Transducing T cells with αβ or γδ TCRs specific for target cancer antigens while simultaneously knocking out the endogenous TCR-β locus using CRISPR/Cas9 increased the surface expression of transgenic TCRs, led to higher sensitivity to cognate antigen, and demonstrated stronger, polyfunctional response in vitro and ex vivo to various blood cancers compared to standard TCR transfer. γδ TCRs recognizing pan-cancer targets are attractive for adoptive cell therapy, as they do not exhibit MHC restriction.
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 pre-existing 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-beta is knocked out from the recipient cells using CRISPR/Cas9 technology, simultaneously with transduction with a cancer-reactive receptor of choice. This TCR replacement strategy resulted in markedly increased surface expression of transgenic alphabeta and gammadelta 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+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 gammadelta TCR used in conjunction with CRISPR/Cas9 knockout of the endogenous alphabeta TCR resulted in more efficient redirection of CD4+ and CD8+ T-cells against a panel of established blood cancers and primary, patient-derived B acute lymphoblastic leukemia blasts compared to standard TCR transfer. Our results suggest that TCR transfer combined with genome editing could lead to new improved generations of cancer immunotherapies.