不同信号肽对嵌合抗原受体T细胞杀伤作用的影响研究

doi:10.19401/j.cnki.1007-3639.2022.02.006

摘要/Abstract

摘要：

背景与目的：信号肽（signal peptide,SP）是一段存在于前体蛋白N-端的短肽链,能够调节前体蛋白的折叠和转移,在蛋白质的分泌过程中扮演着极其重要的角色。近年来,靶向CD19的嵌合抗原受体（chimeric antigen receptor,CAR）T细胞在白血病治疗中取得了重大突破,关于CAR结构的胞内域改造方面也有诸多研究,而对单链可变片段（scFv）的N端SP研究进展缓慢。探讨4种不同SP的CD19-CAR在T细胞表面表达及对CD19⁺靶细胞的杀伤作用。方法：通过基因合成和分子克隆技术,构建含4种不同SP（SP1、SP2、SP3、SP4）的靶向CD19抗原的CAR载体,进行慢病毒包装,将得到的慢病毒转染T细胞,利用流式细胞术检测细胞转染效率,采用钙黄绿素释放法检测该细胞对靶细胞的杀伤作用,采用酶联免疫吸附实验（enzyme-linked immunosorbent assay,ELISA）检测细胞因子IFN-γ和TNF-α的分泌水平。结果： 成功构建4种不同SP的重组慢病毒载体,将4种慢病毒转导T细胞后,结果显示,分别有20.9%、22.6%、31.5%、38.6%的T细胞表面能够表达CD19-CAR（分别命名为SP1-CD19、SP2-CD19、SP3-CD19和SP4-CD19细胞）,进一步杀瘤实验证明,SP4-CD19细胞对CD19⁺肿瘤细胞的杀伤作用显著高于SP1-CD19、SP2-CD19和SP3-CD19细胞（P<0.01）,并且当效靶比为10∶1 共培养24 h 后,与SP1-CD19、SP2-CD19和 SP3-CD19细胞相比,SP4-CD19细胞的IFN-γ和TNF-α的分泌水平显著升高（P<0.05）。此外,4种不同SP的CAR-T对CD19^–肿瘤细胞K562的杀伤作用差异无统计学意义（P>0.05）。结论： SP4-CD19细胞的转染效率、细胞因子分泌水平及对CD19⁺肿瘤细胞的杀伤作用均显著高于SP1-CD19、SP2-CD19和SP3-CD19细胞,该研究成果为CAR-T优化改造及其高效的临床应用奠定了科学基础。

关键词: 信号肽, 嵌合抗原受体, 靶向CD19的嵌合抗原受体（chimeric antigen receptor, CAR）T细胞, 体外杀瘤, 细胞因子

Abstract:

Background and purpose: Signal peptide (SP) is a short peptide chain at the N-terminal of precursor protein, which can regulate the folding and transfer of precursor protein and plays an important role in protein secretion. In recent years, significant breakthroughs have been made in the treatment of leukemia with CD19-targeted chimeric antigen receptor (CAR) T cells, and many achievements have been reported in the intracellular domain modification of CAR structure, while the N-terminal SP of scFv presents less progress. The purpose of this study was to investigate the CD19-CAR expression of four different SP on the surface of T cells and their effect on the killing of CD19⁺ target cells. Methods: The CAR vectors containing four different SP (SP1, SP2, SP3, SP4) targeting CD19 antigen were constructed by gene synthesis and molecular cloning technology, and then packaged into lentivirus. The obtained lentivirus was transfected into T cells. The transfection efficiency of the cells was detected by flow cytometry, the killing effect of the cells on target cells was detected by calcein release assay, and the secretion levels of IFN-γ and TNF-α were detected by ELISA. Results: The recombinant lentiviral plasmids with four different SP were successfully constructed, and the four packaged lentiviruses were transduced into T cells. The results showed that 20.76%, 22.29%, 31.57% and 38.42% of T cells expressed CD19-CAR (named as SP1-CD19, SP2-CD19, SP3-CD19 and SP4-CD19 cells, respectively), respectively. The killing effect of SP4-CD19 on CD19^- tumor cells was significantly higher compared with SP1-CD19, SP2-CD19 and SP3-CD19 cells (P<0.01), and the secretion levels of IFN-γ and TNF-α in SP4-CD19 cells were significantly higher than those in SP1-CD19, SP2-CD19 and SP3-CD19 cells when the effect-target ratio was 10 to 1 for 24 h (P<0.05). There was no significant difference in the killing effect of CAR-T on CD19 negative cells K562 among four different SP (P>0.05). Conclusion: The transfection efficiency and killing effect of SP4-CD19 cells on CD19⁺ tumor cells were significantly higher compared with SP1-CD19, SP2-CD19 and SP3-CD19 cells, which laid a scientific foundation for the optimization and efficient clinical application of CAR-T.

Key words: Signal peptide, Chimeric antigen receptor, CD19-targeted chimeric antigen receptor T cells, Anti-tumor in vitro, Cytokines

中图分类号:

R730.51

李帆, 张琴星, 童祥文, 田高辉, 顾力行, 徐瑶. 不同信号肽对嵌合抗原受体T细胞杀伤作用的影响研究[J]. 中国癌症杂志, 2022, 32(2): 142-151.

LI Fan, ZHANG Qinxing, TONG Xiangwen, TIAN Gaohui, GU Lixing, XU Yao. A study on influence of different signal peptides on anti-tumor effect of chimeric antigen receptor (CAR) T cells[J]. China Oncology, 2022, 32(2): 142-151.

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SP	Sequence	Source
SP1	ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (MALPVTALLLPLALLLHAARP)	Human CD8α
SP2	ATGCTGCTGCTGGTGACCAGCCTGCTTCTGTGCGAACTGCCCCACCCCGCCTTCCTGCTAATCCCC (MLLLVTSLLLCELPHPAFLLIP)	Human CSF2Rα
SP3	ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATGTCTAGAATGGCC (MDFQVQIFSFLLISASVIMSRMA)	Mouse light chain Kappa-K32 precursor
SP4	ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGT (MYRMQLLSCI ALSLALVTNS)	Human IL-2

Primer name	Primer sequence(5'-3')
SP2-forwards	GGATCTATTTCCGGTGAATTCGCCACCATGCTGCTGCTGGTGACC
SP2-reverse	CTGGGTCATCTGGATGTCCGGCCTGGCGGCGTGGA
SP3-forwards	GGATCTATTTCCGGTGAATTCGCCACCATGGATTTTCAGGTG
SP3--reverse	CTGGGTCATCTGGATGTCGGCCATTCTAGACATTAT
SP4-forwards	GGATCTATTTCCGGTGAATTCGCCACCATGTACAGGATGCAAC
SP4-reverse	CTGGGTCATCTGGATGTCACTGTTTGTGACAAGTG
CAR-forwards	GACATCCAGATGACCCAGAC
CAR-reverse	GGGAGGGAGAGGGGCGGATCCTTAGCGAGGGGGCAGGGCC