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 EF1α promoter (CD38 (KO/KI)EF1α). CD38 knockout reduces fratricide and allows the expansion of CAR‐T cells. Meanwhile, CD38 (KO/KI)EF1α 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)EF1α 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)EF1α 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.