无标记的体外检测可预测抗神经节苷脂嵌合抗原受体 T 细胞产品的效力。
Label-free in vitro assays predict the potency of anti-disialoganglioside chimeric antigen receptor T-cell products.
机构信息
Regenerative Bioscience Center, University of Georgia, Athens, Georgia, USA; Division of Neuroscience, Biomedical and Health Sciences Institute, University of Georgia, Athens, Georgia, USA.
Department of Biochemistry & Molecular Biology, Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, USA; Institute of Bioinformatics, University of Georgia, Athens, Georgia, USA.
出版信息
Cytotherapy. 2023 Jun;25(6):670-682. doi: 10.1016/j.jcyt.2023.01.008. Epub 2023 Feb 26.
BACKGROUND AIMS
Chimeric antigen receptor (CAR) T cells have demonstrated remarkable efficacy against hematological malignancies; however, they have not experienced the same success against solid tumors such as glioblastoma (GBM). There is a growing need for high-throughput functional screening platforms to measure CAR T-cell potency against solid tumor cells.
METHODS
We used real-time, label-free cellular impedance sensing to evaluate the potency of anti-disialoganglioside (GD2) targeting CAR T-cell products against GD2+ patient-derived GBM stem cells over a period of 2 days and 7 days in vitro. We compared CAR T products using two different modes of gene transfer: retroviral transduction and virus-free CRISPR-editing. Endpoint flow cytometry, cytokine analysis and metabolomics data were acquired and integrated to create a predictive model of CAR T-cell potency.
RESULTS
Results indicated faster cytolysis by virus-free CRISPR-edited CAR T cells compared with retrovirally transduced CAR T cells, accompanied by increased inflammatory cytokine release, CD8+ CAR T-cell presence in co-culture conditions and CAR T-cell infiltration into three-dimensional GBM spheroids. Computational modeling identified increased tumor necrosis factor α concentrations with decreased glutamine, lactate and formate as being most predictive of short-term (2 days) and long-term (7 days) CAR T cell potency against GBM stem cells.
CONCLUSIONS
These studies establish impedance sensing as a high-throughput, label-free assay for preclinical potency testing of CAR T cells against solid tumors.
背景目的
嵌合抗原受体 (CAR) T 细胞在治疗血液恶性肿瘤方面显示出显著疗效；然而，它们在治疗实体瘤（如胶质母细胞瘤 [GBM]）方面并没有取得同样的成功。因此，我们迫切需要高通量的功能筛选平台来衡量 CAR T 细胞对实体肿瘤细胞的效力。
方法
我们使用实时、无标记细胞阻抗传感技术，在体外 2 天和 7 天的时间内，评估针对 GD2+患者来源的 GBM 干细胞的抗二唾液酸神经节苷脂 (GD2) 靶向 CAR T 细胞产品的效力。我们使用两种不同的基因转移模式来比较 CAR T 产品：逆转录病毒转导和无病毒 CRISPR 编辑。获取并整合终点流式细胞术、细胞因子分析和代谢组学数据，以创建 CAR T 细胞效力的预测模型。
结果
结果表明，与逆转录病毒转导的 CAR T 细胞相比，无病毒 CRISPR 编辑的 CAR T 细胞具有更快的细胞溶解作用，同时伴随着炎症细胞因子释放增加、共培养条件下 CD8+ CAR T 细胞的存在以及 CAR T 细胞渗透到三维 GBM 球体中。计算模型确定，肿瘤坏死因子 α 浓度增加，同时谷氨酰胺、乳酸和甲酸盐减少，这与短期（2 天）和长期（7 天）CAR T 细胞对 GBM 干细胞的效力最具预测性。
结论
这些研究确立了阻抗传感作为一种高通量、无标记的方法，可用于 CAR T 细胞对实体瘤的临床前效力测试。
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