Simultaneous deletion of endogenous TCRαβ for TCR gene therapy creates an improved and safe cellular therapeutic. Creating an improved T-cell therapeutic.

Abstract 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. Knock-out (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 4 clinically relevant TCRs. Firstly, 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 which contained structural modifications to encourage preferential pairing. Finally, increased expression of tgTCR in edited T-cell populations allowed for increased recognition of antigen expressing tumour targets and prolonged control of tumour 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.