CD38-Specific CAR Integrated into CD38 Locus Driven by Different Promoters Causes Distinct Antitumor Activities of T and NK Cells

The robust and stable expression of CD38 in T-cell acute lymphoblastic leukemia (T-ALL) blasts makes CD38 chimeric antigen receptor (CAR)-T/natural killer (NK) a potential therapy for T-ALL. However, CD38 expression in normal T/NK cells causes fratricide of CD38 CAR-T/NK cells. Here a 2-in-1 gene editing strategy is developed to generate fratricide-resistant locus-specific CAR-T/NK cells. CD38-specific CAR is integrated into the disrupted CD38 locus by CRISPR/Cas9, and CAR is placed under the control of either endogenous CD38 promoter (CD38(KO/KI)) or exogenous EF1 & alpha; promoter (CD38(KO/KI)EF1 & alpha;). CD38 knockout reduces fratricide and allows the expansion of CAR-T cells. Meanwhile, CD38(KO/KI)EF1 & alpha; results in higher CAR expression than CD38(KO/KI) in both CAR-T and CAR-NK cells. In a mouse T-ALL model, CD38(KO/KI)EF1 & alpha; CAR-T cells eradicate tumors better than CD38(KO/KI) CAR-T cells. Surprisingly, CD38(KO/KI) CAR-NK cells show superior tumor control than CD38(KO/KI)EF1 & alpha; CAR-NK cells. Further investigation reveals that endogenous regulatory elements in NK cells lead to higher expression of CD38 CAR than in T cells, and the expression levels of CAR affect the therapeutic outcome of CAR-T and CAR-NK cells differently. Therefore, these results support the efficacy of CD38 CAR-T/NK against T-ALL and demonstrate that the 2-in-1 strategy can resolve fratricide and enhance tumor eradication, paving the way for clinical translation.

Automated summary

The stable expression of CD38 in T-cell acute lymphoblastic leukemia (T-ALL) makes CD38 chimeric antigen receptor (CAR)-T/natural killer (NK) a potential therapy for T-ALL. However, normal T/NK cells expressing CD38 cause fratricide of CD38 CAR-T/NK cells. To overcome this, a 2-in-1 gene editing strategy was developed to generate fratricide-resistant CAR-T/NK cells. The strategy involved integrating CD38-specific CAR into disrupted CD38 locus and controlling its expression using endogenous or exogenous promoters.