目的：探索分泌PD-1 scFv的CAR-T细胞对胃癌的抗瘤功效。方法：选择EGFR作为CAR-T细胞的靶标，构建二代EGFR-CAR-T细胞（EGFR BB-z）和分泌PD-1 scFv的四代EGFR-CAR-T细胞（EGFR BB-z/E30），通过体外激活以及靶细胞长期刺激检测其抗肿瘤活性，通过胃癌移植瘤小鼠模型检测其肿瘤抑制能力。结果：胃癌组织及细胞均高表达EGFR（均P<0.01）。通过慢病毒感染的方式成功获得EGFR BB-z和EGFR BB-z/E30细胞。体外实验表明，与EGFR BB-z相比，EGFR BB-z/E30具有更长效的增殖能力及更强的肿瘤杀伤活性（均P<0.01）。体内实验显示了EGFR BB-z/E30在胃癌细胞皮下移植瘤模型和胃癌人源性肿瘤异种移植瘤（patient-derived tumor xenograft，PDX）模型中具有明显的肿瘤抑制能力（均P<0.01），并可显著增加肿瘤部位 T 细胞的浸润（P<0.01），减少 EGFR BB-z/E30 细胞表面 PD-1 的表达（P<0.01）和高分泌 IFN-γ 的水平（P<0.05）。结论：分泌PD-1 scFv的EGFR-CAR-T细胞EGFR BB-z/E30可对胃癌进展产生显著的抑制作用，为胃癌治疗提供一种潜在新策略。

Objective:To explore the anti-tumor effect of CAR-T cells secreting PD-1 scFv on gastric cancer. Method: We selected EGFR as the target of CAR-T cells and constructed second-generation EGFR-CAR-T cells (EGFR BB-z) and fourth-generation EGFRCAR-T cells secreting PD-1 scFv (EGFR BB-z/E30). The anti-tumor activity was examined after in vitro activation and long-term stimulation, and its tumor suppression ability was validated through a mouse gastric cell xenograft model. Results: EGFR was highly expressed in gastric cancer tissues and cells (all P<0.01). EGFR BB-z and EGFR BB-z/E30 cells were successfully obtained by lentivirus infection. In vitro experiments showed that compared with EGFR BB-Z, EGFR BB-Z/E30 had longer long-term proliferation ability and stronger tumor killing activity (all P<0.01). In vivo experiments also validated that EGFR BB-z/E30 had obvious tumor inhibitory function in subcutaneous gastric tumor cell transplanted xenograft model and patient-derived tumor xenograft model (PDX)(all P<0.01). It also significantly increased T cell infiltration in tumor site and decreased the expression level of PD-1 (P<0.01 or P<0.05) on EGFR BB-z/E30 cell surface as well as the high secretion of IFN-γ (P<0.05). Conclusion: EGFR-CAR-T cell EGFR BB-z/E30 secreting PD-1 scFv can significantly inhibit the progression of gastric cancer and provide a potential new strategy for the treatment of gastric cancer.

海南省卫生计生行业科研资助项目（No.1801320712A2180）