The Effect Of C-Jun Overexpression On The Function Of Cord Blood Derived CD19.CAR-T Cells

Chimeric antigen receptor CAR-T cell therapy is effective for hematological malignancies,but tumor control does not last long.Chimeric antigen receptor technology uses gene editing technology to express the artificially designed chimeric antigen receptor(CAR)on the immune cell membrane,so that the activation of T cells is not restricted by MHC and the second signal.Play a powerful killing effect.The efficacy of CAR-T cell drugs is limited by multiple factors(high density antigens,tumor microenvironment,and T cell exhaustion leading to T cell dysfunction,etc.).In the early stage,the research team has successfully expressed CAR molecules on T cells derived from umbilical cord blood,and prepared CAR-T cells that target CD19.In order to effectively improve the expansion and persistence of CAR-T cells,in this study,we introduced exogenous c-Jun gene fragments and CD19.CAR gene into T cells at the same time to complete gene modification,and prepare CD19 with over-expression of c-Jun transcription factor.CAR-T cells,and study the effect of c-Jun overexpression on the activity of cord blood CAR-T cells.Objective:To investigate the effect of c-Jun modification on the function of CAR19 cord blood T cells,and to provide basic theoretical basis for further study of c-Jun modification combined with universal CAR-T cells.Methods:The c-Jun gene fragment was amplified by PCR,and the c-Jun gene fragment was cloned into the linear plasmid pHAGE-SFFV by infusion seamless ligation technology.After lentivirus packaging,preparation,ultrafiltration tube ultrafiltration concentrated virus to obtain high titer virus concentrate.After collecting healthy human umbilical cord blood cells,obtaining Cord Blood Mononuclear Cells(CBMC)from Ficoll separation solution;co-incubating with CBMC with Dynabeads Human T-Activator CD3/CD28 magnetic bead antibody to fully activate T cells.The cord blood T cells were sorted and purified by CD4-Microbeads and CD8-Microbeads.Using lentivirus as a vector,pHAGE-SFFV-CAR19 and pHAGE-SFFV-Jun were co-transfected into human cord blood T lymphocytes according to the multiplicity of infection(MOI)20,and the expression of cord blood T lymphocytes was detected by flow cytometry The situation of CAR molecules and c-Jun molecules.The two groups of CAR-T cells were cultured for 15 days;the proliferation of the two groups of cells transduced with different viruses was compared.On the 10th day,the two groups of CAR-T cells were compared with the target cell model constructed by our laboratory according to the effective target ratio of 1:1,1:0.3,1:0.1 knock out human leukocyte antigen I(HLA-I)and human leukocyte antigen CD19~+Namalwa-DKO target cells of class II(HLA-II)genes were co-cultured,and CD107a expression on CAR-T cells was detected at 4h;target cell killing was detected at 24h;cell supernatants were also collected for enzyme-linked immunosorbent assay(ELISA,Enzyme Linked Immunosorbent Assay)measures the secretion of cytokines IL-2(Interleukin-2)and Interferon-γ.Monitor the proportion of stem cell-like central memory T cells(Stem Memory T cells,Tscm).The anti-tumor activity of the two groups of CAR-T cells was evaluated through the above experiments.Results:We successfully constructed the recombinant plasmid pHAGE-SFFV-Jun lentiviral vector,and packaged the vector in 293FT cells to produce lentiviral particles.Purified cord blood T cells were obtained after Ficoll separation,CD4-Microbeads and CD8-Microbeads sorting.After T cells were activated by Dynabeads Human T-Activator CD3/CD28 coating antibody for 48 hours,pHAGE-SFFV-Jun and pHAGE-SFFV-CAR19lentiviral particles infect healthy human cord blood T lymphocytes.When MOI=20,we found that the transduction rates of both pHAGE-SFFV-Jun and pHAGE-SFFV-CAR19 