Orthotopic T cell receptor (TCR) replacement: A new concept for engineering TCR edited T cells for adoptive immunotherapy

Adoptive transfer of T cells with transgenic antigen-specific T cell receptors (TCRs) has the potential to therapeutic options of infectious diseases and cancer. However, generation of defined T cell products with reliable functionality still poses a major challenge. Conventionally, genetic engineering by viral transduction is used, which leads to un-targeted transgene integration and interference through the endogenous TCR. Furthermore, careful titration of viral transduction doses is necessary to limit potential off-target effects through multiple integrations. We recently reported that CRISPR/Cas9-mediated orthotopic TCR replacement (OTR) offers the possibility of controlled transgene integration and simultaneous removal of the endogenous TCR. By genetic engineering of many different antigen-specific TCRs, we now show that knock-out of the endogenous TCR and targeted insertion through OTR independently contribute to highly defined transgenic TCR expression. In comparison to low-dose virally transduced TCR transgenic T cells, enhanced and more defined TCR expression through OTR leads to higher functionality and to a less variable T cell responses. Therefore, OTR seems indeed advantageous for the generation of more predictable TCR-transgenic T cell products.