It's recommended to maintain both hands on the wheel when driving a CAR. Eyquem et al. used the CRISPR/Cas9 technology to purposely put a CD19-specific CAR coding gene at the TRAC locus in order to increase control in CAR T cell engineering. The purpose of this genetic change was to knock off existing TCR while introducing the CD19-specific CAR and putting it under the dynamic regulatory control of the endogenous promoter, which would otherwise limit TCR expression. In mouse models, this alteration revealed surprising benefits that contributed to maintained T cell activity and better control of pre-B ALL.
TRAC-CAR T cells were used in both in vitro and in vivo investigations:
CARs were expressed uniformly and consistently at an appropriate baseline level, resulting in increased antitumor activity over control CAR T cells with higher or lower CAR expression.
Following single or repeated antigen exposure, the CAR demonstrated reduced tonic activation signaling and established efficient internalization and balanced re-expression, resembling the normal process of antigen-induced TCR internalization and reexpression.
They retained a naive/central memory phenotype while delaying effector T cell differentiation (loss of CD62L) and exhaustion (increase in LAG3, TIM3, and PD-1), making them better effector cells.
This work evaluated numerous versions of CRISPR/Cas9-produced, CD19-specific CAR T Cells at the TRAC and B2M loci under the control of different promoters as controls and comparisons. The CRISPR/Cas9-produced cells were also compared to traditional, retrovirally-produced CD19-specific CAR T cells with and without a TCR deletion.
TRAC-CAR T cells ultimately surpassed all of their competitors. Surprisingly, even though some other CAR T cells in comparison groups expressed more cell surface CARs, TRAC-CAR T cells were more effective, indicating that just boosting CAR expression is not the way to go. According to the findings, the key to antitumor activity is the sensitive regulation of CAR expression that resembles natural TCR expression.
Aside from their increased potency, CRISPR/Cas9 modified T cells have a better safety profile since the lack of TCRs minimizes the risk of TCR-induced autoimmunity and alloreactivity, and the predictable insertion of the CAR coding sequence reduces the risk of insertional oncogenesis.
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