MEK抑制剂显著增强EGFR CAR-T细胞对口腔鳞癌的抗肿瘤活性

目的:构建靶向表皮生长因子受体（epidermal growth factor receptor，EGFR）的二代嵌合抗原受体（chimeric antigen receptor，CAR）-T细胞，以探索MEK抑制剂联合EGFR CAR-T细胞是否可以对口腔鳞癌产生显著的抗肿瘤活性。方法:免疫组化法检测口腔鳞癌患者癌和癌旁组织中EGFR的表达；流式细胞术检测口腔鳞癌细胞系中EGFR的表达；慢病毒感染法构建EGFR CAR-T细胞；生物发光法检测EGFR CAR-T细胞联合MEKi对靶细胞的杀伤活性；流式细胞术检测共孵育后效靶细胞的占比情况；活体成像监测小鼠肿瘤的生长；免疫组化法检测小鼠肿瘤组织中CD3的表达；ELISA法检测肿瘤组织中IFN-γ和GrmB的含量。结果:人口腔鳞癌组织较癌旁组织EGFR显著高表达，大部分口腔鳞癌细胞系高表达EGFR。联合MEKi在短时间的共孵育中并不能显著增强EGFR CAR-T细胞对肿瘤细胞系的杀伤活性，但在较长时间的共孵育后，联合MEKi显著增强EGFR CAR-T细胞的增殖（P＜0.01）和对肿瘤细胞的细胞毒性（P＜0.001）。体内实验也表明，联合MEKi较单独使用EGFR CAR-T细胞而言对肿瘤抑制能力更强（P＜0.01），肿瘤组织中CD3+T细胞的浸润更多（P＜0.001），肿瘤部位细胞因子IFN-γ（P＜0.001）和GrmB（P＜0.001）的含量也更高。结论:联合MEKi可以在体内外条件下显著增强EGFR CAR-T细胞的抗肿瘤活性，有望成为针对口腔鳞癌的潜在治疗策略。

MEK inhibitors significantly enhanced the antitumor activity of EGFR CAR-T cells against oral squamous cell carcinoma

Objective:Constructing second-generation chimeric antigen receptor (CAR)-T cells targeting epidermal growth factor receptor (EGFR) to explore whether MEK inhibitor combined with EGFR CAR-T cells can produce significant antitumor activity against oral squamous cell carcinoma.Methods:The expression of EGFR in carcinoma and adjacent tissues of oral squamous cell carcinoma was detected by immunohistochemistry.The expression of EGFR in oral squamous cell carcinoma cell lines was detected by flow cytometry.EGFR CAR-T cells were constructed by lentivirus infection.The killing activity of EGFR CAR-T cells combined with MEKi against target cells was detected by bioluminescence assay.Flow cytometry was used to detect the proportion of target cells after co-incubation.In vivo imaging was used to monitor tumor growth in mice.The expression of CD3 in mouse tumors tissues was detected by immunohistochemistry.The contents of IFN-γ and GrmB in tumor tissues were determined by ELISA.Results:The expression of EGFR in human oral squamous cell carcinoma tissues was significantly higher than in paracancerous tissues.EGFR was highly expressed in most of the oral squamous cell carcinoma cell lines.The combination of MEKi did not significantly enhance the killing activity of EGFR CAR-T cells against tumor cell lines after a short period of co-incubation,but the combination of MEKi significantly enhanced the proliferation （P＜0.01） and cytotoxicity （P＜0.001） of EGFR CAR-T cells after a longer period of co-incubation.In vivo studies also showed that MEKi combined with EGFR CAR-T cells demonstrated greater tumor inhibition than EGFR CAR-T cells alone,with more CD3+T cell infiltration in tumor tissues （P＜0.001）,and higher levels of IFN-γ （P＜0.001） and GrmB （P＜0.001） in tumor sites.Conclusion:Combined with MEKi can significantly enhance the antitumor activity of EGFR CAR-T cells in vivo and in vitro,which is expected to be a potential therapeutic strategy for oral squamous cell carcinoma.