PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxic T-cells expressing HER2-specific chimeric antigen receptor

Mol Ther. 2011 Dec;19(12):2133-43. doi: 10.1038/mt.2011.131. Epub 2011 Jul 19.


Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) can be modified to function as heterologous tumor directed effector cells that survive longer in vivo than tumor directed T cells without virus specificity, due to chronic stimulation by viral antigens expressed during persistent infection in seropositive individuals. We evaluated the nonviral piggyBac (PB) transposon system as a platform for modifying EBV-CTLs to express a functional human epidermal growth factor receptor 2-specific chimeric antigen receptor (HER2-CAR) thereby directing virus-specific, gene modified CTLs towards HER2-positive cancer cells. Peripheral blood mononuclear cells (PBMCs) were nucleofected with transposons encoding a HER2-CAR and a truncated CD19 molecule for selection followed by specific activation and expansion of EBV-CTLs. HER2-CAR was expressed in ~40% of T cells after CD19 selection with retention of immunophenotype, polyclonality, and function. HER2-CAR-modified EBV-CTLs (HER2-CTLs) killed HER2-positive brain tumor cell lines in vitro, exhibited transient and reversible increases in HER2-CAR expression following antigen-specific stimulation, and stably expressed HER2-CAR beyond 120 days. Adoptive transfer of PB-modified HER2-CTLs resulted in tumor regression in a murine xenograft model. Our results demonstrate that PB can be used to redirect virus-specific CTLs to tumor targets, which should prolong tumor-specific T cell survival in vivo producing more efficacious immunotherapy.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Antigen Presentation
  • Antigens, CD19 / metabolism
  • Brain Neoplasms / immunology*
  • Brain Neoplasms / metabolism
  • Brain Neoplasms / therapy*
  • Cell Line, Tumor
  • Coculture Techniques
  • Enzyme-Linked Immunosorbent Assay
  • Epstein-Barr Virus Infections / immunology
  • Epstein-Barr Virus Infections / metabolism
  • Epstein-Barr Virus Infections / therapy
  • Flow Cytometry
  • Herpesvirus 4, Human / genetics
  • Humans
  • Immunotherapy*
  • Male
  • Mice
  • Mice, Inbred NOD
  • Mice, SCID
  • Receptor, ErbB-2 / genetics*
  • Receptor, ErbB-2 / immunology
  • Receptors, Antigen / genetics*
  • Receptors, Antigen / immunology
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / immunology
  • T-Lymphocytes, Cytotoxic / immunology*
  • Transduction, Genetic
  • Tumor Cells, Cultured


  • Antigens, CD19
  • Receptors, Antigen
  • Recombinant Fusion Proteins
  • Receptor, ErbB-2