Research Papers: Immunology:
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
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Diana Knies1,8,*, Sebastian Klobuch1,9,*, Shao-An Xue2,*, Matthias Birtel3, Hakim Echchannaoui1, Oezlem Yildiz4, Tana Omokoko4, Philippe Guillaume5,10, Pedro Romero6, Hans Stauss2, Ugur Sahin3,4,7, Wolfgang Herr1,9,11, Matthias Theobald1,**, Simone Thomas,1,9,11,** and Ralf-Holger Voss1,3,7**
1 Department of Hematology, Oncology, and Pneumology, University Cancer Center (UCT), University Medical Center (UMC) of Johannes Gutenberg University, Mainz, Germany
2 Institute of Immunity and Transplantation, University College London, Royal Free Hospital, London, United Kingdom
3 TRON-Translational Oncology at the University Medical Center of Johannes Gutenberg University gGmbH, Mainz, Germany
4 Biopharmaceutical New Technologies (BioNTech) Corporation, Mainz, Germany
5 Ludwig Institute for Cancer Research (LICR), Lausanne Branch, Epalinges, Switzerland
6 Translational Tumor Immunology Group, Ludwig Center for Cancer Research of the University of Lausanne, Lausanne, Switzerland
7 Research Center for Immunotherapy (FZI), University Medical Center of Johannes Gutenberg University, Mainz, Germany
8 Municipal Clinic Karlsruhe, Karlsruhe, Germany
9 Department of Internal Medicine III, Hematology and Oncology, University Hospital, Regensburg, Germany
10 TCMetrix, Epalinges, Switzerland
11 Center for Interventional Immunology, University of Regensburg, Regensburg, Germany
* Shared first authorship
** Shared senior authorship
Ralf-Holger Voss, email:
Simone Thomas, email:
Matthias Theobald, email:
Keywords: human, tumor immunity, T-cells, T-cell receptors, gene therapy, Immunology and Microbiology Section, Immune response, Immunity
Received: September 09, 2015 Accepted: March 10, 2016 Published: March 26, 2016
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.
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