| Glioma is the most common primary malignant intracranial tumor,accounting for about 28%of all primary brain tumors.Among them,glioblastoma(GBM)is the most malignant one,whose 5-year survival rate of the patients is only 5.1%.GBM has obvious immunosuppressive characteristics,such as limited lymphocyte infiltration and low expression of immune checkpoint molecules.So far,immunotherapy of glioma remains ineffective.Oncolytic viruses(OVs)are an effective means of treating a variety of tumors.Currently,four oncolytic viruses have been approved for clinical use,among which Delytact(teserpaturev/G47?)has been approved in Japan for the treatment of glioma.In clinical trials,the virus showed a certain inhibitory effect on tumor growth after being stereotactically injected.However,due to the high heterogeneity and immunosuppressive microenvironment of the tumor,the fact that the virus lacks additional treatment payloads,as well as the injection was only proceeded once instead of repeated intravenous injection,the therapeutic effect of G47?on tumors is limited.Vaccinia virus(VV)has many advantageous characteristics that are superior to other oncolytic viruses in terms of therapeutic efficacy and safety.VV does not require specific surface receptors to enter tumor cells,making it more suitable for treating highly heterogeneous tumors;its large viral genome that can accept foreign DNA inserts;its various forms of existence during replication and immune escape characteristics give it potential for intravenous administration for repeated multiple dosing.In addition,VV also has a rapid intracellular life cycle,good safety records while used as smallpox vaccines,the ability of replication in hypoxic environments,activation of immunogenic cell death pathways and induction of vascular collapse in tumor microenvironments.All these indicate that vaccinia virus can be used as an effective and safe biological treatment for tumors.The oncolytic VV Lister strain used in this study has previously undergone rational gene editing to enhance its oncolytic effect,while reducing side effects.Thymidine kinase and N1L gene deletions ensure tumor selectivity and the deletion of N1L provides an additional advantage of significant improvement in induction of anti-tumor immune responses via multiple mechanisms,including improved T cell infiltration to tumors,improved activation of T cells,and increased circulating natural killer(NK)cells.In addition,the virus expresses a second,mutant copy of the viral B5R gene that we have previously shown to be essential for extracellular enveloped virus(EEV)production and therefore long-range spread of the virus.Indeed,after intravenous(i.v.)delivery of the virus with this modification,significantly more viral genome copies were detected in subcutaneous tumors and survival was improved in Syrian hamster models of disseminated pancreatic cancer.In order to further tap into the clinical application potential and optimize intravenous administration effects of VV,we have also previously described an effective systemic delivery platform for VV via transient inhibition of PI3Kd to prevent macrophage uptake of systemically delivered VV.The development of an i.v.delivery platform of VV is crucial for glioma treatment because of its intracranial location,which poses a safety concern for i.t.injection of therapeutics.Interleukin-21(IL-21)is an extremely attractive cytokine in the context of anti-tumor immunotherapy as it can safely and effectively enhance immunity to tumors through multiple mechanisms.IL-21 is an effective inducer of T cell activation in vivo,enhances the antigen affinity of specific CD8~+T cells,inhibits the development of regulatory T cells(Treg),induces the maturation of NK and NKT cells,induces activation and cytolytic potential of NK and NKT cells,promotes the production of tumor-specific Ig G B cells,and inhibits angiogenesis by reducing the expression of VEGFR1 and TIE1 in endothelial cells.31 Most importantly,even high-dose delivery of IL-21 has not resulted in development of toxic side effects often noted with other similarly pleiotropic cytokines.ICI are currently being investigated as a therapeutic option for glioma.Promising therapeutic activity has been noted in preclinical glioblastoma models,but the results of clinical trials in patients with recurrent glioblastoma are disappointing,likely due to the low mutational burden and poor T cell infiltration in GBM.OVs have previously been shown by us and others to synergize effectively with ICI.OVs can efficiently reprogram the TME,recruiting anti-tumor immune cells.However,tumor cells can shut down activation of T cells via rapid activation of the immune checkpoints.The