Description

Targeting CARs to the TRAC Locus Enhances T Cell Potency

M. Sadelain

Memorial Sloan-Kettering Cancer Center, New York, NY, USA

Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumor rejection. The most successful CARs used to date are those targeting CD19, which offer the prospect of complete remissions in patients with chemorefractory/relapsed B cell malignancies. CARs are typically transduced into patient T cells using γretroviral or other randomly-integrating retroviral vectors (RVs), which may result in variegated CAR expression and transcriptional silencing. Recent advances in genome editing enable efficient sequence-specific modifications in human primary cells, including site-specific transgene integration into the CCR5 and AAVS1 loci in T lymphocytes. Using CRISPR/Cas9 to edit human T cells, we have established conditions yielding efficient target gene disruption and CAR insertion in a single step. We have found that directing a CD19 CAR to the human T cell receptor (TCR) alpha chain (TRAC) locus not only results in efficient and uniform CAR expression in human peripheral blood T cells, but, remarkably, also enhances T cell potency, vastly outperforming that of conventionally generated CAR T cells. We further show that CAR gene expression under the control of the TCR alpha promoter minimizes tonic signaling and allows effective regulation of CAR expression. These findings provide important insights into CAR immunobiology and highlight the potential of CRISPR/Cas9 genome editing to advance immunotherapies.