Adoptive T cell therapy (ACT) provides the means to specifically restore immunity against infections or cancer. ACT with donor-derived physiological T cells has shown its safety and efficacy in multiple different clinical settings. However, the availability, accessibility, and quality of donor-derived T cells intended for ACT are drastically restricting the applicability of this therapy to a few, carefully selected patients. The genetic introduction of a transgenic T cell receptor (TCR) can reprogram the specificity of a T cell. Therefore, TCR editing enables us to overcome the limitations of conventional ACT with donor-derived T cells since the desired TCR and the human leukocyte antigen (HLA)-matched T cell can be taken from different sources. So far, clinical trials with TCR-edited T cells showed promising results, yet therapy responses were only moderate and temporary. In this thesis, we elaborate on the complexity of TCR editing and how it might contribute to the observed low clinical response rates. For that, we set out to systematically investigate the two most important aspects of TCR editing: First, the isolation and identification of optimal TCRs for clinical use. Second, the functional consequences introduced by the editing process itself and how it can be improved to produce better TCR-edited T cell products. Particularly the latter aspect was so far little acknowledged by the scientific community. Here, we developed a platform for reliable isolation of paired TCR α- and β-chains facilitating the generation of a comprehensive TCR library consisting of 261 unique virus-specific TCRαβ sequences. Subsequently, we used this TCR library for the development of a cellular platform for functional testing and identification of suitable TCRs for clinical use, as well as for the systematic investigation of different TCR editing methods. The current gold standard for investigation of TCR-intrinsic characteristics is transgenic re-expression in primary T cells. However, this is not only a very laborious and costly process but also faces a high degree of variability due to factors such as T cell activation status, phenotype, or donor origin. In contrast, usage of an immortalized cell line offers more standardized conditions in conjunction with a drastically decreased workload. Here, we SUMMARY 2 developed a Jurkat-based test system for high-throughput and reliable TCR characterization. Most importantly, TCR-intrinsic features such as antigen-HLA specificity and functional avidity determined with this cell line closely paralleled measurements in primary T cells, therefore enabling highly standardized and unbiased assessment of TCR functionality. Furthermore, we investigated the method of TCR editing as a potential factor introducing bias in transgenic TCR function. By in-depth systematic investigation of conventional TCR editing in comparison to novel clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated orthotopic TCR replacement (OTR), we identified substantial functional differences originating from the different editing methods. First, we highlight the importance of full endogenous TCR α- and β-chain knock-out (KO) for the production of safe and functional T cell products. Second, we show that transgenic TCR placement under the endogenous promoter is feasible and results in more physiological TCR regulation upon antigenic stimulation. Third, we demonstrate that random transgene integration and variable copy numbers introduce functional variability, whereas, targeted TCR integration via OTR results in highly defined, homogenous T cell products. Fourth, by comparing OTR T cell products to clinically relevant low copy number conventionally produced T cells, we observed increased TCR surface expression and functional avidity after OTR. Fifth, we provide evidence that OTR T cell products - through a more homogenous TCR surface expression - are more predictable in terms of in vivo functionality. In summary, we developed a test system that proved to be a powerful tool for high-throughput and reliable screening of TCRs intended for clinical use and - more general - for investigation of TCR function and biology. Furthermore, we demonstrate that TCR editing via OTR facilitates the production of highly defined, homogenous TCR-redirected T cell products with an enhanced safety profile, physiologic TCR regulation as well as increased and more predictable functionality in comparison to conventional editing. These novel tools and findings are of utmost importance for the production of next-generation TCR-transgenic T cells and will probably contribute to better and more prolonged therapy responses