目的：探讨共表达淋巴细胞活化基因3（lymphocyte activation gene 3, LAG-3）抗体（LAG-3 antibody，aLAG）的溶瘤腺病毒对成胶质细胞瘤的抗肿瘤活性。方法：在溶瘤腺病毒Ad3的骨架中插入aLAG序列，获得重组溶瘤腺病毒Ad3-aLAG。应用WB法检测感染成胶质细胞瘤GL261细胞中aLAG的表达水平，MTT法检测重组溶瘤腺病毒对GL261细胞的杀伤能力，通过小鼠皮下移植瘤模型评估重组溶瘤腺病毒对成胶质细胞瘤的体内肿瘤生长的抑制效果，免疫组化染色法检测肿瘤浸润T细胞，并通过流式细胞术检测肿瘤浸润T细胞分泌细胞因子TNF-α及IFN-γ的水平。结果：成功构建的重组溶瘤腺病毒可以有效表达aLAG（P<0.01），并且可以在体外杀伤GL261细胞（P<0.01）。小鼠皮下移植瘤模型实验结果显示，重组溶瘤腺病毒Ad3-aLAG较Ad3溶瘤腺病毒而言肿瘤抑制能力更强（P<0.01），并且可以有效增强移植瘤组织中 CD3+ T 细胞的浸润（P<0.01）、增强浸润 T 细胞分泌IFN-γ的能力（P<0.01）。结论：Ad3-aLAG重组溶瘤腺病毒在体内外均显著抑制成胶质细胞瘤细胞的生长，同时增强体内的抗肿瘤免疫反应，有望为成胶质细胞瘤治疗提供一种新的方案。

Objective: To explore the anti-tumor activity of oncolytic adenovirus co-expressing lymphocyte activation gene 3 (LAG-3)antibody (aLAG) against glioblastoma. Methods: aLAG sequence was inserted into the skeleton of oncolytic adenovirus Ad3 to obtain recombinant oncolytic adenovirus (Ad3-aLAG). The expression of aLAG in infected gliblastoma GL261 cells was detected by WB. The cytotoxicity of recombinant oncolytic adenovirus against glioblastoma was detected by MTT method. The tumor inhibitory activity of recombinant oncolytic adenovirus against glioblastoma in vivo was evaluated with mice subcutaneous xenograft model. Tumor infiltrating T cells were detected by immunohistochemical staining, and the levels of cytokines TNF-α and IFN-γ secreted by tumor infiltrating T cells were detected by Flow cytometry. Results: The recombinant oncolytic adenovirus was successfully constructed,which could effectively express aLAG and kill GL261 cells in vitro (all P<0.01). Experimental results of mice subcutaneous xenograft model showed that the tumor inhibition ability of recombinant oncolytic adenovirus Ad3-aLAG was stronger than that of Ad3 oncolytic adenovirus (P<0.01), and Ad3-aLAG could effectively enhance the infiltration of CD3+T cells in tumor tissue (P<0.01) and enhance the IFN- γ secretion ability of infiltrating T cells (P<0.01). Conclusion: Ad3-aLAG recombinant oncolytic adenovirus can significantly inhibit the growth of glioblastoma cells in vivo and in vitro, and enhance the anti-tumor immune response in vivo, which is promising to provide a new scheme for the treatment of glioblastoma.

海南省卫生健康行业科研项目（No. 19A200024）