title: Generation of tumoricidal cytotoxic T lymphocytes from healthy donors after in vitro stimulation with a replication-incompetent vaccinia virus encoding MART-1/Melan-A 27-35 epitope.
abstract: Active specific immunotherapy targeting tumor-associated antigens (TAA) requires reagents of high immunogenicity and safety. To address this issue, we constructed a recombinant vaccinia virus carrying a minigene insert encoding the HLA-A2.1-restricted MART-1/Melan-A27-35 melanoma TAA (rVV-M). To facilitate the entry of the antigenic epitope into the endoplasmic reticulum, a sequence coding for adenovirus E3/19K leader peptide was added. This rVV-M was made replication-incompetent by treatment with psoralen and UV light. Infection with rVV-M rendered HLA-A2.1 EBV-transformed lymphoblastoid cells sensitive to the cytotoxic effects of HLA-class-1-restricted, MART-1/Melan-A27-35-specific cytotoxic T lymphocytes (CTL). The capacity of rVV-M to generate HLA-A2.1-restricted MART-1/Melan A-specific CTL was demonstrated from tumor-infiltrating-lymphocyte (TIL) cultures and from healthy donors' peripheral-blood mononuclear cells (PBMC). MART-1/Melan-A27-35-specific CTL were generated from TIL after 2 weekly stimulation courses. Infection with rVV-M elicited a higher CTL response than addition of exogenous peptide, whereas, when a similar protocol was used to stimulate PBMC of healthy donors, significant and specific cytotoxic activity could be observed only upon rVV-M infection but not upon exogenous peptide addition. All CTL generated upon rVV-M stimulation were also able to efficiently kill melanoma cell lines expressing both MART-1/Melan-A and HLA-A2.1. In addition, TNF-alpha production could be induced in rVV-M-stimulated CTL upon co-culture with COS-7 cells transiently transfected with MART-1/Melan-A and HLA-A2.1 genes. This safe and highly immunogenic reagent could be of use in TAA-targeted clinical immunotherapy.
authors.affiliation: Department of Surgery (F. Harder), University of Basel, Switzerland. zajac@ubaclu.unibas.ch
authors.forename: P
authors.initials: P
authors.lastname: Zajac
authors.forename: D
authors.initials: D
authors.lastname: Oertli
authors.forename: G C
authors.initials: GC
authors.lastname: Spagnoli
authors.forename: C
authors.initials: C
authors.lastname: Noppen
authors.forename: C
authors.initials: C
authors.lastname: Schaefer
authors.forename: M
authors.initials: M
authors.lastname: Heberer
authors.forename: W R
authors.initials: WR
authors.lastname: Marti
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology*; D001483:Base Sequence; D001782:Blood Donors; D019556:COS Cells; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D000939:Epitopes / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology* / Q000473:pathology; D008545:Melanoma / Q000276:immunology* / Q000473:pathology; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D012016:Reference Values; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D014614:Vaccines, Synthetic; D014616:Vaccinia virus / Q000276:immunology* / Q000502:physiology; D014765:Viral Vaccines; D014779:Virus Replication
chemical_list: D000956:Antigens, Viral; D000939:Epitopes; C105787:MART-1-Melan-A(27-35) epitope; D009363:Neoplasm Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D014614:Vaccines, Synthetic; D014765:Viral Vaccines