Synergistic Antitumor Effect Of Shrna Knocking Down Immune Checkpoint Molecules PD-1 Combined With CAR-CD30-T And Process Optimization Of CAR-T

As one of the most promising immunotherapies,CAR-T therapy has shown significant anti-tumor activity and higher rates of clinical remission in treating hematological malignancies.Nowadays,there are still many challenges in CAR-T cell therapy.Single CAR-T cell therapy has low efficacy and short survival time in the treatment of solid tumors and still has a high recurrence rate and the possibility of drug resistance in the treatment of hematoma.The shortage of lentiviral vectors has become the bottleneck of its application in the cellular immunotherapy industry.It is essential to explore a scalable and repeatable lentivirus manufacturing process that conforms to the c GMP(Current Good Manufacture Practices)level.Currently,there are few car-T cell cryopreservation liquids for direct clinical injection on the market.So,the development of clinical-grade cell cryopreservation liquid is also an important part in CAR-T cell therapy.Combination therapies become more and more popular with the development of cellular immunotherapy.However,the most promising therapeutic target CD30 combined with immune checkpoint PD-1 knockdown has not been reported yet.In this study,CD30 was selected as a therapeutic target,combined with PD-1 knockdown technology,and its combined effect on Hodgkin’s lymphoma before clinical treatment was studied,to provide the theoretical basis for clinical treatment.First,the effects of anti-CD3/CD28 magnetic beads and lentivirus on the expression of PD-1 protein in T-cells during the preparation of CAR-T cells were studied.Flow cytometry results indicated that PD-1 expression in Jurkat was significantly increased to 63.5±4.9%(P＜0.01)and 63.0±5.3%(P＜0.01)by anti-CD3/CD28 bead and CAR-CD30 lentivirus.Add 0.5:1 anti-CD3/CD28 magnetic beads to stimulate the expression of PD-1 in Jurkat within 48 h,then the expression of PD-1 protein decreased with time,and decreased to within 4.5±0.3%at 144 h.After CAR-CD30 lentivirus infected Jurkat cells with MOI=3 and stimulated Jurkat cells with complex number,the carrier carried car-CD30 antibody repeatedly bound with CD30 antigen protein of cell line,continuously upregulating PD-1,resulting in over-stimulation of transfected positive cells,and the proportion of positive cells decreased with time.The 0.5:1 ratio of anti-CD3/CD28magnetic beads can temporarily increase the expression of PD-1 protein in T cells by about three times without affecting the T cell viability.CD30 protein expressed in normal human T cells are relatively low,and the experiment proves the CAR-CD30 slow virus,the T cells did not show significant PD-1 raised(P＞0.05).Our experiments do corroborate this hypothesis showed it has not significant up-regulation of PD-1 after CAR-CD30 lentivirus stimulated T cells.The cell peripheral region of the complex tumor microenvironment may stimulate and affect the PD-1 expression on T cell.Meanwhile,this supports the need to combine CAR-T treatments and anti-PD-1drug.In this study,a sequence was selected in vitro that knocked down the expression of PD-1 by about 50%.In vitro treatment of Hodgkin’s lymphoma by lentiviral vector carrying PD-1 interfering RNA combined with CAR-CD30 protein.Experimental results show that the PD-1 on low and the increase of the CAR-CD30 protein,can inhibit the T cells proliferation(P＜0.01),and the influence of the CAR-CD30 protein more significantly.In parallel,experiments confirmed that CAR-CD30-T cells had a specific targeted and lethal effect on L428,while the knockdown of PD-1 inhibited its killing effect to a certain extent.Therefore,experiments suggest that PD-1 interfering RNA may not be suitable for combined anti-tumor therapy with CAR-CD30 cell therapy.293 suspension cells were cultured in a Stirred Tank Bioreactor with L-type bullae ventilation system for large-scale packaging of lentivirus vectors in the study.Four methods were used to domesticate 293T cells and verify their lentivirus packaging ability.After verification of the ability to lentivirus packaging,adapted 293T cell were expanded in flask inoculated,drawing a growth curve and packaging lentivirus into a stirred bioreactor with5.5 L work volume.The results show that 293T suspension cultivation was successful in mixed media(293 CD05 medium and SMM293-TⅡin a mixing ratio of 1:1 by volume.Suspension cells were cultured in a stirred tank bioreactor,and the cell density reached1.5×10~6 cells/m L two days later.Plasmid complexes and feeding medium can then be added,and a crude lentivirus product of 6.5×10~7 TU/m L can be harvested after four days of culture.Compared with the cell factory process,the Stirred Tank Bioreactor lentivirus packaging process has the advantages of low cost and high efficiency.To gain sufficient numbers of viable CAR-T,our study aimed to optimize a clinical-grade and injectable cryopreservation solution for CAR-T.We evaluated the stability,infuse time,osmotic,pressure p H and in vivo and in vitro cryopreservation effect of the cryopreservation solution.The experimental results confirmed that the chemical properties of frozen storage solution prepared from different proportions of raw materials remained stable after being placed in ampoules at 4℃for 60 days.The osmotic pressure of the frozen solution was 1922 m Osm/kg,and the p H was 6.4.In order to reduce the harm caused by high osmotic pressure,the osmotic pressure was 311 m Osm/kg after 40 times dilution to meet the requirements of injection.The experimental results showed that CAR T cells recovered the killing property of RAJI within 1-1.5 h,and CAR T cells also had effective anti-tumor activity in vivo and in vitro.In theory,the cryopreserved liquid can be used for injection,so it has a reasonable prospect of popularization.