Basic Research On Enhanced Tandem CAR T Cells In The Treatment Of B-line Hematological Malignancies

BACKGROUND:Chimeric antigen receptor T cell(CAR T)immunotherapy targeting the CD19 antigen has shown clinical efficacy in patients with leukaemia and lymphoma.However,to date,this treatment has been much less effective for lymphoma than acute lymphoblastic leukaemia(ALL),in part because CAR T cells enter an exhausted state characterized by upregulation of inhibitory receptors and loss of effector function.Despite their activity,CAR T cell approaches have limitations that will need to be addressed,including excessive toxicity,relapses mediated via antigen escape,difficulties overcoming the suppressive tumor microenvironment,among others.METHODS:Part I:Firstly,we designed a series of single-chain bispecific CARs by optimizing the order of anti-CD19 and anti-CD20 sc Fvs,then to screend the best Tan CAR structure based on antitumor experments.Then,the spatial conformational advantage of Tan CAR T in the antitumor process was subsequently assessed by immune synapse(IS)and intracellular calcium influx assays.The molecular mechanism of the anti-tumor advantage of Tan CAR T was initially explored by RNA sequencing.Finally,animal experiments were carried out to evaluate the difference between Tan CAR T and single-target CAR T anti-tumor effects in vivo.Part II:Firstly,we optimized the d CAR T cell production protocol based on the dosage and viability of DAC.Stable cell proliferation and viability determined the production scheme of adding 10-100 n M of DAC in synchronism with CAR gene infection to avoid the possible toxicity of DAC.Subsequently,the cytotoxicity ability,cytokine secretion ability,memory,exhaustion and other phenotype expressions of d CAR T cells were determined by long-term low-efficiency target ratio cell killing experiments and flow cytometry.The methylation level and gene expression differences between d CAR T cells and ordinary CAR T cells before and after antigen stimulation were detected by methylation chip and RNA sequencing.To construct immunodeficient NPG mouse models of human non-Hodgkin’s lymphoma and B-cell leukemia,and to evaluate the tumor-suppressive ability,related cellular functions and cell phenotypic status of d CAR T cells against B-lineage hematological malignancies in vivo.Finally,the intratumoral CAR T cells in animal experiments were sorted for transcriptome sequencing to explore the difference between the anti-tumor effect of d CAR T cells and the gene expression driven by ordinary CAR T cells in vivo,and to explore the possible mechanism of their synergism.RESULTS:Part I:1.Tan CAR7 T cells show dual antigen coverage and elicit a potent and durable antitumor response.2.Tan CAR7 T cells efficiently killed both wild-type and mutant Raji cells.3.Tan CAR7 T cells exhibit a stable IS and stronger and rapid degranulation degranulation than single-targeted CAR T cells.4.Tan CAR7 T cells have contained a higher percentage of central memory T cells and a lower percentage of exhaustion T cells than in single-targeted CAR T cells upon antigen exposure.5.Tan CAR7 T cells showed higher upregulation of gene related to calcium flux,IS structure and T cell activity.6.Tan CAR7 T cells showed superior antitumor activity than single-targeted CAR T cells in vivo using a lymphoma xenograft model in NSG mice.Part II:1.Established a culture technology system for modifying CAR T cells in vitro with low-dose DAC.2.The CD4:CD8 T cell ratio and an elevation in the central memory populations among cultured CAR T cells treated with 10 n M DAC(d CAR T cells)compared to CAR T cells.The addition of DAC at a dose of 10 n M during CAR T cell culture had little or no effect on CAR T cell viability or proliferative capacity.Low-dose,short-term DAC treatment in vitro persistently induced the degradation of DNMT3a in d CART cells.The upregulated expression of memory-and proliferation-associated genes and enhanced downregulation of the expression of T cell inhibitor-,death-and activity/exhaustion-related genes in the d CAR T cells compared to the CAR T cells.3.Upon a coculture at an effector-to-target(E:T)ratio of 1:30 for 64 hours,the d CAR T cells were significantly better at eliminating Raji tumour cells than the CAR T cells.After two weeks of constant stimulation by low-dose Raji cells,CAR T cells exhibited higher proportions of PD1+cells than d CAR T cells did.Compared to CAR T cells,d CAR T cells produced higher levels of cytokines upon restimulation with Raji cells.4.In animal experments,both d CAR T and CAR T cell treatment with 1×10~7 CAR T cells effectively controlled tumour growth.But,in the tumour rechallenge experiment,the d CAR T cells rapidly expanded and controlled tumour growth after tumour cell reinoculation,and with high expression of memory phenotypes.The cell and CAR gene numbers and levels of cytokines related to T cell proliferation were substantially higher in the d CAR T group than in the CAR T group.5.In“stress tests”,the d CAR T cells showed strong tumour control capabilities compared to the CAR T cells.6.We performed RNA-seq analysis of the tumour-infiltrating d CAR T and CAR T cells to further investigate the T cell function-associated gene status of the d CAR T cells and differences compared with the CAR T cells.GSEA revealed upregulated the expression of memory-associated and cell proliferation-related GO items in the d CAR T cells compared with the CAR T cells on days 7 and 14.Compared with tumour-infiltrating CAR T cells,tumour-infiltrating d CAR T cells showed the upregulated expression of activation/exhaustion-associated genes on day 7 and downregulated expression of these genes on day 14 after cell infusion(Fig.10d).CONCLUSION:Part I:We designed a series of tandem CARs(Tan CARs)and found that Tan CAR7 T cells not only showed dual antigen targeting of both CD19 and CD20 but also formed superior and stable immunological synapse(IS)structures,which may be related to their robust antitumor activity.Part II:We demonstrated that CAR T cells underwent DNA reprogramming after DAC treatment,which induced significant sustained cell expansion,cytotoxicity,and cytokine production and reduced exhaustion after antigen exposure.Especially shown by the“stress test”,the d CAR T cells at very low doses could efficiently control tumours with a very large tumour burden.Importantly,the d CAR T cells maintained a higher proportion of cells with a memory phenotype than did the CAR T cells under long-term tumour stimulation.The addition of demethylating drugs may become a convenient and economical means to consistently achieve this goal.