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Enhancing the expression and function of an EBV-TCR on engineered T cells by combining Sc-TCR design with CRISPR editing to prevent mispairing

Cellular & Molecular Immunology volume 17, pages 1275–1277 (2020)Cite this article

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Adoptive transfer of antigen-specific T cells is an effective form of immunotherapy for cancers,1 and genetic modification of T cells provides a rapid way to produce the desired tumor specificity2 with improved T-cell survival3 and functions.4 Recent clinical trials have further demonstrated that TCR-engineered T cells can persist and mediate clinical benefits in late-stage cancer patients.5 However, mispairing between introduced and endogenous TCRs may form TCRs with unknown specificities and potentially cause off-target toxicity.6 To reduce the risk of mispairing, single-chain TCR (Sc-TCR) structures have been proposed,7,8 but these structures are frequently unstable, and there is no systematic comparison to answer if Sc-TCRs are superior to Dc-TCRs. As CRISPR editing has been reported to efficiently knockout endogenous TCR genes,9 we combined these two technologies in this study targeting a tumor-causing virus, EBV, to explore a refined strategy to reduce mispairing aimed at enhancing the expression and functions of EBV-TCR-engineered T cells.

To compare the expression of three different EBV-TCRs, we transduced our EBV-TCR-CD19 vectors into Jurkat cells. By gating on CD19+ T cells, differences in EBV-TCR construct-dependent expression were analyzed by anti-murine Cβ (mCβ) antibody and EBV-pentamer staining. As shown in Fig. 1F, in wild-type Jurkat cells, 68.9% of the EBV-Dc-TCR-CD19-transduced cells expressed mCβ, while only 30.7% of these cells stained positive with the EBV-pentamer. However, the percentage of mCβ+ cells rose moderately to 82.4 and 94.5% in the EBV-Sc-TCR-CD19- and EBV-SSc-TCR-CD19-transduced cells, and the percentage of EBV-pentamer+ cells markedly increased to 63.1 and 84.9%, respectively. These findings demonstrated dramatic improvements in antigen recognition, with the order EBV-SSc-TCR > EBV-Sc-TCR >> EBV-Dc-TCR in wild-type Jurkat cells. The expression of these EBV-TCR-CD19 vectors on TCR− Jurkat cells created by CRISPR knockout shared a pattern similar to that of the TCR+ Jurkat cells but with higher pentamer percentages, most likely due to reduced mispairing (Fig. 1F, lower panels).

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Acknowledgements

This work was supported by Blood Wise (H.J.S. and E.M.), the National Natural Science Foundation of China (NSFC-81972883: S.A.X. and Y.C.), the Social Development & Scientific Technology Key Project of Shaanxi Province (2016SF-079: S.A.X.) and the Scientific Technology Program & Innovation Fund of the Xi’An City Special Project (CXY1531WL12: S.A.X.). We would like to thank Professor Benny Chain for providing Jurkat cells for the study.

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  1. These authors contributed equally: Shao-An Xue, Yuan Chen

Authors and Affiliations

  1. Key Laboratory of Natural Product Development & Anticancer Innovative Drug Research in Qinling, Xi’An. Xi’An University, Xi’An, 710065, P. R. China

    Shao-An Xue, Yuan Chen, Bo Wang, Ke-Ke Chen, Feng-Qin He & Xiao-Xia Cheng

  2. Institute of Immunity & Transplantation, University College London Medical School, Royal Free Hospital, Rowland Hill Street, London, NW3 2PF, UK

    Shao-An Xue, Yuan Chen, Virad Kisan, Laura Scolamiero, Angelika Holler, Liquan Gao, Emma Morris & Hans J. Stauss

  3. Research Center for Immunotherapy (FZI), University Medical Center of Johannes Gutenberg University, Mainz, Germany

    Ralf-Holger Voss

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Contributions

S.A.X., Y.C., V.K., B.W., K.K.C., F.Q.H., X.X.C., L.S., A.H., and L.G. performed the research; S.A.X., Y.C., V.K., and B.W. analyzed the data; S.A.X., R.H.V., and H.J.S. designed the experiments; E.M. and H.J.S. supervised the study; S.A.X. and Y.C. wrote the paper; and R.H.V. and H.J.S. reviewed the paper.

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Correspondence to Shao-An Xue or Hans J. Stauss.

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Competing interests

S.A.X., R.H.V., and H.J.S. are inventors on patents describing the Sc-TCR technology. The remaining authors declare no competing interests.

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Xue, SA., Chen, Y., Voss, RH. et al. Enhancing the expression and function of an EBV-TCR on engineered T cells by combining Sc-TCR design with CRISPR editing to prevent mispairing. Cell Mol Immunol 17, 1275–1277 (2020). https://doi.org/10.1038/s41423-020-0396-9

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