title: T Cells Engineered to Express a T-Cell Receptor Specific for Glypican-3 to Recognize and Kill Hepatoma Cells In Vitro and in Mice.
abstract: OBJECTIVE
  Cancer therapies are being developed based on our ability to direct T cells against tumor antigens. Glypican-3 (GPC3) is expressed by 75% of all hepatocellular carcinomas (HCC), but not in healthy liver tissue or other organs. We aimed to generate T cells with GPC3-specific receptors that recognize HCC and used them to eliminate GPC3-expressing xenograft tumors grown from human HCC cells in mice.
  
  
  METHODS
  We used mass spectrometry to obtain a comprehensive peptidome from GPC3-expressing hepatoma cells after immune-affinity purification of human leukocyte antigen (HLA)-A2 and bioinformatics to identify immunodominant peptides. To circumvent GPC3 tolerance resulting from fetal expression, dendritic cells from HLA-A2-negative donors were cotransfected with GPC3 and HLA-A2 RNA to stimulate and expand antigen-specific T cells.
  
  
  RESULTS
  Peptide GPC3367 was identified as a predominant peptide on HLA-A2. We used A2-GPC3367 multimers to detect, select for, and clone GPC3-specific T cells. These clones bound the A2-GPC3367 multimer and secreted interferon-γ when cultured with GPC3367, but not with control peptide-loaded cells. By genomic sequencing of these T-cell clones, we identified a gene encoding a dominant T-cell receptor. The gene was cloned and the sequence was codon optimized and expressed from a retroviral vector. Primary CD8(+) T cells that expressed the transgenic T-cell receptor specifically bound GPC3367 on HLA-A2. These T cells killed GPC3-expressing hepatoma cells in culture and slowed growth of HCC xenograft tumors in mice.
  
  
  CONCLUSIONS
  We identified a GPC3367-specific T-cell receptor. Expression of this receptor by T cells allows them to recognize and kill GPC3-positive hepatoma cells. This finding could be used to advance development of adoptive T-cell therapy for HCC.
authors.affiliation: Institute of Virology, Technische Universität München, Helmholtz Zentrum München, München, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Dargel
authors.affiliation: Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Michal
authors.initials: M
authors.lastname: Bassani-Sternberg
authors.affiliation: Institute of Virology, Technische Universität München, Helmholtz Zentrum München, München, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Hasreiter
authors.affiliation: Institute for Diabetes and Obesity, Helmholtz Zentrum München, Garching, Germany.
authors.forename: Fabio
authors.initials: F
authors.lastname: Zani
authors.affiliation: Institute of Virology, Technische Universität München, Helmholtz Zentrum München, München, Germany; German Center for Infection Research (DZIF), Munich Site, Germany.
authors.forename: Jan-Hendrik
authors.initials: JH
authors.lastname: Bockmann
authors.affiliation: Institute of Virology, Technische Universität München, Helmholtz Zentrum München, München, Germany; German Center for Infection Research (DZIF), Munich Site, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Thiele
authors.affiliation: Institute of Virology, Technische Universität München, Helmholtz Zentrum München, München, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Bohne
authors.affiliation: Institute of Virology, Technische Universität München, Helmholtz Zentrum München, München, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Wisskirchen
authors.affiliation: Institute of Molecular Immunology, Helmholtz Zentrum München, München, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Wilde
authors.affiliation: I. Medizinische Klinik und Poliklinik, Universitätsmedizin der Johannes Gutenberg-Universität, Mainz, Germany.
authors.forename: Martin F
authors.initials: MF
authors.lastname: Sprinzl
authors.affiliation: Institute of Molecular Immunology, Helmholtz Zentrum München, München, Germany; Clinical Cooperation Groups Antigen Specific Immunotherapy and Immune Monitoring, Technische Universität München, Helmholtz Zentrum München, München, Germany.
authors.forename: Dolores J
authors.initials: DJ
authors.lastname: Schendel
authors.affiliation: Clinical Cooperation Groups Antigen Specific Immunotherapy and Immune Monitoring, Technische Universität München, Helmholtz Zentrum München, München, Germany; 3rd Medical Department, University Hospital Rechts der Isar, Technische Universität München, München, Germany.
authors.forename: Angela M
authors.initials: AM
authors.lastname: Krackhardt
authors.affiliation: Max-Delbrück-Centrum for Molecular Medicine (MDC) and Institute of Biology, Humboldt University Berlin, Berlin-Buch, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Uckert
authors.affiliation: Institute of Molecular Immunology, University Hospital Rechts der Isar, Technische Universität München, München, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Wohlleber
authors.affiliation: Clinical Cooperation Groups Antigen Specific Immunotherapy and Immune Monitoring, Technische Universität München, Helmholtz Zentrum München, München, Germany; Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, München, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Schiemann
authors.affiliation: Institute for Diabetes and Obesity, Helmholtz Zentrum München, Garching, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Stemmer
authors.affiliation: Institute of Virology, Technische Universität München, Helmholtz Zentrum München, München, Germany.
authors.forename: Mathias
authors.initials: M
authors.lastname: Heikenwälder
authors.affiliation: German Center for Infection Research (DZIF), Munich Site, Germany; Clinical Cooperation Groups Antigen Specific Immunotherapy and Immune Monitoring, Technische Universität München, Helmholtz Zentrum München, München, Germany; Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, München, Germany.
authors.forename: Dirk H
authors.initials: DH
authors.lastname: Busch
authors.affiliation: Department of Pediatrics, University Hospital Rechts der Isar, Technische Universität München, München, Germany.
authors.forename: Günther
authors.initials: G
authors.lastname: Richter
authors.affiliation: Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Mann
authors.affiliation: Institute of Virology, Technische Universität München, Helmholtz Zentrum München, München, Germany; German Center for Infection Research (DZIF), Munich Site, Germany; Clinical Cooperation Groups Antigen Specific Immunotherapy and Immune Monitoring, Technische Universität München, Helmholtz Zentrum München, München, Germany. Electronic address: protzer@tum.de.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Protzer
mesh_terms: D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism / Q000637:transplantation*; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D002470:Cell Survival; D018920:Coculture Techniques; D003602:Cytotoxicity, Immunologic; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism*; D005260:Female; D019672:Genes, T-Cell Receptor; D005818:Genetic Engineering / Q000379:methods*; D053673:Glypicans / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015789:HLA-A2 Antigen / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D016056:Immunodominant Epitopes; D016219:Immunotherapy, Adoptive / Q000379:methods*; D007371:Interferon-gamma / Q000276:immunology / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D008213:Lymphocyte Activation; D016513:Mice, SCID; D013997:Time Factors; D014162:Transfection; D023041:Xenograft Model Antitumor Assays
chemical_list: C105937:GPC3 protein, human; D053673:Glypicans; D015789:HLA-A2 Antigen; C573415:IFNG protein, human; D016056:Immunodominant Epitopes; D007371:Interferon-gamma
keywords: Cancer Immunotherapy; Immune Response; Liver Cancer; Tumor-Associated Antigens