Simultaneous Deletion of Endogenous TCRαβ for TCR Gene Therapy Creates an Improved and Safe Cellular Therapeutic

Generation of an optimal T cell therapeutic expressing high frequencies of transgenic T cell receptor (tgTCR) is essential for improving TCR gene therapy. Upon TCR gene transfer, presence of endogenous TCRαβ reduces expression of tgTCR due to TCR mixed-dimer formation and competition for binding CD3. Knockout (KO) of endogenous TCRαβ was recently achieved using CRISPR/Cas9 editing of the TRAC or TRBC loci, resulting in increased expression and function of tgTCR. Here, we adopt this approach into current protocols for generating T cell populations expressing tgTCR to validate this strategy in the context of four clinically relevant TCRs. First, simultaneous editing of TRAC and TRBC loci was reproducible and resulted in high double KO efficiencies in bulk CD8 T cells. Next, tgTCR expression was significantly higher in double TRAC/BC KO conditions for all TCRs tested, including those that contained structural modifications to encourage preferential pairing. Finally, increased expression of tgTCR in edited T cell populations allowed for increased recognition of antigen expressing tumor targets and prolonged control of tumor outgrowth in a preclinical model of multiple myeloma. In conclusion, CRISPR/Cas9-mediated KO of both endogenous TCRαβ chains can be incorporated in current T cell production protocols and is preferential to ensure an improved and safe clinical therapeutic. Morton et al. describe the application of CRISPR/CAS9 genome editing in standard T cell production protocols for TCR gene therapy. Simultaneous editing of the TRAC/TRBC loci produced highly efficient knockout of endogenous TCRαβ. This resulted in increased frequency of T cells expressing transgenic TCR and ultimately an improved cellular therapeutic.