定点突变优化信号肽对嵌合抗原受体T细胞功能的影响

Effect of signal peptide optimized by site-directed mutagenesis on the function of chimeric antigen receptor T cells

摘要:

信号肽(signal peptide, SP)参与调节嵌合抗原受体(chimeric antigen receptor, CAR)结构的分泌水平及跨膜易位过程，对嵌合抗原受体T细胞(chimeric antigen receptor T cell, CAR-T)行使功能至关重要，本研究通过定点突变技术优化SP序列，并研究其对CD19-CAR-T杀伤功能的影响。首先，利用基因合成和分子克隆技术，构建含野生型SP (SP-wtY)、2种突变型SP (SP-muK或SP-muR)的靶向CD19的CAR载体；对构建成功的载体进行慢病毒包装，并使用慢病毒侵染T细胞，利用流式细胞术检测细胞的转染效率，钙黄绿素释放法检测对靶细胞的体外杀瘤能力，酶联免疫吸附(enzyme linked immunosorbent assay, ELISA)技术检测2种细胞因子[干扰素-γ (interferon-γ, IFN-γ)和干扰素-α (interferon-α, TNF-α)]的分泌水平。结果显示，构建了野生型和信号肽突变的重组慢病毒质粒，慢病毒转导T细胞后，携带3种信号肽(SP-wtY、SP-muK或SP-muR)的CD19-CAR转染效率分别为33.9%、35.5%、36.8%。进一步杀伤实验显示，与SP-muK和SP-wtY细胞相比，SP-muR细胞的肿瘤杀伤效果显著提高，当效靶比为10:1时共培养24 h后，SP-muR组CAR-T细胞的细胞因子IFN-γ和TNF-α分泌水平显著高于SP-muK和SP-wtY组。本研究揭示信号肽N端正电荷数的增加可以提高CAR的表达效率和对CD19⁺靶细胞的杀伤作用，为CAR结构的优化改造和临床高效应用提供了科学依据。

关键词:定点突变;信号肽;嵌合抗原受体;CD19-CAR-T细胞;体外杀瘤;细胞因子

Abstract:

Signal peptides (SP) are involved in regulating the secretion level and transmembrane translocation of chimeric antigen receptors (CAR), which is crucial for CAR-T cells. This study aimed to optimize the SP sequence by site-directed mutagenesis and investigate its impact on the killing function of CD19-CAR-T. Firstly, CAR vectors targeting CD19 containing wild-type SP (SP-wtY) or two mutant SP (SP-muK or SP-muR) were constructed using gene synthesis and molecular cloning techniques. The successfully constructed vector was packaged with lentivirus, and T cells were infected. The transfection efficiency of T cells was detected by flow cytometry, while the killing effect on target cells was assessed using the calcein release method. The secretion levels of cytokines interferon-γ (IFN-γ) and interferon-α (TNF-α) were measured using enzyme linked immunosorbent assay (ELISA). The results showed that successful construction of recombinant lentivirus plasmids with wild type and signal peptide mutation. After the transferring the lentivirus into T cells, the transfection efficiency of CD19-CAR carrying three signal peptides (SP-wtY, SP-muK, or SP-muR) were 33.9%, 35.5%, and 36.8%, respectively. Further killing assay showed that the tumor-killing effect of SP-muR cells was significantly higher than that of SP-muK and SP-wtY cells. When the ratio of effector to target was 10:1, the secretion levels of cytokines IFN-γ and TNF-α of CAR-T cells of the SP-muR group were significantly higher than those in SP-muK and SP-wtY groups. In summary, this study revealed that increasing the N-terminal positive charge of the signal peptide can improve the expression efficiency of CAR and promote the killing of CD19⁺ target cells. These findings provide a scientific basis the optimization and clinical application of CAR structure.

Key words:point mutation;signal peptide;chimeric antigen receptor;CD19-CAR-T cells;killing tumor in vitro;cytokines