virus and CD3~+T cells transduced with and only pHAGE-SFFV-CAR19 virus were more than 60%.The CAR19 expression rates of CD3~+,CD4~+and CD8~+T cells in the CD19.CAR-T group were77.7%±1.0%,83.9%±3.4%and 62.9%±2.5%,respectively.The CAR19 expression rates of CD3~+,CD4~+and CD8~+T cells in the CAR19 group were 63.6%±2.4%,72.4%±1.6%and41.4%±6.9%,respectively.The c-Jun expression rate of T cells in JUN-CD19.CAR-T group was more than 75%,and the expression rates of CD3~+T cells,CD4~+T cells and CD8~+T cells were 85.1%±1.9%,88.3%±4.2%and 75.7%±2.9%,respectively.The proportion of T cells that successfully transduced CAR19 and c-Jun virus at the same time was as high as 60%±1.9%.The results showed that we successfully transformed the target c-Jun gene and CAR19 gene through lentiviral particles.It was transduced to T cells,and the transduction efficiency was high enough.After 15days of lentivirus transduction,the CAR19 transduction rate in the CD19.CAR-T group decreased to 47.5%±0.8%,while the CAR19 transduction rate in the JUN-CD19.CAR-T group decreased to 65.7%±2.0%,indicating that the T cells successfully transduced with the c-Jun gene contributed to the stable and lasting expression of the CAR19 gene.The proliferation ability of human umbilical cord blood CD4~+T and CD8~+T cells transduced with c-Jun and CAR19 virus was significantly higher than that of CD19.CAR-T group.CD4~+T cells in non-transduced virus group and CD19.CAR-T group did not continue to proliferate on the 11th day,CD8~+T cells did not continue to proliferate on the 13th day,and JUN-CD19.CAR-T group proliferated to the 15th day.The CD4~+/CD8~+of JUN-CD19.CAR-T group transduced with c-Jun and CAR19 virus was higher than that of CD19..CAR-T group and CBT group;it is suggested that the successful transduction of c-Jun virus CAR19-T has a stronger effect on the proliferation of CD4~+T than that of CD8~+T cells,and that human umbilical cord blood T cells modified by c-Jun gene can change the distribution of CD4~+and CD8~+subsets.JUN-CD19.CAR-T group and CD19.CAR-T group were incubated with Namalwa-DKO CD19 positive cells at different effector-target ratios for 4 hours.Flow cytometry showed that the expression of CD107a in JUN-CD19.CAR-T group was significantly higher than that in CD19.CAR-T group at different effector-target ratios.After 24 hours,the killing rates of JUN-CD19.CAR-T group were 83%,89%and 85%,respectively.The killing rates of CD19.CAR Murt group were 67%,82%and 80%,respectively.The killing rate of negative control T cell group was only 22%,45%and 10%.There was no significant difference between JUN-CD19.CAR-T group and CD19.CAR-T group.The expression of memory-related molecules in the two groups was detected by flow cytometry.The proportion of effector-related molecules in T cells in JUN-CD19.CAR-T group was higher than that in CD19.CAR-T group,suggesting that T cells in this group had higher effector function and enhanced effector cell differentiation.ELISA was used to detect the secretion of cytokines IFN-γand IL-2 in JUN-CD19.CAR-T group and CD19.CAR-T group after co-culture with CD19~+target Namalwa-DKO cells for 24 hours.The results showed that when co-cultured with CD19~+target cells Namalwa-DKO at 3:1 and 1:1,the secretion of IFN-γin JUN-CD19.CAR-T group was significantly higher than that in CD19.CAR-T group(P<0.0001).However,there was no statistical difference in the secretion of IL-2,which was closely related to the presence of IL-2 in X-vivo.15 complete medium.Conclusion:CAR-CBT cells transduced with c-Jun gene and over-expression of c-Jun can enhance the long-term proliferation ability of cord blood-derived CD19.CAR-CBT cells,especially CD4~+T cells;and have higher effector functions,secretion More cytokines such as IFN-γenhance the ability to control the growth of target cells and better play an anti-tumor effect.