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

Abstract

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

CLC Number:

R392.11

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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Effect of co-expression of B2M protein on biological behaviors of third-generation CAR-T cells targeting mesothelin

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