Abstract
The efficient expression of T-cell receptors (TCRs) or chimeric antigen receptors (CARs) in primary human T cells is crucial for preclinical testing of receptor properties for adoptive T-cell therapies. Multiple streams of technological platforms have been developed in the recent decades to genetically modify primary T cells including nonviral platforms such as transposon-based systems (PiggyBac, Sleeping Beauty), TALENs, or CRISPR-Cas9). The production of CAR- or TCR-encoding retroviral vectors, however, is still the most commonly used technique both in preclinical as well as in clinical settings.
In this chapter we describe a comprehensive 12-day protocol for (a) generating high-titered gamma-retroviral vector particles containing the transgene of interest (e.g., TCR , CAR ), (b) the isolation, activation and rapid expansion of primary T cells and (c) the stable genetic engineering of these T cells with the transgene for subsequent characterization.
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References
Zhang J, Wang L (2019) The emerging world of TCR-T cell trials against cancer: a systematic review. Technol Cancer Res Treat 18:153303381983106. https://doi.org/10.1177/1533033819831068
Feins S, Kong W, Williams EF et al (2019) An introduction to chimeric antigen receptor (CAR) T-cell immunotherapy for human cancer. Am J Hematol 94:S3–S9. https://doi.org/10.1002/ajh.25418
Bulcha JT, Wang Y, Ma H et al (2021) Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther 6:53. https://doi.org/10.1038/s41392-021-00487-6
Puig-Saus C, Ribas A (2019) Gene editing: towards the third generation of adoptive T-cell transfer therapies. Immuno Oncol Technol 1:19–26. https://doi.org/10.1016/J.IOTECH.2019.06.001
Clauss J, Obenaus M, Miskey C et al (2018) Efficient non-viral T-cell engineering by sleeping beauty Minicircles diminishing DNA toxicity and miRNAs silencing the endogenous T-cell receptors. Hum Gene Ther 29:569–584. https://doi.org/10.1089/hum.2017.136
Engels B, Cam H, Schüler T et al (2003) Retroviral vectors for high-level transgene expression in T lymphocytes. Hum Gene Ther 14:1155–1168. https://doi.org/10.1089/104303403322167993
Lorenz FKM, Ellinger C, Kieback E et al (2017) Unbiased identification of T-cell receptors targeting immunodominant peptide–MHC complexes for T-cell receptor immunotherapy. Hum Gene Ther 28(12):1158–1168. https://doi.org/10.1089/hum.2017.122
Miliotou AN, Papadopoulou LC (2018) CAR T-cell therapy: a new era in cancer immunotherapy. Curr Pharm Biotechnol 19:5–18. https://doi.org/10.2174/1389201019666180418095526
Zheng Z, Chinnasamy N, Morgan RA (2012) Protein L: a novel reagent for the detection of chimeric antigen receptor (CAR) expression by flow cytometry. J Transl Med 10:29. https://doi.org/10.1186/1479-5876-10-29
Acknowledgments
We would like to thank Wolfgang Uckert for pioneering previous work on this topic as well as Michaela Naschke, Christin Hesse, and Nicole Nussbaum for excellent technical support.
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Edes, I., Clauss, J., Stahn, R., Japp, A.S., Lorenz, F.K.M. (2022). TCR and CAR Engineering of Primary Human T Cells. In: Walther, W. (eds) Gene Therapy of Cancer. Methods in Molecular Biology, vol 2521. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2441-8_5
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DOI: https://doi.org/10.1007/978-1-0716-2441-8_5
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