4.第20天，与PBS组荧光信号强度（p/s/cm2/sr）相比，Control-T（5134800.00±89334.76 vs.4789800.00±45806.11），MesoCAR-T（5134800.00±89334.76vs.4771600.00±50312.03），PD1-MesoCAR-T（5134800.00±89334.76vs.4766600.00±40997.56）组肿瘤增长缓慢，差异有统计学意义（P<0.05）。第30天，与PBS组肿瘤荧光信号强度（p/s/cm2/sr）相比，Control-T （9216600.00±225496.79vs.7371400.00±12088222au
5.48），MesoCAR-T（9216600.00±22549
6.79vs. 4677000.00±50808.46），PD1-MesoCAR-T（9216600.00±225496.79vs.4632800.00±55074.50）组肿瘤增长缓慢，与Control-T组相比，MesoCAR-T（7371400.00±120885.48vs. 4677000.00±50808.46），PD1-MesoCAR-T（7371400.00±120885.48vs.4632800.00±55074.50）组肿瘤增长缓慢，差异有统计学意义（P<0.05）。第40天，与PBS组荧光信号强度（p/s/cm2/sr）相比，Control-T（364260000.00±437331402.00vs.47675000.00±4721924.04），MesoCAR-T（3642600 00.00±437331402.00vs.5899200.00±652853.51），PD1-MesoCAR-T（364260000.00±437331402.00 vs.4432200.00±350740.22）组肿瘤增长缓慢，与Control-T组相比，MesoCAR-T （47675000.00±4721924.04vs.5899200.00±652853.51），PD1-MesoCAR-T（47675000.00±4721924. 04vs.4432200.00±350740.22）组肿瘤增长缓慢，差异有统计学意义（P
<0.05）。到第40天，PBS组死亡小鼠3只，Control-T组死亡小鼠1只。到第50天PBS组死亡小鼠4只，Control-T组死亡小鼠3只，MesoCAR-T组死亡小鼠1只。
结论
生活垃圾处理器1.本组织芯片免疫组化实验研究结果说明相对于肝外胆管癌癌旁组织，间皮素在肝外胆管癌组织中呈高表达，癌细胞胞浆和胞膜中均有差异性表达。相对于胆囊癌癌旁组织，间皮素在胆囊癌组织中呈高表达，癌细胞胞浆，胞膜和胞核中均有差异性表达。对于研究者进一步研究间皮素与胆道恶性肿瘤的发生发展机制及其生物学行为关系提供参考，也为本课题后续实验提供了临床组织学水平研究基础； 2.成功构建了表达PD-1抗体的靶向间皮素嵌合抗原受体修饰T细胞，为后续体外、体内研究及其应用于胆道恶性肿瘤的临床试验奠定基础；
3.PD1-MesoCAR-T细胞和MesoCAR-T细胞在一定的效靶比前提下对胆道肿瘤细胞胞膜间皮素高表达细胞株均有一定的杀伤能力，并且能促进细胞因子在效应细胞胞内的分泌。对肿瘤细胞胞膜间皮素表达较低的细胞株杀伤效果不明显；
4.PD1-MesoCAR-T细胞和MesoCAR-T细胞均对体内间皮素高表达胆管癌细胞株移植瘤有一定的抑制作用。两者间移植瘤的抑制作用暂未发现明显差异，需要弥补本课题的不足，完善研究方案，进一步研究。
关键词：PD-1抗体，间皮素，嵌合抗原受体T细胞，胆道肿瘤
作者：倪庆强
指导教师：吴孟超、杨甲梅、钱其军
A preliminary study of PD-1-antibody-expressing,chimeric antigen-receptor T cells targeting mesothelin in patients with
malignant biliary tumors
Abstract
光触媒滤网Objectives
To construct a programmed cell death1(PD-1)-antibody-expressing,chimeric, antigen-receptor T cell(CAR-T)targeting mesothelin(PD1-MesoCAR-T).We report our preliminary in vitro and in vivo data on the effects of PD1-MesoCAR-T on extrahepatic cholangiocarcinoma and gallbladder carcinoma.Our purpose was to develop new ways in which genetically modified T cells may be used to treat malignant biliary tract tumors.
