| ObjectivesTo construct a programmed cell death 1(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.Methods1.The Shanghai Outdo Biotech Co.Ltd.performed a microarray analysis of extrahepatic cholangiocarcinoma tissues from 27 patients(and nine samples of adjacent normal tissue)and gallbladder carcinoma tissues from 80 patients(and 20 samples of adjacent normal tissue).The expression level and cellular location of mesothelin in carcinoma and adjacent tissues were immunohistochemically explored.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-1 antibody 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 CD137 and 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 tract tumor cell lines EH-GB1,EH-GB2,EH-CA1a,EH-CA1b,and GBC-SD.The Real Time 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 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.WeconstructedEH-CA1a-Luccelllinesthatstablyexpressedthe 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.Results1.Compared to adjacent normal tissue,both the cytoplasm(3.65±2.98 vs.0.89±0.93)and the membrane(2.69±2.68 vs.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±2.73 vs.1.20±1.44),membrane(1.13±1.26 vs.0.25±0.44),and nucleus(0.70±1.25 vs.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).2.PD1-MesoCAR-T and MesoCAR-T[constituted about 30%of all T cells].The exogenous CD3 zeta chain(MesoCAR)was about 65 kDa in size,and the IgG4 Fcγ(of the anti-PD1 antibody)about 55 kDa,as revealed by Western blotting.The concentration of anti-PD1 antibody secreted by PD1-MesoCAR-T was ca.400 ng/mL as measured by ELISA.Compared to Control-T and MesoCAR-T,the anti-PD1 antibody secreted by PD1-MesoCAR-T lowered the levels of the immunosuppressive molecules TIM-3,LAG-3,and PD-1 in activated T cells.The CD3+CD8+and CD3+CD4+positivity levels were 45%and 52%for both PD1-MesoCAR-T and MesoCAR-T,respectively.The level of the CD137 marker was highest in PD1-MesoCAR-T,lower in MesoCAR-T,and lowest in Control-T.The CD28 marker 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.About 86.9%and 66.2%of EH-CA1a and EH-CA1b cells,respectively,were positive for mesothelin.The figures for EH-GB1,EH-GB2,and GBC-SD cells were approximately 18.2%,18.6%,and 9.7%,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 both8:1(0.9333±0.0029 vs.0.4041±0.4042)and 4:1(0.8698±0.0219 vs.0.3285±0.2823)(both P<0.05).MesoCAR-T exerted a lethal effect at the 4:1 ratio only(0.6825±0.0400 vs.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.After 20 days of growth,compared to the tumor fluorescence signal strength of the control(PBS)group(5134800.00±89334.76p/s/cm~2/sr),allofControl-T(4789800.00±45806.11 p/s/cm~2/sr),MesoCAR-T(4771600.00±50312.03 p/s/cm~2/sr),and PD1-MesoCAR-T(4766600.00±40997.56 p/s/cm~2/sr)significantly inhibited tumor growth(P<0.05).After 30 days,the tumor fluorescence signal strength of the PBS group was9216600.00±225496.79 p/s/cm~2/sr and those of the Control-T,MesoCAR-T,and PD1-MesoCAR-Tgroups7371400.00±120885.48,4677000.00±50808.46and4632800.00±55074.50 p/s/cm~2/sr,respectively.All of the novel T cell lines significantly inhibited tumor growth(P<0.05).After 40 days,the tumor fluorescence signal strength for thePBS,Control-T,MesoCAR-T,andPD1-MesoCAR-Tgroupswere364260000.00±437331402.00,47675000.00±4721924.04,5899200.00±652853.51,4432200.00±350740.22 p/s/cm~2/sr,respectively;all of the novel T cell lines significantly inhibited tumor growth(P<0.05).Up to 40 days,three mice died in the PBS and one in the Control-T group 1.Up to 50 days,four mice died in the PBS,three in the Control-T,and one in the MesoCAR-T group.Conclusions1.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. |
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