靶向CD30的CAR-T细胞慢病毒转导条件优化研究

doi:10.19401/j.cnki.1007-3639.2023.07.002

中国癌症杂志 ›› 2023, Vol. 33 ›› Issue (7): 646-654.doi: 10.19401/j.cnki.1007-3639.2023.07.002

靶向CD30的CAR-T细胞慢病毒转导条件优化研究

田高辉¹(), 张琴星¹, 史江舟¹, 赵芬芳¹, 王宁¹, 赵家旋², 卢玉琳¹, 徐瑶¹()

1.武汉科技大学生命科学与健康学院生物医学研究院，湖北武汉 430081
2.天津科技大学生物工程学院，天津 300457

收稿日期:2022-12-09 修回日期:2023-05-04 出版日期:2023-07-30 发布日期:2023-08-10
通信作者: 徐瑶（ORCID: 0000-0001-7926-3791），博士，副教授，武汉科技大学生命科学与健康学院生物医学研究院主任。
作者简介:田高辉（ORCID: 0009-0007-8700-2779），硕士。
基金资助:
湖北省科学技术厅支持企业创新发展项目(2021BAB126)

A study on optimized lentiviral transduction conditions in CAR-T cells targeting CD30

TIAN Gaohui¹(), ZHANG Qinxing¹, SHI Jiangzhou¹, ZHAO Fenfang¹, WANG Ning¹, ZHAO Jiaxuan², LU Yulin¹, XU Yao¹()

1. Institute of Biology and Medicine, College of Life Science and Health, Wuhan University of Science and Technology, Wuhan 430081, Hubei Province, China
2. College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China

Received:2022-12-09 Revised:2023-05-04 Published:2023-07-30 Online:2023-08-10
Contact: XU Yao.

摘要/Abstract

摘要：

背景与目的：嵌合抗原受体T（chimeric antigen receptor T，CAR-T）细胞技术在血液肿瘤治疗领域已被广泛应用，慢病毒转导是CAR-T细胞制备的关键环节，与CAR-T细胞的质量密切相关，因此慢病毒转导过程涉及的各项参数仍需进一步优化。本研究旨在探讨携带CD30抗体序列的慢病毒转导T细胞的感染复数（multiplicity of infection，MOI）、温育密度、转导前活化时间和转导体系高度对抗CD30 CAR-T细胞的影响，优化CAR-T细胞的转导条件，提高转导效率和CAR-T细胞功能。方法：采用不同的MOI、温育密度、转导前活化时间和转导体系高度等对人外周血来源的T细胞进行转导优化，转导后分别检测抗CD30 CAR-T细胞的增殖能力、转导效率、细胞存活率和体外杀伤效率等，以确定最优的T细胞转导条件。结果：MOI为1.00、1.50和3.00时转导效率和有效细胞数显著高于0.00、0.25和0.50组。在温育密度大于1.0×10⁷个/mL时，温育密度对T细胞转导效率无影响。活化时间为72 h组细胞存活率低于80%，显著低于其他组；24、48 h的转导效率显著高于0、8、16 h组；48 h组CAR-T细胞的增殖速率显著高于24 h组。转导体系高度为0.16 mm时转导效率和增殖倍数都显著高于0.53 mm组，但对CAR-T细胞的体外杀伤效率无影响。结论：通过对CAR-T细胞功能的综合评估，确定慢病毒的最佳转导条件为MOI=1、温育密度为1.0×10⁷个/mL、转导前活化48 h、转导体系高度为0.16 mm。

关键词: 慢病毒载体, 抗CD30嵌合抗原受体T细胞, 转导条件

Abstract:

Background and purpose: Chimeric antigen receptor T (CAR-T) cell technology has been widely used in the field of blood tumor treatment. Lentivirus transduction is a key link in the preparation of CAR-T cells, which is closely related to the quality of CAR-T cells. Therefore, the parameters involved in the lentivirus transduction process still need to be further optimized. This study aimed to investigate the effects of multiplicity of infection (MOI), incubation density, activation time before transduction and transduction system height on anti-CD30 CAR-T cells in lentiviral transduction T cells carrying CD30 antibody sequences, optimize the transduction conditions of CAR-T cells, and improve transduction efficiency and CAR-T cells function. Methods: Different MOI, incubation density, activation time before transduction and transduction system height were used to optimize the transduction of human peripheral blood-derived T cells, then proliferation capacity, transduction efficiency, cell viability and killing efficiency in vitro of anti-CD30 CAR-T cells were detected. Results: When the MOI was 1.00, 1.50 and 3.00, the transduction efficiency and effective cell number were significantly higher than those in the 0.00, 0.25 and 0.50 groups. When the incubation density was greater than 0.5×10⁷ cells/mL, the incubation density had no significant effect on the transduction efficiency of T cells. The cell viability rate of the 72 h group was lower than 80%, which was lower compared with other groups significantly, the transduction efficiency of 24 and 48 h was significantly higher compared with the 0 h, 8 h and 16 h groups, and the expansion rate of CAR-T cells in the 48 h group was significantly higher compared with the 24 h group. The transduction efficiency and proliferation capacity of 0.16 mm in height of transduction system were significantly higher compared with the 0.53 mm group, however, had no significant effect on killing efficiency of CAR-T cells in vitro. Conclusion: Through the comprehensive evaluation of CAR-T cells function, the optimal transduction conditions for lentivirus include MOI of 1.00, incubation density of 1.0×10⁷ cells/mL, activation before transduction for 48 h and transduction system height of 0.16 mm.

Key words: Lentiviral vectors, Anti-CD30 chimeric antigen receptor T cell, Transduction conditions

中图分类号:

R730.54

田高辉, 张琴星, 史江舟, 赵芬芳, 王宁, 赵家旋, 卢玉琳, 徐瑶. 靶向CD30的CAR-T细胞慢病毒转导条件优化研究[J]. 中国癌症杂志, 2023, 33(7): 646-654.

TIAN Gaohui, ZHANG Qinxing, SHI Jiangzhou, ZHAO Fenfang, WANG Ning, ZHAO Jiaxuan, LU Yulin, XU Yao. A study on optimized lentiviral transduction conditions in CAR-T cells targeting CD30[J]. China Oncology, 2023, 33(7): 646-654.

图/表 5

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靶向CD30的CAR-T细胞慢病毒转导条件优化研究

A study on optimized lentiviral transduction conditions in CAR-T cells targeting CD30

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