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. 2022:2521:85-93.
doi: 10.1007/978-1-0716-2441-8_5.

TCR and CAR Engineering of Primary Human T Cells

Inan Edes  1 , Julian Clauss  2 , Rainer Stahn  2 , Alberto Sada Japp  2 , Felix K M Lorenz  2
Affiliations

TCR and CAR Engineering of Primary Human T Cells

Inan Edes et al. Methods Mol Biol. 2022.

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.

Keywords: Chimeric antigen receptor; Gamma-retrovirus; Genetic engineering; Primary human T cells; Retroviral vector; T-cell receptor; Transduction; Transfection.

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