Second-generation And Third-generation GPC3-CAR Modified T Lymphocytes Targeting Hepatocellular Carcinoma In Vivo

Background and ObjectiveThe chimeric antigen receptors?CARs?are chimeric immunoglobulin T cell receptor?TCR?molecules that are derived from transgenes encoding single-chain variable fragments?scFv?,Combined with immunoreceptor tyrosine-based activation motifs of T cells,CARs can specifically recognize and bind to antibodies from tumor-associated antigens?TAA?,induce changes in cell conformation,cause the expression of transcription factors as well as the release of cytokines,and therefore inducing T lymphocytes to produce cytotoxic activity against tumor cells.In recent years,researchers have tried to improve the intensity and duration of the transmitting signals transmitted by chimeric antigen molecules by reforming the intracellular signaling domain of CARs.The CAR-T which has undergone changes from the first generation to the fifth generation,with constant and significant renovations,the 5^th generation CAR-T has becomes a new hot spot for immunotherapy.Hepatocellular carcinoma?HCC?is one of the most common cancers in the world.Much as the treatment to it has improved at the level of clinic,the survival rate remains as low as 25%to 39%among those in range of over 5 years.Glypican-3?GPC3?is a member of the glypican family.It's reported that the possibility in its expression is as high as 80%in primary hepatocellular carcinoma,but it is not expressed in normal liver tissue.Due to the specificity and high sensitivity of GPC3 to HCC,GPC3 is expected to be a new target for the diagnosis and treatment of hepatocellular carcinoma.In the previous study,GPC3-CAR was introduced into T cells and GPC3-CARs of the second and the third generation were constructed.T-lymphocytes were successfully transfected and in vitro killing experiments were carried out to obtain the preliminary results.Based on the previous studies,we set up experiments in vivo in order to further verify the effectiveness of GPC3-CAR in the targeted therapy of hepatocellular carcinoma.GPC3-CAR with good targeting,strong lethality and safety is the foundation for the clinical application of GPC3-CAR adoptive T cell therapy in the treatment of hepatocellular carcinoma.Methods1.Cultivation of liver cancer cell linesIn our previous study,we have screened out HepG2 and GPC3-overexpressing Sk-Hep-1 hepatocellular carcinoma cell lines with high GPC3 expression in hepatoma cells.HepG2 and Sk-Hep-1 hepatocellular carcinoma cell lines were cultured and massively expanded with 10%complete medium?fetal bovine serum:MEM medium=1:9?.2.Construction of tumor-bearing model of mouse hepatocellular carcinoma6-8 weeks-old NOD/SCID mice?purchased from Shanghai Lingchang Biotechnology Company,No.2013001828737?were selected for our experiment.The hepatocellular carcinoma cell lines were firstly cultivated to a certain number.Then we extracted the hepatoma cells in the logarithmic phase of cell growth diluted to 5×10 ⁶/0.2 ml and injected into the mouse's right thigh subcutaneously to establish a HepG2 and Sk-Hep-1 subcutaneous hepatocellular carcinoma tumor-bearing mouse model.3.Preparation of GPC3-CAR gene modified T cellsPeripheral blood was collected from healthy individuals.T cells were divided into magnetic beads to sort T lymphocytes,and T cells were optimally cultured with cytokines?300 U/mL?.The second generation of GPC3-CAR?GBBz?,the second generation of GPC3-CAR?G28BBz?and the empty vector Mock were used to infect T lymphocytes to make Mock-T and GBBz CAR-T and G28BBz CAR-T cultured in vitro.After 28 days of in vitro culture,a certain number of patients are reached for follow-up treatment.4.Adoptive immunotherapy of GPC3-CAR in tumor-bearing mice with hepatocellular carcinomaWhen the tumor size was 100 to 200 mm³,the HepG2 and Sk-Hep-1 mouse models were randomly divided into 4 groups?n=5?:sterile saline group,Mock-T group,GBBz-T and G28BBz-T,8×10⁶ T cells were injected into the tail vein of each mouse for adoptive immunotherapy in