分泌型PD-1抗体可提高c-Met CAR-T细胞对胰腺癌细胞的杀伤作用

doi:10.12122/j.issn.1673-4254.2024.10.16

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (10): 1976-1984.doi: 10.12122/j.issn.1673-4254.2024.10.16

• • 上一篇

分泌型PD-1抗体可提高c-Met CAR-T细胞对胰腺癌细胞的杀伤作用

闵静婷¹(), 彭上¹, 杜娜娜¹, 安然², 甄翔程³, 曹佳威³, 周陈航⁴, 李正红⁵()

^1.蚌埠医科大学，基础医学院，安徽蚌埠 233000
^2.蚌埠医科大学，公共基础学院，安徽蚌埠 233000
^3.蚌埠医科大学，临床医学院，安徽蚌埠 233000
^4.蚌埠医科大学，口腔医学院，安徽蚌埠 233000
^5.蚌埠医科大学，生命科学学院，安徽蚌埠 233000

收稿日期:2024-06-20 出版日期:2024-10-20 发布日期:2024-10-31
通讯作者: 李正红 E-mail:429118736@qq.com;lzhbbmc@162.com
作者简介:闵静婷，硕士，讲师，E-mail: 429118736@qq.com
基金资助:
安徽高校自然科学研究项目(2023AH051991);国家级大学生创新创业训练计划项目(202310367011)

Enhanced tumoricidal activity of PD-1 antibody-secreting c-Met CAR-T cells against pancreatic cancer cells

Jingting MIN¹(), Shang PENG¹, Nana DU¹, Ran AN², Xiangcheng ZHEN³, Jiawei CAO³, Chenhang ZHOU⁴, Zhenghong LI⁵()

^1.College of Basic Medical Sciecens, Bengbu Medical University, Bengbu 233000, China
^2.School of Basic Sciences, Bengbu Medical University, Bengbu 233000, China
^3.Clinical Medical College, Bengbu Medical University, Bengbu 233000, China
^4.School of Stomatology, Bengbu Medical University, Bengbu 233000, China
^5.College of Life Sciences, Bengbu Medical University, Bengbu 233000, China

Received:2024-06-20 Online:2024-10-20 Published:2024-10-31
Contact: Zhenghong LI E-mail:429118736@qq.com;lzhbbmc@162.com

摘要/Abstract

摘要：

目的设计并制备能够分泌PD-1抗体和靶向c-Met的CAR-T细胞，以消除肿瘤对CAR-T细胞的免疫抑制作用，从而提高CAR-T细胞对胰腺癌的治疗效果。方法采用Kaplan-Meier Plotter、GEPIA和Timer2.0生物信息学数据库，分析c-Met在胰腺癌中的表达、生存期及免疫浸润。免疫组化检测胰腺癌临床样本c-Met和PD-L1表达，流式细胞术验证胰腺癌细胞Aspc-1 c-Met和PD-L1表达通过基因编辑将PD-1分泌型抗体和HIS标签连接至2代c-Met CAR分子后，构建PD-1/c-Met CAR质粒并包被慢病毒，慢病毒感染至活化T细胞内，通过流式细胞技术检测CAR-T阳性率和细胞亚群；Western blotting检测分泌型PD-1抗体在细胞上清液中的存在；体外功能试验中，通过LDH释放实验检测CAR-T对靶细胞的杀伤效率，CCK-8检测靶细胞存在下PD-1抗体对CAR-T增殖的促进作用。ELISA检测PD-1/c-Met CAR-T和c-Met CAR-T活化后细胞因子的分泌量。结果生信分析结果显示，胰腺癌组织c-Met表达高于正常组织（P<0.01）；c-Met表达水平与胰腺癌患者生存期呈负相关（P<0.01）。c-Met的表达可能与多种免疫细胞浸润成正相关。免疫组化结果显示，胰腺癌c-Met和PD-L1表达均高于癌旁组织（P<0.01）；流式细胞术结果显示，Aspc-1细胞c-Met和PD-L1表达量为90.7%和57.7%，琼脂糖凝胶电泳显示成功制备四代PD-1/c-Met CAR分子；流式细胞术和Western blotting显示，成功构建PD-1/c-Met CAR-T且PD-1抗体可顺利分泌。体外功能验证中LDH结果显示，PD-1/c-Met CAR-T对肿瘤细胞杀伤效率在效靶比为20：1时高于c-Met CAR-T（P<0.01）；CCK-8实验结果显示，靶细胞刺激72 h后增殖效率高于c-Met CAR-T（P<0.01）； ELISA结果显示，PD-1/c-Met CAR-T分泌的细胞因子IL-2和TNF-α高于c-Met CAR-T（P<0.01）。结论 PD-1抗体分泌型c-Met CAR-T可成功构建，并在体外胰腺癌细胞上显示出优于c-Met CAR-T肿瘤杀伤效率和增殖效率。

关键词: 嵌合抗原受体T细胞, c-Met, PD-1分泌型抗体, 胰腺癌

Abstract:

