Effection Of Akt Inhibitor Combined With Cytokine(IL-2/7/15/21) On The Biological Activity Of CAR-T Cells

Background and purposeSince immunotherapy began to be applied to cancer in the 19th century,more and more immunotherapies have emerged and have demonstrated a strong ability to eradicate tumor cells,such as immune checkpoint inhibitors(ICI),tumor-vaccines,CAR-T cells,etc.It has achieved an unprecedented therapeutic effect in which CD 19 CAR-T cells are particularly effective in treating hematological malignancies derived from B cells.However,the in vitro expansion of CAR-T mainly depends on IL-2.Most of the end-stage effector cells are obtained.The proliferative capacity and immune memory ability are weak,and the long-term anti-tumor effect cannot be exerted.The patient needs to infuse CAR multiple times.-T,may increase the burden on patients,and even bring strong tox_ic side effects.Memory cells have the ability of self-renewal and differentiation,and can kill tumor cells more lastingly.Therefore,increasing the proportion of memory cells is the key to optimizing CAR-T immunotherapy.The strong synergy between IL-7,IL-2 and IL-15 can preserve the phenotype of memory cells and enhance CAR-T cytotoxicity;Akt inhibitor(Akti)will not affect the proliferation of CD 19 CAR-T cells,but will also Differentiated CTL transcription rearranged and transformed into memory cells.The purpose of this study is to explore the effects of Akti combined with mixed cytokines(IL-2/7/15/21)on the biological function of CD19 CAR-T,and to find a culture that effectively enhances CAR-T function and prolongs its presence in vivo System to provide useful clues for the follow-up CAR-T cell research and clinical application.Method1.Using molecular biology method to construct CD 19 CAR lentivirus,extract human peripheral blood mononuclear cells and purify into CD3+T cells,transfect lentivirus into CD3+T cells,respectively in IL-2,Akti and IL-2.Akti combined with mixed cytokine(IL-2/7/15/21)three systems to culture CAR-T cells,and on the 9th day,the flow cytometer was used to detect the T cell lentivirus transfection rate.2.The number of CAR-T cells on the cell counting plate,and draw CAR-T cell growth curves under three culture conditions.3.On the 10th day after CAR-T expansion in vitro,the phenotypes of three groups of CAR-T memory cells were detected by flow cytometry.4.On the 11th day after CAR-T expansion in vitro,CAR-T cells and tumor cells were co-cultured according to the effective target ratio(E:T)1:1,and flow cytometry was used to evaluate the tumor cell kill rate of the three groups after 6h and 24h.The difference is that ELISA method detects the release of cytokines.5.SPSS 21.0 analyzes the experimental data statistically and plots with Graphpad Prism8.P<0.05 was considered statistically significant.Result1.There is no significant difference in the CAR-T transfection rate between the three culture methods.2.In terms of cell proliferation,CAR-T cultured with Akti combined with cytokines had a stronger effect of expanding CAR-T cells than the first two groups.There was no significant difference between IL-2 group and Akti combined with IL-2 group.3.In terms of memory phenotype,the ratio of early memory cells formed by Akti combined with cytokine group in CD4+early memory cells was significantly higher than that in the first two groups,but there was no significant difference in the first two groups.In CD8+early memory cells,the ratio of Akti combined cytokine group was higher than Akti group than IL-2 group.4.In terms of killing effect,the three groups of CAR-T cells were stronger than the control T cell group;the Akti combined cytokine group was stronger than the IL-2 group but there was no significant difference from the Akti group.ConclusionIn this experiment,CAR-T cells cultured under conventional IL-2,Akti combined IL-2,and Akti combined mixed cytokine(IL-2/7/15/21)systems were studied.CAR-T cells have the best effects on in vitro expansion,central memory and stem cell memory phenotype formation,and cytotoxicity.Provide an experimental basis for the subsequent improvement of the clinical role of CAR-T cell immunotherapy.