title: PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxic T-cells expressing HER2-specific chimeric antigen receptor.
abstract: 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.
authors.affiliation: Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: Yozo
authors.initials: Y
authors.lastname: Nakazawa
authors.forename: Leslie E
authors.initials: LE
authors.lastname: Huye
authors.forename: Vita S
authors.initials: VS
authors.lastname: Salsman
authors.forename: Ann M
authors.initials: AM
authors.lastname: Leen
authors.forename: Nabil
authors.initials: N
authors.lastname: Ahmed
authors.forename: Lisa
authors.initials: L
authors.lastname: Rollins
authors.forename: Gianpietro
authors.initials: G
authors.lastname: Dotti
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Gottschalk
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Wilson
authors.forename: Cliona M
authors.initials: CM
authors.lastname: Rooney
mesh_terms: D000818:Animals; D017951:Antigen Presentation; D018941:Antigens, CD19 / Q000378:metabolism; D001932:Brain Neoplasms / Q000276:immunology* / Q000378:metabolism / Q000628:therapy*; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D020031:Epstein-Barr Virus Infections / Q000276:immunology / Q000378:metabolism / Q000628:therapy; D005434:Flow Cytometry; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D007167:Immunotherapy; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000276:immunology; D011946:Receptors, Antigen / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D018941:Antigens, CD19; D018719:Erb-b2 Receptor Tyrosine Kinases; D011946:Receptors, Antigen; D011993:Recombinant Fusion Proteins