SerpinB9 Antagonizes T Cell Endogenous Granzyme Activity To Improve CAR-T Anti-tumor Activity

Background and Objective: The Chimeric antigen receptor T cells(CAR-T)immunotherapy utilizes ex vivo genetically modified T cells from cancer patients that express a tumor-targeting CAR molecule to treat cancer.Currently,CAR-T therapy is highly effective in treating hematological malignancies but lacks efficacy in solid tumors.Prolonging CAR-T cell persistence and effector functions in tumor microenvironment is critical for its application in solid tumor.Granzyme B(GzmB)is a critical cytotoxic factor in T cells that is highly expressed in CD8+ T cells.However,studies showed that high GzmB expression in T cells can also lead to their own demise,preventing hyper-activation of T cells and occurrence of autoimmunity.But this would also result in reduction in T cell number and selective survival of T cell populations with low cytotoxic functions,and thus potentially hinder the anti-tumor responses of T cells.Therefore,we hypothesize that antagonizing T cell endogenous GzmB activity could facilitate CAR-T survival and effector function upon tumor antigen stimulation and enhance CAR-T anti-tumor activity.SERPINB9(SB9)is the natural inhibitor of GzmB.By forming covalent interaction with GzmB,SerpinB9 inhibit its proteolytic activity.Hence,we over-expressed SB9 gene in CAR-T cells using retroviral vector,antagonizing the endogenous GzmB activity and evaluated how SB9 influence T cell apoptosis,proliferation and effector functions.In addition,using tumor models,we assess whether forced SB9 expression could improve CAR-T cell anti-tumor activity.Our research will pave way for the development of improved CAR-T therapy against solid tumors.Methods: First,we analyzed public single cell RNA sequencing database for SB9 expression in cancer patients’ T cells and assess the relationship between the expression of SB9 and genes involved in T cell effector functions and proliferation.Next,we constructed retroviral vector for SB9 and produce retroviral supernatant from 29 T cells for transduction.We transduced primary human T cell and confirmed the overexpression of SB9 through q PCR,Western Blot and flow cytometry.Then,we measured T cell proliferation(by CFSE and Ki67 staining)as well as apoptosis(by Annexin V/7AAD staining)and validated the phenotypic observation with RNA-Seq transcriptomics analysis and q PCR.Last,we over-expressed SB9 in CAR-T cells and assessed their anti-tumor activity in ex vivo coculture assay and in vivo tumor models.Results:(1)Single cell RNA sequencing analysis showed that the expression of several cytotoxic molecules and proliferation marker gene is higher in T cells with high SB9 expression than those with low SB9 expression.(2)over-expression of SB9 can significantly improve the survival and proliferation of T cells.(3)Through RNA-Seq transcriptomics analysis,we found that SB9 over-expressed T cells showed elevated expression of genes involved in T cell effector functions and in glycolysis and cholesterol metabolism.(4)SB9 over-expressed CAR-T cells showed prolonged cytotoxicity against tumor in vitro.(5)SB9 over-expressed CAR-T cells showed superior anti-tumor activity in vivo.Conclusion:(1)over-expression of SB9 in T cells can suppress activity of endogenous GzmB and the GzmB mediated apoptosis,protecting the survival of highly functional T cell population.(2)Expressing SB9 in CAR-T can improves its anti-tumor activity in tumor models.