title: Transgenic antigen-specific, HLA-A*02:01-allo-restricted cytotoxic T cells recognize tumor-associated target antigen STEAP1 with high specificity.
abstract: Pediatric cancers, including Ewing sarcoma (ES), are only weakly immunogenic and the tumor-patients' immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology, targetable tumor-associated antigens (TAA) are identified and exploited for engineered T-cell therapy. Here, the specific recognition and lytic potential of transgenic allo-restricted CD8(+) T cells, directed against the ES-associated antigen 6-transmembrane epithelial antigen of the prostate 1 (STEAP1), was examined. Following repetitive STEAP1(130) peptide-driven stimulations with HLA-A*02:01(+) dendritic cells (DC), allo-restricted HLA-A*02:01(-) CD8(+) T cells were sorted with HLA-A*02:01/peptide multimers and expanded by limiting dilution. After functional analysis of suitable T cell clones via ELISpot, flow cytometry and xCELLigence assay, T cell receptors' (TCR) α- and β-chains were identified, cloned into retroviral vectors, codon optimized, transfected into HLA-A*02:01(-) primary T cell populations and tested again for specificity and lytic capacity in vitro and in a Rag2(-/-)γc(-/-) mouse model. Initially generated transgenic T cells specifically recognized STEAP1(130)-pulsed or transfected cells in the context of HLA-A*02:01 with minimal cross-reactivity as determined by specific interferon-γ (IFNγ) release, lysed cells and inhibited growth of HLA-A*02:01(+) ES lines more effectively than HLA-A*02:01(-) ES lines. In vivo tumor growth was inhibited more effectively with transgenic STEAP1(130)-specific T cells than with unspecific T cells. Our results identify TCRs capable of recognizing and inhibiting growth of STEAP1-expressing HLA-A*02:01(+) ES cells in vitro and in vivo in a highly restricted manner. As STEAP1 is overexpressed in a wide variety of cancers, we anticipate these STEAP1-specific TCRs to be potentially useful for immunotherapy of other STEAP1-expressing tumors.
authors.affiliation: Children's Cancer Research Center and Department of Pediatrics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany and Comprehensive Cancer Center Munich (CCCM) , Munich, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Schirmer
authors.affiliation: Laboratory for Pediatric Sarcoma Biology, Institute of Pathology of the LMU Munich , Munich, Germany.
authors.forename: Thomas G P
authors.initials: TG
authors.lastname: Grünewald
authors.affiliation: Medical Department III, Hematology and Oncology , Munich, Germany.
authors.forename: Richard
authors.initials: R
authors.lastname: Klar
authors.affiliation: Children's Cancer Research Center and Department of Pediatrics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany and Comprehensive Cancer Center Munich (CCCM) , Munich, Germany.
authors.forename: Oxana
authors.initials: O
authors.lastname: Schmidt
authors.affiliation: Institute of Molecular Immunology/Experimental Oncology, Klinikum rechts der Isar, Technische Universität München , Munich, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Wohlleber
authors.affiliation: Laboratory for Pediatric Sarcoma Biology, Institute of Pathology of the LMU Munich , Munich, Germany.
authors.forename: Rebeca Alba
authors.initials: RA
authors.lastname: Rubío
authors.affiliation: Max Delbrück Center for Molecular Medicine , Berlin, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Uckert
authors.affiliation: Children's Cancer Research Center and Department of Pediatrics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany and Comprehensive Cancer Center Munich (CCCM) , Munich, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Thiel
authors.affiliation: Institute of Virology, Technische Universität München, Helmholtz Zentrum München , Munich, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Bohne
authors.affiliation: Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München , Munich, Germany.
authors.forename: Dirk H
authors.initials: DH
authors.lastname: Busch
authors.affiliation: Medical Department III, Hematology and Oncology , Munich, Germany.
authors.forename: Angela M
authors.initials: AM
authors.lastname: Krackhardt
authors.affiliation: Children's Cancer Research Center and Department of Pediatrics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany and Comprehensive Cancer Center Munich (CCCM) , Munich, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Burdach
authors.affiliation: Children's Cancer Research Center and Department of Pediatrics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany and Comprehensive Cancer Center Munich (CCCM) , Munich, Germany.
authors.forename: Günther H S
authors.initials: GH
authors.lastname: Richter
keywords: Allo-restricted T cells; Ewing sarcoma; STEAP1; TCR-transgenic T cells