共表达B2M蛋白对靶向间皮素的3代CAR-T细胞生物学行为的影响

doi:10.13481/j.1671-587X.20260504

摘要/Abstract

摘要：

目的探讨共表达β2微球蛋白（B2M）与嵌合抗原受体（CAR）对靶向间皮素（MSLN）的嵌合抗原受体T细胞（CAR-T）生物学行为的影响，阐明B2M在增强CAR-T细胞对MSLN阳性实体瘤疗效中的作用。方法构建共表达B2M与CAR的3代抗MSLN CAR-T（B2M-28bbz-T）细胞和对照传统抗MSLN CAR-T（28bbz-T）细胞。构建稳定过表达MSLN的293T细胞（293T-MSLN）。采用实时荧光定量PCR（RT-qPCR）法检测各组CAR-T细胞中B2M、重链可变区（VH）和轻链可变区（VL）mRNA表达水平，流式细胞术检测各组CAR-T细胞中B2M蛋白和CAR分子表达情况及T淋巴细胞增殖能力。以293T-MSLN细胞和MSLN阳性卵巢癌SK-OV-3细胞为靶细胞，以效靶比（E/T）为1∶1分别将未转导的T淋巴细胞、28bbz-T细胞和B2M-28bbz-T细胞与靶细胞共孵育72 h，孵育后的靶细胞分别为T组、28bbz-T组和B2M-28bbz-T组。采用细胞毒性实验检测CAR-T细胞作用下各组靶细胞活性，酶联免疫吸附试验（ELISA）法检测各组靶细胞上清中干扰素γ（IFN-γ）和肿瘤坏死因子α（TNF-α）水平。选取20只雌性NSG小鼠随机分为磷酸盐缓冲液（PBS）组、28bbz-T组、B2M-28bbz-T组和蓝色荧光蛋白（BFP）-28bbz-T组，每组5只。将SK-OV-3细胞皮下植入小鼠构建卵巢癌细胞系来源异种移植（CDX）模型，于第7天经尾静脉注射相应CAR-T细胞，观察结束后处死小鼠并测量各组小鼠肿瘤体积和质量。结果成功构建B2M-28bbz-T细胞和28bbz-T细胞。RT-qPCR法，与28bbz-T组比较，B2M-28bbz-T组细胞中B2M mRNA表达水平明显升高（P<0.01），VH和VL mRNA表达水平均无明显变化，差异无统计学意义（P>0.05）。流式细胞术，2组细胞CAR分子表达和增殖能力无明显差异。细胞毒性实验，与28bbz-T细胞比较，B2M-28bbz-T组293T-MSLN细胞和SK-OV-3细胞活性均明显降低（P<0.01或P<0.001）。ELLSA法，与28bbz-T组比较，B2M-28bbz-T组293T-MSLN细胞和SK-OV-3细胞上清中IFN-γ及TNF-α水平均明显升高（P<0.05）。体内实验，与PBS组比较，28bbz-T组和B2M-28bbz-T组小鼠的肿瘤生长均受到抑制，其中B2M-28bbz-T组1只小鼠肿瘤被完全清除。28bbz-T组小鼠肿瘤质量明显高于B2M-28bbz-T组（P<0.01）；28bbz-T组与BFP-28bbz-T组小鼠肿瘤质量比较差异无统计学意义（P>0.05）。结论 B2M与CAR分子共表达可通过促进T淋巴细胞活化和促炎细胞因子分泌，增强靶向MSLN的CAR-T细胞毒性及其体内抗肿瘤活性。

关键词: 嵌合抗原受体T细胞, 间皮素, β2微球蛋白, 细胞治疗, 肿瘤免疫, 细胞系来源异种移植模型

Abstract:

Objective To discuss the effect of co-expression of beta-2-microglobulin （B2M） protein and chimeric antigen receptor （CAR） molecule on the biological behaviors of mesothelin （MSLN）-targeted chimeric antigen receptor T cell（CAR-T）， and to clarify the role of B2M in enhancing the therapeutic efficacy of CAR-T cells against MSLN-positive solid tumors. Methods The B2M and CAR co-expressing third-generation anti-MSLN CAR-T （B2M-28bbz-T） cells and control traditional anti-MSLN CAR-T （28bbz-T） cells were constructed. The 293T cells stably overexpressing MSLN （293T-MSLN） were constructed. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of B2M， variable region of heavy chain （VH）， and variable region of light chain （VL） mRNA in the CAR-T cells in various groups. Flow cytometry was used to detect the expressions of B2M protein and CAR molecule in the cells and the proliferation abilities of the CAR-T cells in various groups. The 293T-MSLN cells and MSLN-positive ovarian cancer SK-OV-3 cells were selected as target cells. Target cells were co-incubated with untransduced T lymphocytes， 28bbz-T cells， and B2M-28bbz-T cells at an effector-to-target ratio （E/T） of 1∶1 for 72 h. After co-incubation， the target cells were divided into T group， 28bbz-T group， and B2M-28bbz-T group. Cytotoxicity assay was used to detect the activities of target cells in various groups under the action of CAR-T cells. Enzyme-linked immunosorbent assay （ELISA） method was used to detect the levels of interferon-γ （IFN-γ） and tumor necrosis factor-α （TNF-α） in the supernatants of target cells in various groups. Twenty female NSG mice were randomly divided into four groups： phosphate-buffered saline（PBS） group， 28bbz-T group， B2M-28bbz-T group， and blue fluorescent protein （BFP）-28bbz-T group， with 5 mice in each group. The SK-OV-3 cells were subcutaneously implanted into the mice to construct ovarian cancer cell line-derived xenograft （CDX） models. The corresponding CAR-T cells were injected into the mice in various groups via the tail vein on the 7th day. After the observation， the mice were sacrificed， and the tumor volumes and weights of the mice in various groups were measured. Results The B2M-28bbz-T cells and 28bbz-T cells were successfully constructed. The RT-qPCR method results showed that compared with 28bbz-T group， the expression level of B2M mRNA in B2M-28bbz-T group was significantly increased （P<0.01）， while no significant differences were observed in the expression levels of VH and VL mRNA （P>0.05）. The flow cytometry results showed that no significant differences were observed in CAR molecule expression and proliferation ability of cells between 28bbz-T group and B2M-28bbz-T group. The cell toxicity assay results showed that compared with 28bbz-T group， the activities of 293T-MSLN cells and SK-OV-3 cells in B2M-28bbz-T group were significantly decreased （P<0.01 or P<0.001）. The ELISA method results showed that compared with 28bbz-T group， the levels of IFN-γ and TNF-α in the supernatant of 293T-MSLN cells and SK-OV-3 cells in B2M-28bbz-T group were significantly increased （P<0.05）. The in vivo experiment results showed that compared with PBS group， the tumor growth was inhibited in the mice in 28bbz-T group and B2M-28bbz-T group， and the tumor of one mouse in B2M-28bbz-T group was completely cleared. Compared with 28bbz-T group， the tumor weight of the mice in B2M-28bbz-T group was significantly decreased （P<0.01）， while no statistically significant difference was observed in the tumor weight of the mice in BFP-28bbz-T group （P>0.05）. Conclusion Co-expression of B2M and CAR molecules may enhance the cytotoxicity and in vivo anti-tumor activity of MSLN-targeting CAR-T cells by promoting T lymphocyte activation and pro-inflammatory cytokine secretion.

Key words: Chimeric antigen receptor T cell, Mesothelin, Beta-2-microglobulin, Cell therapy, Tumor immunity, Cell-line-derived xenograft model

中图分类号:

R392.11

李佳鑫,胡雄兵,李娟. 共表达B2M蛋白对靶向间皮素的3代CAR-T细胞生物学行为的影响[J]. 吉林大学学报(医学版), 2026, 52(5): 1224-1233.

Jiaxin LI,Xiongbing HU,Juan LI. Effect of co-expression of B2M protein on biological behaviors of third-generation CAR-T cells targeting mesothelin[J]. Journal of Jilin University(Medicine Edition), 2026, 52(5): 1224-1233.

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