无病毒 CRISPR-CAR T 细胞的制备及鉴定及其诱导实体瘤消退的能力
Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression.
机构信息
Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin, USA.
出版信息
J Immunother Cancer. 2022 Sep;10(9). doi: 10.1136/jitc-2021-004446.
BACKGROUND
Chimeric antigen receptor (CAR) T cells have demonstrated high clinical response rates against hematological malignancies (e.g., CD19+ cancers) but have shown limited activity in patients with solid tumors. Recent work showed that precise insertion of a CAR at a defined locus improves treatment outcomes in the context of a CD19 CAR; however, it is unclear if such a strategy could also affect outcomes in solid tumors. Furthermore, CAR manufacturing generally relies on viral vectors for gene delivery, which comprise a complex and resource-intensive part of the manufacturing supply chain.
METHODS
Anti-GD2 CAR T cells were generated using CRISPR/Cas9 within 9 days using recombinant Cas9 protein and nucleic acids, without any viral vectors. The CAR was specifically targeted to the T cell receptor alpha constant gene (). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, targeted next-generation sequencing, scRNA-seq, spectral cytometry, and ELISA assays, respectively. Functionality was evaluated in an NSG™ xenograft neuroblastoma model.
RESULTS
In comparison to retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit -targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory-like phenotype, and low tonic signaling prior to infusion arising in part from the knockout of the T cell receptor. On exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibit specific cytotoxicity against GD2+ cells and induce solid tumor regression . VFC-CAR T cells demonstrate robust homing and persistence and decreased exhaustion relative to retroviral CAR T cells against a human neuroblastoma xenograft model.
CONCLUSIONS
This study leverages virus-free genome editing technology to generate CAR T cells featuring a -targeted CAR, which could inform manufacturing of CAR T cells to treat cancers, including solid tumors.
背景
嵌合抗原受体 (CAR) T 细胞在对抗血液恶性肿瘤（例如，CD19+癌症）方面显示出高临床反应率，但在实体瘤患者中表现出有限的活性。最近的研究表明，在 CD19 CAR 的背景下，精确地将 CAR 插入到一个定义的基因座中可以改善治疗效果；然而，尚不清楚这种策略是否也能影响实体瘤的结果。此外，CAR 制造通常依赖于用于基因传递的病毒载体，这是制造供应链中复杂且资源密集型的一部分。
方法
使用 CRISPR/Cas9 在 9 天内生成抗-GD2 CAR T 细胞，使用重组 Cas9 蛋白和核酸，而不使用任何病毒载体。CAR 被特异性靶向到 T 细胞受体 alpha 恒定基因 (). 使用 CHANGE-seq、靶向下一代测序、单细胞 RNA 测序、光谱细胞术和 ELISA 测定分别在基因组、转录组、蛋白质组和分泌组水平上对 T 细胞产物进行了表征。在 NSG™异种神经母细胞瘤模型中评估了功能。
结果
与逆转录病毒 CAR T 细胞相比，无病毒 CRISPR CAR（VFC-CAR）T 细胞表现出靶向 CAR 转基因的基因组整合、与记忆样表型相关的转录和蛋白质特征的升高、以及输注前部分由 T 细胞受体缺失引起的低紧张信号。在暴露于 GD2 靶抗原后，抗-GD2 VFC-CAR T 细胞对 GD2+细胞表现出特异性细胞毒性，并诱导实体瘤消退。与逆转录病毒 CAR T 细胞相比，VFC-CAR T 细胞在针对人神经母细胞瘤异种移植模型时具有更强的归巢和持久性，并降低了衰竭。
结论
本研究利用无病毒基因组编辑技术生成了具有靶向 CAR 的 CAR T 细胞，这可能为治疗癌症（包括实体瘤）的 CAR T 细胞制造提供信息。
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