Objective To construct c-Met CAR-T cells secreting PD-1 antibodies to reduce immune inhibitory effect of tumor cells and enhance the efficacy of CAR-T cell therapy against pancreatic cancer. Methods Kaplan-Meier Plotter, GEPIA, and Timer 2.0 bioinformatics databases were used to analyze c-Met expression in pancreatic cancer and its correlation with survival and immune infiltration status. In clinical samples of pancreatic cancer and pancreatic cancer Aspc-1 cells, c-Met and PD-L1 expressions were detected using immunohistochemistry or flow cytometry. Using gene editing technology, PD-1 secretory antibodies and HIS tags were linked to second-generation c-Met CAR molecules to construct PD-1/c-Met CAR plasmids, which were then packaged into lentiviruses for infection of activated T cells. The positive rate and cell subset distribution of CAR-T cells were analyzed with flow cytometry, and secretory PD-1 antibodies in cell supernatants were detected using Western blotting. The target cell killing efficiency and proliferative activity of the modified CAR-T cells were evaluated after activation, and cytokine secretion was analyzed using ELISA. Results The expression of c-Met was significantly higher in pancreatic cancer than in normal tissues, and its expression level was negatively correlated with the patients' survival and positively correlated with immune cell infiltration. The clinical samples of pancreatic cancer tissues expressed significantly higher levels of c-Met and PD-L1 than the adjacent tissues, and 90.7% and 57.7% of Aspc-1 cells were positive for c-Met and PD-L1, respectively. The constructed PD-1/c-Met CAR-T cells were capable of secreting PD-1 antibodies and showed a significantly higher killing efficiency against tumor cells than c-Met CAR-T cells at an effector-to-target ratio of 20:1, with also a higher proliferative activity after target cell stimulation and higher levels of IL-2 and TNF-α secretin. Conclusion PD-1/c-Met CAR-T cells have higher killing efficiency against pancreatic cancer cells with also higher proliferative activity than c-Met CAR-T cells.

Key words: chimeric antigen receptor T-cell therapy, c-Met, secretary PD-1 antibody, pancreatic cancer

闵静婷, 彭上, 杜娜娜, 安然, 甄翔程, 曹佳威, 周陈航, 李正红. 分泌型PD-1抗体可提高c-Met CAR-T细胞对胰腺癌细胞的杀伤作用[J]. 南方医科大学学报, 2024, 44(10): 1976-1984.

Jingting MIN, Shang PENG, Nana DU, Ran AN, Xiangcheng ZHEN, Jiawei CAO, Chenhang ZHOU, Zhenghong LI. Enhanced tumoricidal activity of PD-1 antibody-secreting c-Met CAR-T cells against pancreatic cancer cells[J]. Journal of Southern Medical University, 2024, 44(10): 1976-1984.

图/表 8

图1 基于生物信息学分析c-Met基因在胰腺癌中的表达及临床意义

Fig.1 Bioinformatics analysis of c-Met expression in pancreatic cancer and its clinical significance. A: Analysis of expression level of c-Met protein in PAAD tissues and adjacent normal tissues based on data from GEPIA database. *P<0.01 vs normal tissues. B: Expressions of MET, EGFR, MUC1 and GAS6 in esophageal cancer and normal tissues. C: Expression level of c-Met protein in PAAD patients in different clinicopathological stages. D: c-Met expression level in PAAD is negatively correlated with overall survival (OS). E: Expression level of c-Met in PAAD is negatively correlated with progression-free survival (PFS). F: High expression of c-Met is associated with low survival rate in stage 2 patients. G: High expression of c-Met is associated with poor survival in stage 3 patients. H: Expression of c-Met is positively correlated with CD8+ T cells, dendritic cells, NK cells and macrophages.

图2 免疫组化检测胰腺癌组织样本c-Met 、PD-L1蛋白的表达

Fig.2 Expression of c-Met and PD-L1 protein in pancreatic cancer tissue samples detected by immunohistochemistry. A: c-Met immunohistochemical staining of clinical specimens of pancreatic cancer tissues (×400). B: PD-L1 immunohistochemical staining of pancreatic cancer tissues (×400). C: c-Met immunohistochemical staining of adjacent tissues (×400). D: PD-L1 immunohistochemical staining of adjacent tissues (× 400 ). E, F: Statistical chart of C-met and PD-L1 pathological scores.

图3 流式细胞术检测Aspc-1表面c-Met、PD-L1蛋白的表达

Fig.3 Flow cytometry for analysis of c-Met and PD-L1 protein expressions on Aspc-1 cell surface.

图4 CAR-T设计、制备与检测

Fig.4 CAR-T cell design, construction and detection. A: CAR core structure diagram. B: Electrophoresis of BamHI digestion products of CAR plasmid. C: CAR-T transfection positive rate. D: CAR-T cell subset distribution. ***P<0.001 vs T cells.

图5 细胞上清液中HIS标记PD-1 Scfv免疫印记

Fig. 5 Immunoblotting of HIS-labeled PD-1 Scfv in the cell supernatant.

图6 靶细胞刺激下效应细胞增殖变化

Fig.6 Changes in proliferative activity of the effector cells stimulated by the target cells. *P<0.01 vs T cells; #P<0.01 vs CD19 CAR-T; &P<0.01 vs c-Met CAR-T.

图7 不同效靶比下各效应细胞对靶细胞的杀伤效率

Fig.7 Efficiency of the effector cells for target cell killing in different effector-target ratios. *P<0.01 vs T cells; #P<0.01 vs CD19 CAR-T; &P<0.01 vs c-Met CAR-T.

图8 CAR-T细胞因子释放柱状图

Fig.8 CAR-T cell cytokine release determined using ELISA. #P<0.01 vs T cells; *P<0.01 vs CD19 CAR-T; &P<0.01 vs c-Met CAR-T group.

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分泌型PD-1抗体可提高c-Met CAR-T细胞对胰腺癌细胞的杀伤作用

Enhanced tumoricidal activity of PD-1 antibody-secreting c-Met CAR-T cells against pancreatic cancer cells

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