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

Abstract

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 (CD38KO/KI) or exogenous EF1α promoter (CD38KO/KIEF1α). CD38 knockout reduces fratricide and allows the expansion of CAR‐T cells. Meanwhile, CD38KO/KIEF1α results in higher CAR expression than CD38KO/KI in both CAR‐T and CAR‐NK cells. In a mouse T‐ALL model, CD38KO/KIEF1α CAR‐T cells eradicate tumors better than CD38KO/KI CAR‐T cells. Surprisingly, CD38KO/KI CAR‐NK cells show superior tumor control than CD38KO/KIEF1α 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.

Identification of a Prognostic Model Based on NK Cell-Related Genes in Multiple Myeloma Using Single-Cell and Transcriptomic Data Analysis

“…These therapies have been employed in treating diseases such as multiple myeloma, acute myeloid leukemia, acute lymphoblastic leukemia, and non-Hodgkin lymphoma. [12][13][14][15] Recently, a team has utilized single-cell sequencing data to study the significance of NK cell-related gene models in cancer prognosis. Xiang et al screened and validated NK cell marker genes in hepatocellular carcinoma (HCC) cells, as well as to develop an NK-cell-related gene signature to predict the prognosis of HCC patients.…”

Nicotinamide-Expanded Allogeneic Natural Killer Cells with CD38 Deletion, Expressing an Enhanced CD38 Chimeric Antigen Receptor, Target Multiple Myeloma Cells

“…This phenomenon not only drastically reduced the viability of these NK cells but also fundamentally altered their functionality. It is noteworthy that similar works attempting to express anti-CD38 CAR on NK cells had avoided using anti-CD38 CARexpressing NK cells as a control in their experiments, likely due to similar challenges and observations [46,47].…”

“…Functional assays were performed in all NK cell groups in the presence or absence of Daratumumab. A control group presenting anti-CD38 CAR-expressing NK cells without CD38 knock-out was excluded from the experimental design due to high toxicity, which manifested in vast fratricide as previously described [46,47] and as presented in Figure 5C, thus rendering it impossible to obtain the starting conditions required for a specific lysis assay. Testing included two independent methods of specific lysis assays and quantification of activation markers following coculture with various target cells expressing different levels of CD38.…”

Broadening the horizon: potential applications of CAR-T cells beyond current indications

“…A similar technique was employed by Liao et al. for the generation of CD38-specific CAR-T cells ( 48 ). Similar to this is the editing of the T-cell receptor α constant (TRAC) locus ( 49 ).…”