Mechanism Of Targeting Glutamine To Regulate Glucose Metabolism Reprogramming And Overcome Immune Escape Of CD19 CAR-T Cells In The Treatment Of Acute Lymphoblastic Leukemia

BackgroundChimeric antigen receptor T cells(CAR-T)are the most epoch-making new tumor immunotherapy technology in this century.Different from traditional T cell therapy,CAR-T cells have the characteristics of strong targeting,non-HLA-restricted and self-expanding ability.The remission rate of B-ALL treated with CD 19 CAR-T cells can reach more than 90%,but the one-year recurrence risk is 17%-57%.The high recurrence risk limits the long-term efficacy of CD 19 CAR-T cells.Therefore,the mechanism of relapse after CAR-T therapy and how to reduce the recurrence risk are currently the focus of research.The mechanisms of relapse may include limited persistence of CAR-T cells,tumor antigen escape,changes in tumor microenvironment,high tumor load,and nibbling,among which limited persistence and decreased function of CAR-T cells are one of the main causes of relapse.Many studies have attempted to reduce the risk of recurrence by improving the structure of CAR-T,dual-target CAR-T,CAR-T bridge transplantation,and combination of other therapies after CAR-T,but it is difficult to fundamentally solve the durability problem of CAR-T itself.Early T cells(Tn,Tscm,Tcm)have longer survival time and stronger anti-tumor effect after differentiation.Recent scientific studies have shown that glutamine(Gln)is a metabolic checkpoint for tumor immunotherapy.Our previous study found that Gln was significantly increased in patients who relapsed after CD 19 CAR-T treatment with B-ALL,and abnormal changes in genes related to glucose metabolism were observed.We propose that targeting glutamine regulates glucose metabolism reprogramming to overcome immune escape in the treatment of acute lymphoblastic leukemia with CD 19 CAR-T,a mechanism that has not been reported in CAR-T immunotherapy for hematologic tumors.This study focuses on the frontier,explores a new path from the metabolic pathway,explores a new method to overcome the relapse after CAR-T therapy,and provides a new idea for the prevention and treatment of relapse after tumor immunotherapy,which has great research significance and value.MethodsFlow cytometry(FCM)was used to detect T cell depletion indicators in patients with recurrent acute B lymphoblastic leukemia treated with CD 19 CAR-T immunotherapy.Liquid chromatography-mass spectrometry(LC-MS)and NA-Seq sequencing were used to analyze the levels of glutamine and glucose metabolism in patients with remission and recurrence of acute B-lymphoblastic leukemia after CD 19 CAR-T cell therapy.Gln antagonist DON on acute B lymphoblastic leukemia cell line(Nalm6)was detected by Seahorse XF96 and FCM.FCM,Western blot(WB)and Seahorse XF96 were used to detect the depletion,differentiation and metabolism of CD 19 CAR-T under the influence of DON.Apoptosis and cytokine secretion levels of CAR-T cells co-cultured with Nalm6 under DON were detected by FCM.ResultsThe levels of glutamine and intermediate metabolites of glycolysis in patients with relapsed B-ALL after CD 19 CAR-T cell therapy were significantly higher than those in remission stage.DON can increase the proportion of Tn and Tscm in CD 19 CAR-T cells,decrease the expression of depletion indexes(such as PD-1,LAG3,TIM3),and decrease the expression of glycolytic related proteins LDHA,GLUT1,HK2 and the related signaling pathway c-MYC in CD 19 CAR-T cells.The glycolysis level of tumor and CAR-T cells was decreased,and the oxidative phosphorylation level of CD 19 CAR-T cells was increased.At the same time,DON cooperated with CAR-T cells to enhance the function of killing tumor cells,and the cytokine secretion was not significantly changed.ConclusionsThis study showed that by blocking Gln in vitro,on the one hand,the glycolytic pathway of tumor cells was inhibited to prevent the proliferation of tumor cells;on the other hand,the glucoconeogenesis,pentose phosphate pathway and oxidative phosphorylation levels of CAR-T cells were up-regulated by reducing the level of CAR-T glycolytic.Thus,CD 19 CAR-T cells were induced to reverse to stem cell memory T cells and central memory T cells,and the depletion of CD 19 CAR-T cells was reduced,and the persistence of CD19 CAR-T cells was prolonged and the antitumor effect was improved.Therefore,the Gln antagonist DON is a potential target for CD 19 CAR-T cells to treat B-ALL immune escape.