Methods
1.The Shanghai Outdo Biotech Co.Ltd.performed a microarray analysis of extrahepatic cholangiocarcinoma tissues from27patients(and nine samples of adjacent normal tissue)and gallbladder carcinoma tissues from80patients(and20samples of adjacent normal tissue).The expression level and cellular location of mesothelin in carcinoma and adjacent tissues were immunohistochemically explored.
阀门手轮
2.PD1-MesoCAR and MesoCAR genes were synthesized and cloned into expression plasmids.PD1-MesoCAR-T was constructed using the electroblot method.Western blotting and enzyme-linked immunosorbent assays(ELISAs)were used to measure the expression levels of anti-PD-1antibody and MesoCAR in the T cells.Cells expressing the immunosuppressive molecule PD1,the T cell immunoglobulin domain,mucin domain-3 (Tim-3),and lymphocyte activation gene-3(LAG-3)were detected by flow cytometry. This technique was also used to measure the proportions of CD3+CD8+and CD3+CD4+ cells among CAR-T,the levels of the T cell activation markers CD137and CD28,and the levels of the memory T cell markers CD62L and CCR7.
3.Western blotting and flow cytometry were used to measure mesothelin expression by the biliary tra
ct tumor cell lines EH-GB1,EH-GB2,EH-CA1a,EH-CA1b,and GBC-SD. The RealTime Cellular Analysis system and the lactose dehydrogenase method were employed to explore whether the novel T cell lines exerted lethal effects on the tumor cell
等离子割
lines listed above.Cytokine secretion by the novel T cell lines was determined by flow cytometry after co-culture of CAR-T and EH-CA1a cells.
4.We constructed EH-CA1a-Luc cell lines that stably expressed the luciferase reporter.EH-CA1a Luc cells were intra-tumorally injected into NOD/SCID mice, followed by MesoCAR-T and PD1-MesoCAR-T.We conducted in vivo imaging and measured tumor fluorescence signal strength.
Results
1.Compared to adjacent normal tissue,both the cytoplasm(3.65±
2.98vs.0.89±0.93) and the membrane(2.69±2.68vs.0.22±0.67)of extrahepatic bile duct carcinoma tissue expressed significantly greater levels of mesothelin(both P<0.05)(cytoplasm: 47.31±38.29%vs.6.56±10.29%;membrane:22.96±30.88%vs.0.11±0.33%;both P<0.05). Compared to adjacent normal tissue,all of the cytoplasm(2.15±2.73vs.1.20±1.44), membrane(1.13±1.26vs.0.25±0.
44),and nucleus(0.70±1.25vs.0.15±0.67)of gallbladder carcinoma tissue expressed significantly higher levels of mesothelin(all P<0.05) (cytoplasm:30.72±39.80%vs.10.45±22.18%;membrane:5.72±11.60%vs.1.10%±2.59%; both P<0.05).
触摸屏手套2.PD1-MesoCAR-T and MesoCAR-T[constituted about30%of all T cells].The exogenous CD3zeta chain(MesoCAR)was about65kDa in size,and the IgG4Fcγ(of the anti-PD1antibody)about55kDa,as revealed by Western blotting.The concentration of anti-PD1antibody secreted by PD1-MesoCAR-T was ca.400ng/mL as measured by ELISA.Compared to Control-T and MesoCAR-T,the anti-PD1antibody secreted by PD1-MesoCAR-T lowered the levels of the immunosuppressive molecules TIM-3,LAG-3, and PD-1in activated T cells.The CD3+CD8+and CD3+CD4+positivity levels were45% and52%for both PD1-MesoCAR-T and MesoCAR-T,respectively.The level of the CD137marker was highest in PD1-MesoCAR-T,lower in MesoCAR-T,and lowest in Control-T.The CD28marker levels did not differ significantly among the cell lines (CD137:PD1-MesoCAR-T,MesoCAR-T,Control-T:22.9%,15.4%,6.3%；CD28：PD1-MesoCAR-T,MesoCAR-T:99.0%,98.6%;PD1-MesoCAR-T,Control-T:99.0%, 99.0%;MesoCAR-T,Control-T:98.6%,99.0%).Both PD1-MesoCAR-T and MesoCAR-T stimulated the formation of central memory T cells and effector memory T cells to an equal extent(CCR7+CD62L+:PD1-MesoCAR-T,MesoCAR-T:15.2%,15.8%;PD1-MesCAR-T, Control-
T:15.2%,8.2%;MesoCAR-T,Control-T:15.8%,8.2%;CCR7-CD62L-:PD1-Mes oCAR-T,MesoCAR-T:28.5%,26.0%;PD1-MesoCAR-T,Control-T:28.5%,12.6%;
MesoCAR-T,Control-T:26.0%,12.6%).