addition of ICI therapy to the regime is therefore likely to be of additional therapeutic benefit by preventing the inactivation of newly recruited T cells.This study first investigated in vitro the replication ability and cytotoxicity of VVLΔTK-STCΔN1L-m IL-21 and VVLΔTK-STCΔN1L in GBM cells.After virus infection for 24,48,72,and 96 hours in multiple GBM cell lines,the virus titer in the culture dish was detected respectively to calculate the efficiency of virus replication in different GBM cell lines.The expression level of m IL-21 carried by virus in supernatant was detected by ELISA method.Then MTS method was used to detect the percentage of surviving tumor cells after viral infection with different dilutions to calculate the cytotoxicity of virus on various GBM cell lines.The results showed that both oncolytic viruses can replicate efficiently in mouse glioma cells.VVLΔTK-STCΔN1L-m IL-21 can synthesize a large amount of m IL-21 in infected tumor cells.Both oncolytic viruses have strong killing ability against different mouse GBM cell lines in vitro and there is no significant difference between them.To further study the therapeutic effect of VVLΔTK-STCΔN1L and VVLΔTK-STCΔN1L-m IL-21 alone or in combination withα-PD1 on GBM in vivo,we established C57BL/6 mouse subcutaneous tumor and orthotopic transplantation tumor models using mouse GBM cell line GL-261.VVLΔTK-STCΔN1L-m IL-21 and its control virus VVLΔTK-STCΔN1L without m IL-21 were locally injected into tumors or intravenously injected after oral gavage of CAL101 alone or in combination withα-PD1 for treatment.By recording the survival status,tumor growth curve,survival time and other means of animal models,we evaluated the safety and efficacy of VVLΔTK-STCΔN1L-m IL-21 combined withα-PD1 therapy for subcutaneous or orthotopic transplantation mouse GBM tumor models.The results showed that VVLΔTK-STCΔN1L-m IL-21 combined withα-PD1 had the best therapeutic effect,which could significantly eliminate subcutaneous tumors(tumor clearance rate:80%for local injection and 50%for intravenous injection)and orthotopic transplantation tumors(tumor clearance rate:71.4%),prolonging the survival time of mouse models and improving survival rate with outstanding performance in safety and efficacy.On this basis,we used mainly flow cytometry to reveal the immunological changes behind this therapeutic effect.After grouping the mouse GL-261subcutaneous tumor model,PBS,VVLΔTK-STCΔN1L,VVLΔTK-STCΔN1L-m IL-21 and VVLΔTK-STCΔN1L-m IL-21+α-PD1 were respectively used for treatment.The day of the start of treatment is called D1,followed by D3 and D5 for a total of 3 treatments.On D7,14,and 21,three mice were randomly selected from each group to collect their lymph nodes,spleen,subcutaneous tumors,and serum.Flow cytometry was used to analyze immune cell subsets.The release of IFN-γafter co-culturing spleen cells with different stimuli was measured to evaluate the level of mouse anti-tumor immune memory activation.Immunohistochemistry was used to detect PD-L1 expression in tumor samples,and ELISA was used to detect m IL-21 levels in serum.The results of the study confirmed that VVLΔTK-STCΔN1L and VVLΔTK-STCΔN1L-m IL-21 effectively activated local and systemic anti-tumor immunity in the mouse subcutaneous tumor model,increased the proportion of CD4~+,CD8~+TCM cells in spleen,lymph nodes and tumors,up-regulated CD122 expression,induced anti-tumor stem memory T cells,promoted polarization of macrophages towards anti-tumor M1 phenotype direction,promoted DC and NK cell activation.Such effect was more significant in the virus in combination withα-PD1 group.After oncolytic virus therapy,the mouse model effectively expressed its anti-tumor factor payload m IL-21,and PD-L1 expression in subcutaneous tumor tissue was significantly increased.The spleen cells in the virus treatment group showed a significant increase in IFN-γsecretion after stimulation with virus protein B8R,GL-261 cells and lysed GL-261 cells,indicating that mice produced strong immune memory and response against viruses and tumors.In summary,we have confirmed that VVLΔTK-STCΔN1L-m IL-21 has significantly reshaped the tumor immune micro-environment after intratumoral and intravenous administration,induced a powerful anti-tumor immune response,thereby controlling tumor growth in mouse subcutaneous and in situ tumor models and prolonging their survival time.Combined withα-PD1 application,the therapeutic effect was significantly improved.More importantly,this study also revealed that after virus treatment,stem memory T cells were produced which exerted strong and persistent anti-tumor activity. |
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