CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells

Adoptive transfer of T cells genetically modified to express a cancer-specific T-cell receptor (TCR) has shown significant therapeutic potential for both hematological and solid tumors. However, a major issue of transducing T cells with a transgenic TCR is the preexisting expression of TCRs in the recipient cells. These endogenous TCRs compete with the transgenic TCR for surface expression and allow mixed dimer formation. Mixed dimers, formed by mispairing between the endogenous and transgenic TCRs, may harbor autoreactive specificities. To circumvent these problems, we designed a system where the endogenous TCR-β is knocked out from the recipient cells using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) technology, simultaneously with transduction with a cancer-reactive receptor of choice. This TCR replacement strategy resulted in markedly increased surface expression of transgenic αβ and γδ TCRs, which in turn translated to a stronger, and more polyfunctional, response of engineered T cells to their target cancer cell lines. Additionally, the TCR-plus-CRISPR–modified T cells were up to a thousandfold more sensitive to antigen than standard TCR-transduced T cells or conventional model proxy systems used for studying TCR activity. Finally, transduction with a pan-cancer–reactive γδ TCR used in conjunction with CRISPR/Cas9 knockout of the endogenous αβ TCR resulted in more efficient redirection of CD4+ and CD8+ T cells against a panel of established blood cancers and primary, patient-derived B-cell acute lymphoblastic leukemia blasts compared with standard TCR transfer. Our results suggest that TCR transfer combined with genome editing could lead to new, improved generations of cancer immunotherapies. •Endogenous TCR knockout increases the expression and functional activity of simultaneously transduced TCR (TCR replacement).•TCR replacement results in superior targeting of hematological malignancies by T cells transduced with a non–HLA-restricted γδ TCR.