| Objective The lentiviral expression vector with HN3 as a single-chain antibody was constructed,and the difference in targeting and effectiveness of T cells modified with lentiviral expression vector with single-chain antibody of GC33 was compared for the purpose of selecting more optimized GPC3-CAR-T used in the treatment of liver cancer,laying the foundation of the clinical application of GPC3-CAR.Methods 1 HN3-4-1BB-CD3? and HN3-CD28-4-1BB-CD3? gene sequences(HBB?,H28BB? for short,the second generation and third generation GPC3-CAR-T cells with GC33 as single-chain antibody constructed in the previous group are referred to as GBB? and G28BB?)was synthesized by gene synthesis technology.The CAR gene with HN3 as a single-chain antibody was synthesized,and the plasmid of interest was prepared.The CAR-T cells constructed with different single-chain antibodies against GPC3 were obtained by lentiviral transfection,The expression of GPC3-CAR on T cells was detected by Western blot and flow cytometry.The percentage of CD3+CD8+ T cells and CD3+CD4+ T cells in 4 groups of CAR-T and control Mock cells was detected by flow cytometry;2 Detection of GPC-3 expression in multiple hepatoma cells was made by Q-PCR,Western-blot and flow cytometry,we selected hepatoma cell lines with different degrees of expression of GPC-3;using cytotoxicity assay to detect the difference in killing efficiency of different single-chain antibodies constructed GPC3-CAR-T cells against different GPC3 expression levels' liver cancer cells;cytokine secretion after GPC3-CAR-T co-culture with tumor cells was detected by ELISA;3 The NCG mouse liver cancer model was established by GPC3 positive liver cancer cells,and GPC3-CAR-T cells were injected into the tail vein.The ability of CAR-T cells to inhibit liver cancer cells in vivo was evaluated by measuring the volume of subcutaneous tumors in mice.The homing ability of CAR-T was evaluated by pathological immunohistochemical staining.The TUNEL Apoptosis Kit detects apoptosis of tumor cells to evaluate the efficacy of CAR-T cells.Results 1 Lentivirus-infected T cells,flow detection results show that CAR-T infection rate is 30%-80%,Western-blot confirmed that chimeric antigen receptor targeting GPC3 can be expressed on T cells;flow detection CAR-T phenotype,It was found that the CD8+ T cells that played a major killing effect in GPC3-CAR-T gradually increased after in vitro culture;2 By means of QPCR,flow cytometry and Western-blot,we found GPC3 was highly expressed in HepG2,Huh-7,Hep3 B and two primary hepatoma cells 1214,1222,has 70%-98.8% positive rate,PLC/PRF/ 5,SMMC7721 is low expression,does not express GPC3 liver cancer cell lines are SK-HEP-1,QGY7701,Bel7402,97L;3 In vitro killing experiments showed that the killing efficiency of GPC3-CAR-T cells against GPC3-positive hepatoma cells increased when with the effective target ratio increase.The killing efficiency of CAR-T cells constructed with GC33 as single-chain antibody was more significant in the same algebraic CAR-T cells.The third generation CAR-T cells have better killing efficiency than the second generation when constructed with the same single-chain antibody.However,when the target cells were GPC3-negative liver cancer cells,there was no significant difference between the groups(P>0.05);the co-culture supernatant detected higher levels of cytokine secretion in CAR-T with GC33 as a single-chain antibody,especially in Hep-G2 that with high expression of GPC3;4 The liver cancer NCG mouse model was successfully established by subcutaneous injection of GPC3 positive hepatoma cells.The GPC3-CAR-T cell treatment group had slower tumor growth rate and average tumor volume than that of the MOCK cell group.They were statistically different(P<0.05).HE staining showed that GPC3-CAR-T cells had no obvious side effects on important organs of mice.Immunohistochemical staining suggested that more CD3+CD8+ T cells infiltrated tumor tissues,which was significantly higher than that of the control group.The apoptosis rate of tumor cells was detected by TUNEL method,the apoptosis rate of GPC3-CAR-T cell treatment group was higher than that of MOCK group,which was statistically different(P<0.05).Conclusions 1 The lentivirus carrying the CAR gene with HN3 as a single-chain antibody was successfully synthesized.The CAR protein was stably expressed on the surface of T cells after transfection.2 GPC3-CAR-T cells cultured in vitro could proliferate in large amounts and were mostly converted into CD8 + effector T cells;3 In vitro,the third-generation GPC3-CAR-T cells were more efficient than the second-generation CAR-T cells when they were constructed with the same single-chain antibody,in the same generation of GPC3-CAR-T cells,the CAR-T cells constructed with GC33 showed stronger killing ability.CAR-T cells constructed with GC33 as a single-chain antibody secrete higher levels of cytokines when co-cultured with target cells in vitro;4 GPC3-CAR-T cells in each group can inhibit the growth of GPC3-positive liver cancer mouse model,but the difference between the groups is not obvious. |
PDF Full Text Request