3.About86.9%and66.2%of EH-CA1a and EH-CA1b cells,respectively,were positive for mesothelin.The figures for EH-GB1,EH-GB2,and GBC-SD cells were approximately18.2%,18.6%,and9.7%,respectively.The lethal effects of the new T-cell clones on EH-GB1,EH-GB2,EH-CA1b,and GBC-SD cells were in the order PD1-MesoCAR-T,MesoCAR-T,and Control-T.Compared with the Control-T group,the lethal effect of PD1-MesoCAR-T was higher when the effector/target cell ratios were both 8:1(0.9333±0.0029vs.0.4041±0.4042)and4:1(0.8698±0.0219vs.0.3285±0.2823)(both P<0.05).MesoCAR-T exerted a lethal effect at the4:1ratio only(0.6825±0.0400vs.
0.3285±0.2823)(P<0.05).The intracellular levels of the cytokines IL-2,IL-4,IL-6,IL-10, TNF-α,and IFN-r were higher after co-culture of PD1-MesoCAR-T/MesoCAR-T and EH-CA1a cells than after co-culture of Control-T and EH-CA1a cells(both P<0.05). Compared with MesoCAR-T,the intracellular levels of the cytokines IL-4,IL-6,IL-10, and TNF-αwere higher after co-culture of PD1-MesoCAR-T and EH-CA1a cells(P<0.05).
4.After20days of growth,compared to the tumor fluorescence signal strength of the control(PBS)group(5134800.00±89334.76p/s/cm2/sr),all of Control-T (4789800.00±45806.11p/s/cm2/sr),MesoCAR-T(4771600.00±50312.03p/s/cm2/sr),and PD1-MesoCAR-T(4766600.00±40997.56p/s/cm2/sr)significantly inhibited tumor growth (P<0.05).After30days,the tumor fluorescence signal strength of the PBS group was 9216600.00±225496.79p/s/cm2/sr and those of the Control-T,MesoCAR-T,and PD1-MesoCAR-T groups7371400.00±12088
5.48,4677000.00±50808.46and 4632800.00±55074.50p/s/cm2/sr,respectively.All of the novel T cell lines significantly inhibited tumor growth(P<0.05).After40days,the tumor fluorescence signal strength for the PBS,Control-T,MesoCAR-T,and PD1-MesoCAR-T groups were 364260000.00±437331402.00,47675000.00±4721924.04,5899200.00±652853.51, 4432200.00±350740.22p/s/cm2/sr,respectively;all of the novel T cell lines significantly inhibited tumor growth(P<0.05).Up to40days,three mice died in the PBS and one in the Control-T group1.Up to50days,four mice died in the PBS,three in the Control-T,and one in the MesoCAR-T group.
Conclusions
1.We immunohistochemically analyzed a tissue microarray.Compared to adjacent normal tissue,the mesothelin expression level was greatly elevated in extrahepatic bile duct carcinoma tissue;the levels differed between the cytoplasm and membrane.This was
also true for gallbladder carcinoma,in which tissue mesothelin was also expressed in the nucleus.These data encourage further study of correlations between mesothelin expression levels and the occurrence,development,and biological behavior of malignant biliary tract tumors.Clinical histological data would also be useful.
2.We successfully constructed PD1-MesoCAR-T,thus laying the foundation for future in vitro/vivo studies and clinical trials seeking to treat malignant biliary tract tumors.
3.At certain effector-target ratios,both PD1-MesoCAR-T and MesoCAR-T killed mesothelin-overexpressing,malignant,bile duct tumor cells,and promoted intracellular cytokine production by effector T cells.In contrast,the transgenic T cell lines exerted no obvious effects on malignant,bile duct tumor cells that expressed only low levels of mesothelin.
4.Both PD1-MesoCAR-T and MesoCAR-T similarly inhibited tumor growth after injection of mesothelin-overexpressing cholangiocarcinoma cell lines into nude mice. Further study is required.
Key words:anti-PD-1antibody,mesothelin,chimeric antigen-receptor T cells, malignant biliary tract tumors
Written by：Qingqiang Ni
Supervised by：Mengchao Wu,Jiamei Yang,Qijun Qian
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