Lymphoma Society; MKB is supported by a Fayez Sarofim Chair and a CPRIT award (RP110553); GD is supported by NIH R01 CA142636, a Leukemia and Lymphoma Society Translational Research grant and W81XWH-10-10425 Department of Defense, Technology/Therapeutic Development Award; BS is supported by NIH R01 CA131027, a Leukemia and Lymphoma Society Translational Research grant and a CPRIT award (RP120298). We also appreciate the support of shared resources in the cancer center support grant P30CA125123.

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Correspondence to B Savoldo.

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Arber, C., Abhyankar, H., Heslop, H. et al. The immunogenicity of virus-derived 2A sequences in immunocompetent individuals. Gene Ther 20, 958–962 (2013).

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  • 2A sequences
  • polycistronic vectors
  • T cell gene transfer
  • immunogenicity

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