When it comes to controlling CARs it’s best to keep both hands on the wheel. In an effort to increase control in CAR T cell engineering, Eyquem et al. have utilized the CRISPR/Cas9 method to deliberately place a CD19-specific CAR coding sequence at the TRAC locus. The goal of this genetic modification was to knock out existing T cell receptors (TCR), while knocking in the CD19-specific CAR and placing it under the dynamic regulatory control of the endogenous promoter that would otherwise moderate TCR expression. This modification revealed unanticipated benefits that contributed to sustained T cell function and better control of pre-B ALL in mouse models.
Across in vitro and in vivo experiments, TRAC-CAR T cells:
expressed CARs homogeneously and consistently at an optimal baseline level that offered improved antitumor efficacy over control CAR T cells with higher or lower CAR expression.
showed reduced tonic activation signaling and established effective internalization and balanced re-expression of the CAR following single or repeated antigen exposure, reminiscent of the natural process of antigen-induced TCR internalization and reexpression.
retained a naive/central memory phenotype and delayed effector T cell differentiation (loss of CD62L) and exhaustion (buildup of LAG3, TIM3, and PD-1), making them better effector cells.
For controls and comparisons, this study tested several variations of CRISPR/Cas9-produced, CD19-specific CAR T Cells at the TRAC and B2M loci, under the control of various promoters. It also pitted the CRISPR/Cas9-produced cells against conventional, retrovirally-produced CD19-specific CAR T cells with and without a TCR knockout.
Ultimately, TRAC-CAR T cells outperformed all of their counterparts. Surprisingly, researchers noted that even though some other CAR T cells in comparative groups expressed more cell surface CARs, the TRAC-CAR T cells were more effective, suggesting that simply maximizing CAR expression is not the way to go. The data suggests that the sensitive regulation of CAR expression that mimics natural TCR expression is the key to antitumor efficacy.
In addition to their higher potency, CRISPR/Cas9 modified T cells offer an improved safety profile, as the lack of TCRs reduces the risk of TCR-induced autoimmunity and alloreactivity, and the predictable insertion of the CAR coding sequence reduces the risk for insertional oncogenesis.
by Lauren Hitchings
