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

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. [Display omitted] T cell product function and safety is directly affected by editing methodConventional TCR editing introduces variability through uncontrolled gene insertionTargeted TCR editing results in homogenous and physiological TCR expressionT cell product homogeneity enables predictable in vivo function for clinical use Müller et al. perform in-depth comparison of conventional TCR editing methods with CRISPR/Cas9-mediated orthotopic TCR replacement. Analysis of TCR transgene genomic insertion, mRNA transcription, and protein surface expression demonstrate that targeted TCR gene editing facilitates the production of homogenous TCR-transgenic T cell products with predictable in vivo functionality.