靶向CD99的CAR-T细胞扩增优化研究

王伊玄; 于淼; 赵家旋; 赵芬芳; 曾毅; 王友湧; 祝海川; 张同存; 史江舟

doi:10.19401/j.cnki.1007-3639.2024.07.003

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靶向CD99的CAR-T细胞扩增优化研究

论著 | 更新时间：2025-12-31

- 靶向CD99的CAR-T细胞扩增优化研究
- Optimization study of CAR-T cell expansion targeting CD99
- 中国癌症杂志 2024年34卷第7期页码：639-649
- 作者机构：
  
  1. 天津科技大学生物工程学院，天津 300457
  2. 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
- 作者简介：
  
  [ "王伊玄（ORCID: 0009-0002-2463-3606），硕士。" ]
  史江舟（ORCID: 0009-0009-8185-4043），博士。
- 基金信息：
  
  国家自然科学基金(82100193)
- DOI：10.19401/j.cnki.1007-3639.2024.07.003
  中图分类号： R730.51
- 收稿：2023-11-25，
  
  修回：2024-02-22，
  
  纸质出版：2024-07-30
- 稿件说明：
移动端阅览
王伊玄, 于淼, 赵家旋, 等. 靶向CD99的CAR-T细胞扩增优化研究[J]. 中国癌症杂志, 2024,34(7):639-649.

Yixuan WANG, Miao YU, Jiaxuan ZHAO, et al. Optimization study of CAR-T cell expansion targeting CD99[J]. China Oncology, 2024, 34(7): 639-649.
王伊玄, 于淼, 赵家旋, 等. 靶向CD99的CAR-T细胞扩增优化研究[J]. 中国癌症杂志, 2024,34(7):639-649. DOI： 10.19401/j.cnki.1007-3639.2024.07.003.

Yixuan WANG, Miao YU, Jiaxuan ZHAO, et al. Optimization study of CAR-T cell expansion targeting CD99[J]. China Oncology, 2024, 34(7): 639-649. DOI： 10.19401/j.cnki.1007-3639.2024.07.003.

摘要

背景与目的：

嵌合抗原受体T（chimeric antigen receptor T，CAR-T）细胞疗法对血液和淋巴系统肿瘤的治疗效果显著，但对实体瘤效果较差，这与靶点选择的因素有关。针对尤因肉瘤（Ewing sarcoma，ES），CD99可作为CAR-T细胞潜在的靶点。由于T细胞自身表达CD99蛋白，靶向CD99的CAR-T细胞存在体外扩增能力有限的问题。本研究旨在通过增加CD99敲低的短发夹RNA（short hairpin RNA，shRNA）、优化慢病毒转导的感染复数（multiplicity of infection，MOI）、筛选培养CAR-T细胞的培养基及培养容器，以获得CD99 CAR-T细胞制备的最优条件。

方法：

筛选shRNA序列，提高CD99 CAR-T细胞的扩增能力。采用不同的MOI、培养基和培养容器，分别检测在不同条件下CAR-T细胞的转导效率、细胞存活率、增殖能力、特异性杀伤能力及干扰素-

（interferon-

，IFN-

）释放水平等，筛选出最优的细胞制备条件。

结果：

通过shRNA敲低得到的KO-CD99 CAR-T细胞扩增水平显著高于CD99 CAR-T细胞[（16.40±0.40）

（6.33±1.53），

0.01

]

。转导MOI为0.25 ~ 1.0、培养基为OptiVitro时细胞的扩增效果最优。在透气瓶中培养的CAR-T细胞扩增倍数显著高于在培养袋中培养的细胞[MOI=0.25：（50.23±3.32）

（13.02±4.82）；MOI=0.50：（49.96±0.83）

（18.25±2.88）；MOI=1.00：（48.27±5.08）

（13.16±6.26）；

0.01

]

，且细胞分型更优、特异性杀伤更高。

结论：

通过shRNA技术得到的KO-CD99 CAR-T细胞可实现稳定扩增。从扩增条件优化结果看，MOI为0.25 ~ 1.00，培养基为OptiVitro，培养容器为透气瓶的条件下

，KO-CD99 CAR-T细胞可获得更优的扩增能力、更多比例的记忆T细胞，为后续开展CD99 CAR-T细胞治疗ES的临床试验奠定了坚实基础。

Abstract

Background and purpose:

Chimeric antigen receptor T (CAR-T) cell therapy has shown remarkable efficacy in treating hematological and lymphatic system tumors

but its effectiveness in solid tumors is relatively poor

which is partly attributed to target selection. For Ewing sarcoma (ES)

CD99 can be a potential target for CAR-T cells. However

due to T cells’ endogenous expression of CD99 protein

CAR-T cells targeting CD99 face limitations in their expansion capacity in vitro. This study aimed to identify the optimal conditions for preparing CD99 CAR-T cells by incorporating CD99 knockdown short hairpin RNA (shRNA)

optimizing the multiplicity of infection (MOI) for lentiviral transduction

and screening for the best culture medium and container for CAR-T cell expansion.

Methods:

shRNA sequences were screened to enhance the expansion capacity of CD99 CAR-T cells. Different MOI

culture media

and containers were used to assess CAR-T cell transduction efficiency

cell viability

proliferation capacity

specific killing ability

and interferon-γ (IFN-γ) release levels under various conditions

in order to identify the optimal cell preparation conditions.

Results:

The expansion level of KO-CD99 CAR-T cells obtained through shRNA knockdown was significantly higher than that of CD99 CAR-T cells [(16.40±0.40)

(6.33±1.53)

0.01

]

. The optimal expansion effect was observed when the transduction MOI was between 0.25 and 1.0

and OptiVitro was used as the culture medium. CAR-T cells cultured in ventilated flasks exhibited significantly higher expansion rates compared to cells cultured in bags [MOI=0.25: (50.23±3.32)

(13.02±4.82); MOI=0.50: (49.96±0.83)

(18.25±2.88); MOI=1.00: (48.27±5.08)

(13.16±6.26);

0.0

]

with better cell phenotype and higher specific killing ability.

Conclusion:

KO-CD99 CAR-T cells obtained through shRNA technology can achieve stable expansion. Based on the optimization of expansion conditions

KO-CD99 CAR-T cells exhibit superior expansion capacity and a higher proportion of memory T cells when the MOI is between 0.25 and 1.00

OptiVitro is used as the culture medium

and ventilated flasks are used as the culture container. These findings lay a solid foundation for further clinical trials of CD99 CAR-T cell therapy for ES.

关键词

Keywords

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