Extracellular Vesicles Armed With Target Antigen Or Cytokines Drive Efficacy Of Chimeric Antigen Receptor-T Cells

Objective: Chimeric antigen receptor(CAR)T cells have achieved very impressive efficacy.Many preclinical models and clinical trials are actively tackling the issue of poor expansion or persistence of CAR-T cells by delivering target antigens to provide the signal 1 required for their activation;or have conducted to promote anti-tumor effects of CAR-T cells by coadministering cytokines to enhance their signal 3 transduction.In order to enhance the transduction of signal 1 and signal 3,which were required for CAR-T cells to exert their efficacy,extracellular vesicles(EVs)bearing CAR target antigens or cytokines on their surface were prepared to investigate the potential of this method for increasing CAR-T cell activation signaling and promoting their proliferation and functional persistence in vivo.Methods: Firstly,HEK293 T monoclonal cell line with CD19 antigen or IL-12 expression on the cell surface was constructed.CD19 EVs or Il-12 EVs in the supernatant were collected to co-incubated with CAR-T cells respectively,the proliferation,cytokine secretion,CD107 a expression,tumor killing,subsets,and immune checkpoint expression of CAR-T cells were measured to assess the effect of target antigen or cytokines modified EVs on CAR-T cell function.In addition to in vitro experiments,a lymphoma xenograft mouse model was created to investigate the impact of CD19 EVs or IL-12 EVs on the expansion,persistence,and anti-tumor activities of CAR-T cells in vivo.Flow cytometry and digital PCR were used to detect CAR-T cells in the peripheral blood of mice,tumor volumes were continuously monitored,and the occurrence of adverse events was studied to determine the safety of applying CD19 EVs or IL-12 EVs in vivo.Furthermore,the transcriptome of CAR-T cells treated with IL-12 EVs was sequenced,differential gene analysis and pathway enrichment analysis were used to identify the important genes and relevant pathways of IL-12 EVs affecting CAR-T cell function,as well as to conduct a preliminary study on the mechanisms involved in the combination of CAR-T cells and IL-12 EVs.Results: HEK293 T cell-derived EVs expressing CD19 or IL-12 were successfully prepared.CAR-T cells prepared from healthy donors and patients-derived T cells could be both antigen-specifically activated by CD19 EVs,hence expanded more efficiently,emitted higher levels of cytokines,and improved cytotoxicity because of increased CAR expression,while CAR-T cells differentiated into the effector-memory phenotype and increased immune checkpoint expression.Consistent with in vitro datas,CD19 EVs-pretreated CART cells demonstrated higher levels of proliferation and complete tumor clearance in mice.In a mouse relapse model,in vivo application of CD19 EVs could promote functional persistence of CAR-T cells,reduce tumor recurrence and greatly prolong the survival of mice with tumor relapse.IL-12 EVs effectively boosted the anti-tumor activities of CAR-T cells both in vivo and in vitro.Transcriptome sequencing of CAR-T cells treated with IL-12 EVs showed that IL-12 EVs prompted the upregulation of cytokine related pathways in CAR-T cells,and enhanced the expression of CAR-T function-related genes,including IFNG,GZMK,GZMB,JUN.Both EVs were safe to utilize in vivo and caused no adverse effects.Conclusion: It is feasible to artificially modify EVs membranes to express target antigens or cytokines to provide the signals required for CAR-T cells to optimally function,and it is effective and safe to promote CAR-T cell function with this strategy.