嵌合抗原受体T细胞靶向LMP1抗原治疗EB病毒阳性淋巴瘤的功能研究

OBJECTIVE: This study aimed to create a type of CAR-T cells that targets LMP1 antigen and study its immunotherapeutic effect on LMP1-positive hematological malignancies. METHODS: To generate LMP1 CAR-T cells, a plasmid expressing LMP1 CAR was created using molecular cloning technology, and T cells were infected with LMP1 CAR lentivirus. The effects of LMP1 CAR-T cells on specific cytotoxicity against LMP1-positive tumor cell lines infected with the EB virus had been confirmed. RESULTS: ① LMP1 protein expressing on EB virus-positive lymphoma cells surface was verified. ② The LMP1 CAR-expressing plasmid was created, and LMP1 CAR-T cells were obtained by infecting T cells with a lentivirus packaging system, with an infection efficiency of more than 80％. ③LMP1 CAR-T cells have a 4∶1 effect-to-target ratio in killing LMP1-positive lymphoma cells. The killing effect of LMP1 CAR-T cells on Raji cells was enhanced after 48 h of coculture, but there was no significant killing effect on Ramos, which are LMP1-negative lymphoma cells. ④After coculture with LMP1-positive lymphoma cells at a ratio of 1∶1 for 5 h, the degranulation effect was enhanced. The proportion of CD107a(+) T cells in the LMP1 CAR-T cell treatment group was significantly higher than that in the vector-T cell group［（13.25±2.94）％ vs（1.55±0.05）％, t＝3.972, P＝0.017］. ⑤After coculture with LMP1-positive lymphoma cells, the proportion of CD69(+) and CD25(+) T cells in the LMP1 CAR-T cell group was significantly higher than that in vector-T cell group［（7.40±0.41）％ vs（3.48±0.47）％, t＝6.268, P＝0.003;（73.00±4.73）％ vs（57.67±2.60）％, t＝2.842, P＝0.047］. ⑥After coculture with LMP1-positive lymphoma cells, cytokine secretion in the LMP1 CAR-T cell group was higher than that in the vector-T cell group［interferon-gamma:（703±73）ng/L vs（422±87）ng/L, t＝2.478, P＝0.068; tumor necrosis factor-alpha:（215±35）ng/L vs（125±2）ng/L, t＝2.536, P＝0.064］. CONCLUSION: In this study, we found that the LMP1 protein is only found on the surface of the EBV-positive tumor cell. Simultaneously, we created an LMP1 CAR-expressing plasmid and obtained LMP1 CAR-T cells by infecting T cells with a lentivirus packaging system. Furthermore, we demonstrated that LMP1 CAR-T cells could specifically kill LMP1-positive tumor cells in vitro. The degranulation and activation effects of LMP1 CAR-T cells were enhanced after coculture with LMP1-positive tumor cells, indicating a potential clinical application.