| Objective EGFRvIII-CART cells were constructed to increase the targeting of T cells to GBM,and PD1CD28 chimeric molecule were expressed on this CART cells in order to convert T-cell inhibitory signals induced by PD-L1 binding to PD-1 to activation signal delivered by CD28.The activation signal reduces the disability of CART cells in the immunosuppressive microenvironment and enhances their anti-tumor ability.Methods1ConstructionoflentiviralvectorEGFRvⅢ-CARand EGFRvⅢ-CAR-P2A-PD1CD281)Synthesis of target gene plus XbaI and SgrAI restriction sites on the first and last of EGFRvⅢscFV sequence and adding the corresponding protective base,then synthesis the sequence.2)Construction of EGFRvIII-CAR vector XbaI/SgrAI restriction enzymes were used to abtain the target fragment(EGFRvⅢscFV)and CD137-CD3 linear vector,the two fragments were ligated to obtain the lentiviral vector EGFRvIII-CAR.3)Construction of EGFRvⅢ-CAR-P2A-PD1 vector SfiI/MluI restriction enzymes were used to abtain PD1 extracellular sequence and PLVX-EGFRvIII linear vector,the two fragments were ligated to obtain the lentiviral vector EGFRvⅢ-CAR-PD1.4)Construction of EGFRvⅢ-CAR-P2A-PD1CD28 vector ApaI/MluI-HF restriction enzymes were used to abtain CD28 intracellular sequence and EGFRvⅢ-CAR-P2A-PD1 linear vector,the two fragments were ligated to obtain the lentiviral vector EGFRvⅢ-CAR-P2A-PD1CD28.2 Lentiviral production,concentration and titration1)transduction of lentiviral expression vector and two packaging plasmids into 293FT cells using Calcium phosphate.2)48hrs,72hrs cell supernatants were collected and the cell debris was removed by filtration.The lentiviral was centrifuged to the bottom of the tube using a ultracentrifuge and dissolved in RIPM 1640 medium.Placed in-80℃refrigerator after aliquot.3)The lentiviral concentrate was diluted 10-fold,100-fold before added to 293FT cells.After 48 hrs,determined the percentage of positive cells by FACS to calculate the biological titer of the lentiviral.3 PBMCs transduction1)Isolated human peripheral blood mononuclear cells(PBMCs)from healthy people.CD3/CD28 magnetic beads were added to stimulate T cells.2)After 48 hrs,pull the virus(MOI=30)and PBMCs together and centrifuged.3)72 hrs after transduction,the expression of CAR molecules and PD1CD28 on CD3~+T cells were detected by western-blot and flow cytometry.4 CRISPR-PD-L1 vector construction,lentiviral production and functional identification1)Designed three pairs of sgRNAs for the PD-L1 coding region.2)Three pairs of sgRNAs were synthesized,phosphorylated and annealed respectively.3)ligated the lentiCRISPRv2 vector with different sgRNAs after BsmBI restriction enzyme digestion.4)Target cells(EGFRvIII+U87 MG)was transduced by the most efficient CRISPR-PD-L1 lentiviral vector.5)72 hrs after transduction,PD-L1was detected by flow cytometry.5InvitrofunctionalassayofEGFRvⅢCARTcellsand EGFRvⅢ-P2A-PD1CD28 CART cells1)EGFRvⅢCART cells and EGFRvⅢ-P2A-PD1CD28 CART cells were co-cultured with target cells respectively,comparing the killing effect.2)EGFRvⅢCART cells and EGFRvⅢ-P2A-PD1CD28 CART cells were co-cultured with target cells respectively,comparing the levels of IL-2 and IFN-γ.6InvivofunctionalassayofEGFRvⅢCARTcellsand EGFRvⅢ-P2A-PD1CD28 CART cells1)EGFRvⅢ~+U87 MG cells were injected subcutaneously into the right back of mice to establish NCG mouse model of human Glioblastoma xenografts.2)When the tumor size reached about 50-100mm~3,mice were randomly divided into5 groups,each group contain 6 mice.3)Resuspended T cells or CART cells in 400μl PBS and inject into the tail vein of mice.4)After infusion,observe the state of the mice on the next day,monitor mice weight,and measure the length and width of the tumor with a vernier caliper.Result1 Construction of lentiviral expression vector EGFRvⅢ-CAR and EGFRvⅢ-CAR-P2A-PD1CD28After double enzyme digestion identification,choose the correct colonies and sequencing,the sequence was as expected.2 Transduction of EGFRvⅢ-CAR,EGFRvⅢ-CAR-P2A-PD1CD28 CART cells1)The titer of EGFRvIII-CAR lentiviral was 2.16×10~8 TU/ml,the titer of EGFRvⅢ-CAR-P2A-PD1CD28 lentiviral was 1.04×10~8TU/ml.2)The efficiency of transduction of EGFRvⅢCART cells was about 50%,the efficiency of transduction of EGFRvⅢ-P2A-PD1CD28 CART cells was about 30%.3 Construction of CRISPR-PD-L1 vector to knockout PD-L1 of target cells Obtained the plasmid capable of effectively knocking out PD-L1.Produced lentiviral titers was 2×10~8TU/ml,successfully infected cells of interest and knockout PD-L1.4 Detection of EGFRvⅢCART cells and EGFRvⅢ-P2A-PD1CD28 CART cells in vitro and in vivo1)In vitro experiments showed that CART cells have an antigen-dependent activation and specific killing effect on EGFRvIII~+U87 MG cells.There was no significant difference between EGFRvⅢCART cells and EGFRvⅢ-P2A-PD1CD28 CART cells in vitro specific killing effect.However,EGFRvIII-P2A-PD1CD28 CART secreted more IL-2 than EGFRvIII CART cells after co-cultured with EGFRvIII~+U87 MG cells.There was no significant difference in IFN-γsecretion between the two kinds of CART cell.2)Anti-tumoreffectsofEGFRvIII-P2A-PD1CD28CARTcellson glioblastoma-bearing mice.In the human glioma xenograft tumor model,On the 21st day after treatment,EGFRvⅢCART cells and EGFRvⅢ-P2A-PD1CD28 CART cells had no significant difference in the control of tumor growth,and both were effective in eliminating tumors.However,On the 28st day after treatment,tumors relapsed in 3 mice in the EGFRvIII CART cells group(3/6).There was no recurrence in the EGFRvIII-P2A-PD1CD28 CART cell group.On the 51st day after treatment,the recurrence rate was 33%in the EGFRvⅢ-P2A-PD1CD28 CART cells group and 50%in the EGFRvⅢCART cells group;the recurrent tumor size in the EGFRvⅢ-P2A-PD1CD28 CART cells group was smaller than in the EGFRvIII CART cells group.The median survival of the EGFRvⅢ-P2A-PD1CD28 CART cells group(75d)was longer than the EGFRvⅢCART cells group(51.5d).So,EGFRvⅢ-P2A-PD1CD28 CART cells demonstrated a stronger ability to control tumors in vivo.ConclusionBoth EGFRvIII CART cells and EGFRvⅢ-P2A-PD1CD28 CART cells have a specific cytotoxic effect on tumor cells both in vitro and in vivo.EGFRvⅢ-P2A-PD1CD28 CART cells has stronger antigen-dependent activation ability in vitro and has strong ability of controlling tumor in vivo. |
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