Removal of Endogenous TCR Chains for Tcr-based Immunotherapies

Abstract Text: The aim of any cancer therapy is to target and kill cancer cells without affecting normal cells. Tumor neoantigens provide targets for therapies based on adoptive transfer of T cell expressing specific TCRs. However, a major issue in transducing T cells with a transgenic TCR is the preexisting expression of TCRs in the recipient lymphocytes. These endogenous TCRs compete with the transgenic TCR for surface expression and allow mixed dimer formation, potentially causing autoreactivity. To circumvent these problems, we developed a novel CRISPR-based method that successfully removes both endogenous TCR α and β chains without affecting the transgenic TCRs. We show that the combination of our guide RNAs successfully removes the endogenous TCRαβ chains with an efficiency of more than 80% in primary T cells. We also demonstrate that this CRISPR strategy results in markedly increased surface expression of the correct transgenic TCR, which positively correlates with a robust killing activity in vitro. We validated the beneficial effect of endogenous TCRαβ knockout in a human immune system mouse model using a clinically relevant TCR targeting a melanoma antigen recognized by T cells (MART-1). In preliminary studies, removal of the endogenous TCR limited the graft-versus-host response without compromising effector function. Ongoing studies will determine whether GvHD inhibition is complete. Together, our studies provide evidence that the simultaneous removal of both endogenous TCRαβ chains improves anti-tumor performance in a preclinical model for human metastatic melanoma. If confirmed, the reduced risk of GvHD will be highly relevant for “off-the-shelf” allogeneic cell therapy.