Dataset Information

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 (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.

SUBMITTER: Liao C

PROVIDER: S-EPMC10520621 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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

Liao Chan C Wang Yajie Y Huang Yanjie Y Duan Yanting Y Liang Yan Y Chen Jiangqing J Jiang Jie J Shang Kai K Zhou Chun C Gu Ying Y Liu Nan N Zeng Xun X Gao Xiaofei X Tang Yongmin Y Sun Jie J

Advanced science (Weinheim, Baden-Wurttemberg, Germany) 20230723 27

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 ...[more]

PMID: 37485647

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Project description:Adoptive transfer of natural killer (NK) cells with augmented antibody-dependent cellular cytotoxicity (ADCC) capabilities and resistance to CD38 targeting has the potential to enhance the clinical anti-myeloma activity of daratumumab (DARA). Therefore, we sought to develop an efficient CRISPR/Cas9-based gene editing platform to disrupt CD38 expression (CD38 knockout (KO)) in ex vivo expanded NK cells and simultaneously arm CD38KO NK cells with a high-affinity CD16 (CD16-158V) receptor. CD38KO human NK cells were generated using Cas9 ribonucleoprotein complexes. The platform was expanded by incorporating messenger RNA (mRNA) transfection of CD38KO NK cells and targeted gene insertion at the CD38 locus to mediate gene knockin (KI). The capacity of these gene-edited NK cells to persist and mediate ADCC in the presence of DARA was tested in vitro and in a MM.1S xenograft mouse model. Highly efficient CD38 gene disruption was achieved in ex vivo expanded NK cells without affecting their proliferative or functional capacity. CD38 KO conferred resistance to DARA-induced NK cell fratricide, enabling persistence and augmented ADCC against myeloma cell lines in the presence of DARA in vitro and in a MM.1S xenograft mouse model. CD38KO NK cells could be further modified by transfection with mRNA encoding a CD16-158V receptor, resulting in augmented DARA-mediated ADCC. Finally, we observed that a homology-directed repair template targeted to the CD38 locus facilitated an efficient 2-in-1 CD38 KO coupled with KI of a truncated CD34 reporter and CD16-158V receptor, with CD38KO/CD16KI NK cells demonstrating a further enhancement of DARA-mediated ADCC both in vitro and in vivo. Adoptive immunotherapy using ex vivo expanded CD38KO/CD16KI NK cells has the potential to boost the clinical efficacy of DARA. By incorporating complementary genetic engineering strategies into a CD38 KO manufacturing platform, we generated NK cells with substantially augmented CD38-directed antitumor activity, establishing a strong rationale for exploring this immunotherapy strategy in the clinic.

Dataset Information

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

Publications

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

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