Justin Eyquem
Postdoctoral Fellow, Michel Sadelain’s Lab
Center for Cell Engineering - MSKCC, USA
Seminar Information
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 γ-retroviral or other randomly-integrating retroviral vectors (RVs), which may result in variegated CAR expression and transcriptional silencing. Therefore, a site-specific CAR gene integration approach is expected to improve CAR T-cell performance.
Using CRISPR/Cas9 to edit human T cells, we established conditions yielding in highly efficient target gene disruption and CAR insertion in a single step. We targeted the CAR transgene into 9 different loci/promoter combinations, including three strategies that placed CAR expression under the control of the endogenous TCR alpha (TRAC), TCR beta and B2M promoters, which resulted in a palette of different expression levels. We have found that directing a 1928z CAR to the TRAC locus not only results in uniform and predictable 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 demonstrated that CARs are internalized and degraded upon antigen interaction and showed that CAR cell-surface replenishment is transcriptionally controlled. The optimal cell-surface CAR expression afforded by the TRAC promoter offsets tonic signaling during ex vivo expansion and slows undesirable differentiation and exhaustion in vivo. These results greatly improved the CAR biology understanding by establishing the first link between CAR expression and CAR T cell function. We will also present that TRAC-targeting benefits goes beyond CD28-based CARs and also improves 4-1BB-based CARs as well as tumor-specific recombinant TCR. Finally, we will report our progress in optimizing and scaling up the generation of TRAC-CARs in GMP conditions for clinical application.
Justin Eyquem received his PhD from the University of Paris-Diderot in collaboration with the biotech company Cellectis. During his PhD, he participated to the development of genome editing tools such as Meganuclease or TALEN in primary human cells and notably identified genomic location for safe integration of therapeutic genes. In 2014, he joined Michel Sadelain’s lab at the MSKCC, who is a pioneer in the Chimeric Antigen Receptor (CAR) T cells field. He is now using CRISPR/Cas9 to engineer CAR T cells and his recent work showed how precise genome editing can augment CAR T cell efficacy, advance CAR immuno-biology and facilitate T cell manufacturing.