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Oncotarget. 2016 Apr 19;7(16):21199-221. doi: 10.18632/oncotarget.8385.

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.

Author information

1
Department of Hematology, Oncology, and Pneumology, University Cancer Center (UCT), University Medical Center (UMC) of Johannes Gutenberg University, Mainz, Germany.
2
Municipal Clinic Karlsruhe, Karlsruhe, Germany.
3
Department of Internal Medicine III, Hematology and Oncology, University Hospital, Regensburg, Germany.
4
Institute of Immunity and Transplantation, University College London, Royal Free Hospital, London, United Kingdom.
5
TRON-Translational Oncology at the University Medical Center of Johannes Gutenberg University gGmbH, Mainz, Germany.
6
Biopharmaceutical New Technologies (BioNTech) Corporation, Mainz, Germany.
7
Ludwig Institute for Cancer Research (LICR), Lausanne Branch, Epalinges, Switzerland.
8
TCMetrix, Epalinges, Switzerland.
9
Translational Tumor Immunology Group, Ludwig Center for Cancer Research of the University of Lausanne, Lausanne, Switzerland.
10
Research Center for Immunotherapy (FZI), University Medical Center of Johannes Gutenberg University, Mainz, Germany.
11
Center for Interventional Immunology, University of Regensburg, Regensburg, Germany.

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.

KEYWORDS:

Immune response; Immunity; Immunology and Microbiology Section; T-cell receptors; T-cells; gene therapy; human; tumor immunity

PMID:
27028870
PMCID:
PMC5008279
DOI:
10.18632/oncotarget.8385
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

R.-H. Voss, S. Thomas, M. Theobald, S.A. Xue, and H. Stauss are inventors on patents and patent applications, which cover parts of this article. U. Sahin, T. Omokoko, O. Yildiz are employees at BioNTech AG (Mainz, Germany), and U. Sahin is consultant for and stock owner of Ganymed Pharmaceuticals. H. Stauss is consultant for Cell Medica and Sofinnova and share holder at Cell Medica.

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