In Vitro Study Of Chimeric Antigen Receptor Modified T Cells Targeting IL-13R?2 Against Glioblastoma

Objective:Glioblastoma(GBM)is the most common primary malignant brain tumor with high invasiveness,fatality and recurrence.Routine therapeutic methods include surgical resection,local or systemic chemotherapy,radiation therapy,antiangiogenic agents and alternating electric felds etc.Nevertheless,the prognosis of GBM patients is still poor,and the 5-year survival rate is less than 10%.It is imperative to develop a new safe and effective alternative treatment to prolong the patients' life and improve the prognosis.Numerous studies have proved that immunotherapy using host immune system to guide immune response is the future direction of GBM treatment.At present,gene transfer technology is used to introduce anti-tumor chimeric antigen receptor(CAR)into autologous T cells to guide high specific recognition and killing of tumor cells.Adoptive CAR-T cell therapy with strong specific killing effects has been observed in preclinical study and clinical trials targeting GBM,which provides a hope for the prognosis of the patients.However,at this stage,the viral vector transfection technique for CAR-T production faces several problems including insertion mutation,genotoxicity,strong immunogenicity and high manufacturing cost,which affects the practical application to some extent.A non-viral vector called minicircle DNA(mcDNA)is a novel small-loop super-spiral expression frame,which has no mutation risk,no immune origin,low manufacturing cost,lack of bacterial sequences such as resistance marker genes and replication origin and more persistent and higher genes in vivo,has emerged as an ideal gene delivery vehicle.After consulting a large number of literatures,it is known that the strategy targeting IL-13R?2 has emerged in pre-clinical studies and clinical trials in the treatment of GBM.Therefore,present study aimed to use electroporation technology to deliver mcDNA into autologous Tcellstoproduce CAR-T cells targeting IL-13Ralpha2.Its antitumor biological functions were demonstrated by detecting the killing efficiency of CAR-T cells and the secretion of cytokines IFN-? and IL-2.Methods:1.CNKI,PMC and PubMed databases were used to review the principles of the treatment of cancer by adoptive CAR-T cells and the related research of minicircle DNA vectors,to understand the expression of IL-13R?2 in GBM and the mechanism of promoting the occurrence and development of GBM.2.Minicircle DNA plasmids containing IL-13 CAR target gene fragments were extracted from parental minicircle DNA plasmids preserved in our laboratory.The basic structure of CAR was IL-13-CD28-4-1BB-CD3?.3.minicircle DNA plasmids were transfected into T cells by electroporation.Survival and transfection effects of T cells and whether the molecular phenotype CD3/4/8 of T cells changed were observed.The expression of IL-13 CAR was detected by using Flow Cytometry and Western Blot respectively.4.Flow Cytometry was used to detect the expression of IL-13R?1 and IL-13R?2 on U251 and A549 cells.In vitro,IL-13 CART cells were co-cultured with U251 glioma cells and A549 lung cancer cells to verify the killing effect of IL-13CAR-T at different efficiencies and target ratios.At the same time,the secretion of cytokines IFN-gamma and IL-2 was detected by ELISA.Results1.Successfully induced and extracted the minicircle DNA plasmid containing the target fragment IL13 CAR.2.The IL13 CAR minicircle DNA plasmid was successfully transfected into autologous T cells by electroporation.The survival rate of T cells after electroporation was about 58%,and the expression of IL-13 CAR on the surface of T cells was up to 38.8%.Western Blot showed clear expression of IL-13 CAR protein on surface of T cells.3.Transfection of minicircle DNA into T cells by electroporation does not affect the molecular phenotypes of T cells such as CD3,CD4,and CD8.4.IL-13CAR-T cells have high killing efficiency for U251 cells with high expression of IL-13R?2 and relatively low expression of IL-13R?1.There is no obvious killing efficiency of A549 cells that do not express IL-13R?2 and only express IL-13R?1.5.U251 cells significantly stimulated IL-13CAR-T cells to secrete large amounts of IFN-? and IL-2.A549 cells did not significantly stimulate the secretion of IFN-? and IL-2 by IL-13CAR-T cells.Conclusions:1.The minicircle DNA plasmid containing IL-13 CAR target gene was successfully prepared and transfected into T cells by electroporation.2.IL-13CAR-T cells with relatively high expression of IL-13 CAR were obtained by electroporation.3.In vitro killing experiments revealed that IL-13CAR-T cells rely on specific antigens to exert cytotoxic responses.The killing efficiency,the secretion of cytokines IFN-?,IL-2 directly or indirectly reflect the strength of anti-tumor activity in vitro.