title: An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells.
abstract: Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/β T-cell receptors (TCRα/β). However, potential mispairing of introduced TCRα/β-chains with endogenous β/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/β-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vβ-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vβ. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive T-cells.
authors.affiliation: Department of Hematology, Oncology, and Pneumology, University Cancer Center (UCT), University Medical Center (UMC) of Johannes Gutenberg University, Mainz, Germany.|Municipal Clinic Karlsruhe, Karlsruhe, Germany.
authors.forename: Diana
authors.initials: D
authors.lastname: Knies
authors.affiliation: Department of Hematology, Oncology, and Pneumology, University Cancer Center (UCT), University Medical Center (UMC) of Johannes Gutenberg University, Mainz, Germany.|Department of Internal Medicine III, Hematology and Oncology, University Hospital, Regensburg, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Klobuch
authors.affiliation: Institute of Immunity and Transplantation, University College London, Royal Free Hospital, London, United Kingdom.
authors.forename: Shao-An
authors.initials: SA
authors.lastname: Xue
authors.affiliation: TRON-Translational Oncology at the University Medical Center of Johannes Gutenberg University gGmbH, Mainz, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Birtel
authors.affiliation: Department of Hematology, Oncology, and Pneumology, University Cancer Center (UCT), University Medical Center (UMC) of Johannes Gutenberg University, Mainz, Germany.
authors.forename: Hakim
authors.initials: H
authors.lastname: Echchannaoui
authors.affiliation: Biopharmaceutical New Technologies (BioNTech) Corporation, Mainz, Germany.
authors.forename: Oezlem
authors.initials: O
authors.lastname: Yildiz
authors.affiliation: Biopharmaceutical New Technologies (BioNTech) Corporation, Mainz, Germany.
authors.forename: Tana
authors.initials: T
authors.lastname: Omokoko
authors.affiliation: Ludwig Institute for Cancer Research (LICR), Lausanne Branch, Epalinges, Switzerland.|TCMetrix, Epalinges, Switzerland.
authors.forename: Philippe
authors.initials: P
authors.lastname: Guillaume
authors.affiliation: Translational Tumor Immunology Group, Ludwig Center for Cancer Research of the University of Lausanne, Lausanne, Switzerland.
authors.forename: Pedro
authors.initials: P
authors.lastname: Romero
authors.affiliation: Institute of Immunity and Transplantation, University College London, Royal Free Hospital, London, United Kingdom.
authors.forename: Hans
authors.initials: H
authors.lastname: Stauss
authors.affiliation: TRON-Translational Oncology at the University Medical Center of Johannes Gutenberg University gGmbH, Mainz, Germany.|Biopharmaceutical New Technologies (BioNTech) Corporation, Mainz, Germany.|Research Center for Immunotherapy (FZI), University Medical Center of Johannes Gutenberg University, Mainz, Germany.
authors.forename: Ugur
authors.initials: U
authors.lastname: Sahin
authors.affiliation: Department of Hematology, Oncology, and Pneumology, University Cancer Center (UCT), University Medical Center (UMC) of Johannes Gutenberg University, Mainz, Germany.|Department of Internal Medicine III, Hematology and Oncology, University Hospital, Regensburg, Germany.|Center for Interventional Immunology, University of Regensburg, Regensburg, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Herr
authors.affiliation: Department of Hematology, Oncology, and Pneumology, University Cancer Center (UCT), University Medical Center (UMC) of Johannes Gutenberg University, Mainz, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Theobald
authors.affiliation: Department of Hematology, Oncology, and Pneumology, University Cancer Center (UCT), University Medical Center (UMC) of Johannes Gutenberg University, Mainz, Germany.|Department of Internal Medicine III, Hematology and Oncology, University Hospital, Regensburg, Germany.|Center for Interventional Immunology, University of Regensburg, Regensburg, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Thomas
authors.affiliation: Department of Hematology, Oncology, and Pneumology, University Cancer Center (UCT), University Medical Center (UMC) of Johannes Gutenberg University, Mainz, Germany.|TRON-Translational Oncology at the University Medical Center of Johannes Gutenberg University gGmbH, Mainz, Germany.|Research Center for Immunotherapy (FZI), University Medical Center of Johannes Gutenberg University, Mainz, Germany.
authors.forename: Ralf-Holger
authors.initials: RH
authors.lastname: Voss
mesh_terms: D019264:Adoptive Transfer; D000818:Animals; D014408:Biomarkers, Tumor; D017252:CD3 Complex / Q000235:genetics / Q000276:immunology*; D002462:Cell Membrane; D049109:Cell Proliferation; D006801:Humans; D016219:Immunotherapy, Adoptive; D015458:Leukemia, T-Cell / Q000235:genetics / Q000276:immunology* / Q000473:pathology; D051379:Mice; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics / Q000276:immunology*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D014408:Biomarkers, Tumor; D017252:CD3 Complex; D016693:Receptors, Antigen, T-Cell, alpha-beta
keywords: Immune response; Immunity; Immunology and Microbiology Section; T-cell receptors; T-cells; gene therapy; human; tumor immunity