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

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CD38-Specific CAR Integrated into CD38 Locus Driven by Different Promoters Causes Distinct Antitumor Activities of T and NK Cells
Advanced Science ( IF 14.3 ) Pub Date : 2023-07-23 , DOI: 10.1002/advs.202207394
Chan Liao ₁ , Yajie Wang _{2,

3,

4} , Yanjie Huang _{5,

6} , Yanting Duan _{2,

3,

4} , Yan Liang ₂ , Jiangqing Chen _{2,

3,

4} , Jie Jiang _{2,

3,

4} , Kai Shang _{2,

3,

4} , Chun Zhou ₇ , Ying Gu ₈ , Nan Liu ₂ , Xun Zeng ₉ , Xiaofei Gao ₅ , Yongmin Tang ₁ , Jie Sun _{2,

3,

4}

Affiliation

Department of Hematology-oncology, Children's Hospital, Zhejiang University School of Medicine, Pediatric Leukemia Diagnostic and Therapeutic Technology Research Center of Zhejiang Province National Clinical Research Center for Child Health, Hangzhou, 310003, China.
Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou, 311121, China.
Bone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, 310058, China.
Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China.
Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, 310058, China.
School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China.
School of Public Health and Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China.
Institute of Genetics, Zhejiang University and Department of Genetics, Zhejiang University school of medicine, Hangzhou, 310058, China.
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China.

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/KIEF1α). CD38 knockout reduces fratricide and allows the expansion of CAR-T cells. Meanwhile, CD38^KO/KIEF1α 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/KIEF1α 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/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.

中文翻译：

由不同启动子驱动的 CD38 特异性 CAR 整合到 CD38 位点导致 T 细胞和 NK 细胞具有不同的抗肿瘤活性

CD38 在 T 细胞急性淋巴细胞白血病 (T-ALL) 母细胞中的强劲和稳定表达使得 CD38 嵌合抗原受体 (CAR)-T/自然杀伤 (NK) 成为 T-ALL 的潜在疗法。然而，正常T/NK细胞中的CD38表达会导致CD38 CAR-T/NK细胞自相残杀。这里开发了一种“二合一”基因编辑策略来生成抗自相残杀的基因座特异性 CAR-T/NK 细胞。CD38 特异性 CAR 通过 CRISPR/Cas9 整合到破坏的CD38基因座中，并且 CAR 置于内源 CD38 启动子 ( CD38 ^KO/KI ) 或外源 EF1α 启动子 ( CD38 ^KO/KI EF1α)的控制之下。CD38 敲除可减少自相残杀并允许 CAR-T 细胞扩增。同时，在 CAR-T 和 CAR-NK 细胞中，CD38 ^KO/KI EF1α 导致比CD38 ^{KO/KI更高的 CAR 表达。}在小鼠 T-ALL 模型中，CD38 ^KO/KI EF1α CAR-T 细胞比CD38 ^KO/KI CAR-T 细胞更好地根除肿瘤。令人惊讶的是，CD38 ^KO/KI CAR-NK 细胞比CD38 ^KO/KI EF1α CAR-NK 细胞表现出更好的肿瘤控制能力。进一步研究发现，NK细胞中的内源性调控元件导致CD38 CAR的表达高于T细胞，并且CAR的表达水平对CAR-T和CAR-NK细胞的治疗结果产生不同的影响。因此，这些结果支持了CD38 CAR-T/NK对抗T-ALL的功效，并证明“二合一”策略可以解决自相残杀并增强肿瘤根除，为临床转化铺平道路。

更新日期：2023-07-23

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