tumor-bearing mice.5.Observe the survival of tumor-bearing mice and tumor growthTumor-bearing mice were observed spirit,diet,defecation and body weight and other general survival,tumor growth in vivo and survival of tumor-bearing mice,tumor-bearing mice choose CAR-T adoptive immunotherapy opportunity to observe the following indicators:?1?observe spirit,diet,defecation and body weight of mice;?2?observe the size of the tumor;?3?execute the mice,take out the tumor and organs such as liver,spleen,heart,lung,kidney and brain and then to observe the damage of vital organs and infiltration of inflammatory cells,and evaluate the safety of GPC3-CAR T cells in the treatment of liver cancer.?4?take the tumor paraffin sections do CD3 immunohistochemistry to observe the CAR-T cell migration in the tumor;?5?use TUNEL to detect survival of tumor cell.?6?Peripheral blood of mice was taken and the secretion of INF-?and IL-2 of blood supernatant was detected by ELISA.Results1.A mouse model of hepatocellular carcinoma with high expression of GPC3 and no expression of GPC3 was successfully constructed.After injection of hepatoma cell suspension for about 7 to 10 days,the tumorigenesis rate of the HepG2 and the Sk-Hep-1is approximately 75%to 90%,and nearly 100%,respectively.2.After treatment of the second and third generation of GPC3-CAR T,the mice were generally in good condition with their body weight fluctuating.Compared with those of the control group mice,the liver,spleen,heart,lung,kidney,brain and other organs of the tumor-bearing mice,and there is no the microscopic structure of each organ tissue was clear and complete,with no diffuse neutrophils and lymphocyte infiltration,no congestion,edema or fibrosis therein.3.After treatment of the second and third generations of GPC3-CAR T,the GPC3-high expressed HepG2 group was significantly reduced in tumor volume and weight?**P<0.01?,while the GBBz and the G28BBz groups has no significant difference both in tumor volume and weight?P>0.05?,comparing with the control group,the Sk-Hep-1 group that did not express GPC3,there was no significant difference in tumor volume and weight among each the T cell groups?P>0.05?.4.Infiltration of T cells in tumor tissues was observed by CD3immunohistochemistry.Light microscope observations showed that there were a large number of positive cells in the G28BBz and GBBz groups of HepG2tumor-bearing mice,and five positive cells in the high-power field.The numbers were 170.8±8.7 and 132.8±12.8,respectively,which were higher than the Mock group 10.2±4.3?**P<0.01?.5.TUNEL was used to detect the apoptosis of tumor cells.The percentage of positive cells was counted by fluorescence microscope in 5 high-power fields.HepG2 group compared with the control group,GBBz vs Mock?*P=0.0274,*P<0.05?;G28BBz vs Mock?*P=0.0193,*P<0.05?;GBBz vsG28BBz?P=0.05254>0.05?.A few of apoptotic cells were observed in Sk-Hep-1group,and there was no significant difference between each T cell group?P>0.05?.6.ELISA was used to detect the secretion of cytokines INF-?and IL-2 from peripheral blood of mice.The levels of IL-2 and INF-?cytokines were extremely low in mice.In the HepG2 group,relatively high levels of cells were secreted after adoptive treatment of second-generation and third-generation GPC3-CAR T lymphocytes.Factor,higher than Mock group?*P<0.05?.The Sk-hep-1 group that did not express GPC3 had no ability to secrete cytokine IL-2 and INF-?,and there was no statistical difference between the each group?P>0.05?.ConclusionsWe constructed a hepatocellular carcinoma xenografts of NOD/SCID mice with high expression of GPC3 and no expression of GPC3.After adoptive immunotherapy of second-generation and third-generation GPC3-CAR T lymphocytes,the residual tumors of high expression GPC3 in hepatocellular carcinoma was smaller than no expression of GPC3 groups.The rats had significant killing,no significant damage to primary organs,and no cytokine storms in the body.It suggests that adoptive immunotherapy of GPC3-CAR T cells can be an effective treatment for HCC.