Targeted T cell receptor gene editing provides predictable T cell product function for immunotherapy

Thomas R Müller , Sebastian Jarosch 1, Monika Hammel , Justin Leube , Simon Grassmann  , Bettina Bernard 1, Manuel Effenberger , Immanuel Andrä , M Zeeshan Chaudhry , Theresa Käuferle , Antje Malo , Luka Cicin-Sain , Peter Steinberger , Tobias Feuchtinger , Ulrike Protzer , Kathrin Schumann , Michael Neuenhahn , Kilian Schober , Dirk H Busch 
Source: Cell Rep Med
Publication Date: (2021)
Issue: 2(8): 100374
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
CD4+, human
Species: human
Tissue Origin: blood
CD8+, human
Species: human
Tissue Origin: blood
4D-Nucleofector® 96-well Systems
4D-Nucleofector® X-Unit

CRISPR/Cas9-mediated KO and KI
Either column selected CD4+ and CD8+ T cells mixed in a 1:1 ratio or bulk PBMCs were activated for two days in RPMI with CD3/CD28 Expamer (provided by Juno Therapeutics, activation with plate-bound aCD3/aCD28 as described above is also possible and gives similar results), 300 IU ml-1 recombinant human IL-2, 5 ng ml-1 recombinant human IL-7 (Peprotech, reference #200-07) and 5 ng ml-1 IL-15. Expamer stimulus was removed by incubation with 1 mM D-biotin (Sigma, reference #D1411-1G). 1x106 cells were electroporated (pulse code EH100) with Cas9 ribonucleoprotein and DNA templates in 20 ml Nucleofector Solution P3 (Lonza, reference #V4SP-3096) with a 4D Nucleofector X unit (Lonza). After electroporation, cells were cultured in RPMI supplemented with 180 IU ml-1 IL-2 until a first FACS analysis on day five after editing.


Adoptive transfer of T cells expressing a transgenic T cell receptor (TCR) has the potential to revolutionize immunotherapy of infectious diseases and cancer. However, the generation of defined TCR-transgenic T cell medicinal products with predictable in vivo function still poses a major challenge and limits broader and more successful application of this "living drug." Here, by studying 51 different TCRs, we show that conventional genetic engineering by viral transduction leads to variable TCR expression and functionality as a result of variable transgene copy numbers and untargeted transgene integration. In contrast, CRISPR/Cas9-mediated TCR replacement enables defined, targeted TCR transgene insertion into the TCR gene locus. Thereby, T cell products display more homogeneous TCR expression similar to physiological T cells. Importantly, increased T cell product homogeneity after targeted TCR gene editing correlates with predictable in vivo T cell responses, which represents a crucial aspect for clinical application in adoptive T cell immunotherapy.