CXCL9 Enhances The CAR T Cell Infiltration And Anti-tumor Efficacy

BackgroundMalignant tumors seriously endanger human health,and about 10 million patients die of cancer every year in the world.The prognosis of advanced cancer patients is still not ideal,although great progress has been made in the multidisciplinary treatment of cancer,such as surgery,radiotherapy,chemotherapy and molecular targeted therapy.So far there is still lack of very effective treatment methods.Therefore,it is particularly important to explore a new therapeutic method to treat malignant tumors.With the development of tumor immunology and molecular biology,immunotherapy,which can inhibit and kill tumor cells by mobilizing or stimulating the body’s own immune function,has become a new hotspot in the field of malignant tumor therapy.Chimeric antigen receptor(CAR)T cell is a kind of targeted antitumor immunotherapy,which can specifically recognize and kill cancer cells expressing tumor-associated antigen,and can also proliferate and activate in vivo,but has no side effect on cells that not expressing tumor-associated antigen.CAR T cells have demonstrated excellent efficacy in hematologic malignancies.CD19-targeted CAR T cells achieve 90%complete response rates in relapsed and refractory B-cell acute lymphoblastic leukemia(B-ALL).Kymriah,Yescarta and Tecartus have been approved by the U.S.Food and Drug Administration(FDA)for the treatment of B-ALL and DLBCL.Although CAR T has been shown to be effective in hematologic tumors,there are many difficulties in the treatment of solid tumors.The number of tumor infiltrating lymphocytes is closely related to patient prognosis.Increasing the accumulation of CAR T cells in the tumor microenvironment is critical for tumor therapy.There is an urgent need to develop new CAR T designs to overcome the barriers of insufficient immune cell infiltration.CXCL9 can increase T cell infiltration into tumor and inhibit tumor angiogenesis.Therefore,we designed CAR T cells(CARTmeso-CXCL9)expressing CXCL9,and confirmed that CARTmeso-CXCL9 can improve the migration,infiltration and anti-tumor efficacy through cell model and animal model,thus providing a new therapeutic strategy for the treatment of solid tumors.1 Effect of CXCL9 on T cell functionObjectiveTo detect the effects of CXCL9 on T cell migration,killing,differentiation and angiogenesis function.Methods(1)The expression of CXCL9 and CXCR3 before and after T cell activation was detected by flow cytometry.(2)Transwell assay was used to detect the effect of CXCL9 on T cell migration function.(3)Flow cytometry was used to detect the effects of CXCL9 on T cell killing,activation and differentiation.(4)The correlation between CXCL9 expression and T cell-related genes in lung cancer was analyzed by TCGA database.(5)Tube formation assay was used to detect the effect of CXCL9 on human umbilical vein endothelial cells angiogenesis.Results(1)CXCR3 increased significantly after T cell activation.(2)CXCL9 increased T cell migration function.(3)CXCL9 promotes the secretion of functional factors of T cells.(4)CXCL9 promoted the differentiation of T cells into Th1.(5)TCGA data analysis showed that the expression of CXCL9 was related to T cell infiltration and the activation and function of T cells in lung cancer tissues.(6)CXCL9 reduced endothelial angiogenesis.ConclusionCXCL9 can enhance T cell migration and effector function.The expression of CXCL9 in lung cancer was positively correlated with T cell infiltration and activation.CXCL9 inhibits angiogenesis.2 CXCL9 secreted CAR T cells enhance anti-tumor ability in vitroObjectiveCARTmeso cells and CARTmeso-CXCL9 cells co-expressing chemokine CXCL9 targeting mesothelin(MSLN)were constructed to verify the effector function and migration function of two CAR T cells on lung cancer cell lines in vitro.Methods(1)The expression of target antigen MSLN in lung cancer tissues was detected by immunohistochemical;the expression of target antigen MSLN in lung cancer cell lines was detected by flow cytometry.(2)CARTmeso cells targeted at MSLN and CARTmeso-CXCL9 cells co-expressing chemokine CXCL9 were respectively constructed by lentivirus transfection method.(3)CXCL9 expression in CARTmeso and CARTmeso-CXCL9 was detected by flow cytometry and Cytometric Bead Array.(4)Flow cytometry and enzyme-linked immunosorbent assay were used to detect the effect function of CARTmeso and CARTmeso-CXCL9 on MSLN positive target cells.(5)Recruitment function of CARTmeso and CARTmeso-CXCL9 cells was detected by transwell assay.Results(1)Immunohistochemical results showed that MSLN was highly expressed in lung cancer tissues.Flow cytometry showed that lung cancer cell line H322 expressed MSLN highly,while A549 expressed MSLN low.(2)Flow cytometry results showed that CARTmeso cells and CARTmeso-CXCL9 cells could be successfully constructed,and CXCL9 expression in CARmeso-CXCL9 cells increased significantly.(3)Flow cytometry showed that CARTmeso-CXCL9 T cells could kill H322 lung cancer cells more effectively than CARTmeso cells,and ELISA showed that CARTmeso-CXCL9 cells could secrete more effector cytokine IFN-y.(4)Transwell results showed that CARTmeso-CXCL9 cells had stronger recruitment function than CARTmeso cells.(5)Tube formation assay showed that the culture supernatant of CARTmeso-CXCL9 could inhibit angiogenesis.ConclusionCARTmeso-CXCL9 cells have stronger killing and migration functions in vitro than traditional CARTmeso cells and could inhibit angiogenesis.3 Effects of CXCL9 on the anti-tumor ability of CARTmeso in a mouse xenograft tumor modelObjectiveMouse xenograft tumor model was established to investigate the anti-tumor ability of CXCL9-modified CARTmeso-CXCL9 cells in NOD/SCID mouse tumor model.Methods(1)The effects of CXCL9-modified CARTmeso-CXCL9 cells on the migration,infiltration,proliferation,differentiation and killing function of T cells were detected in a mouse xenograft subcutaneous tumor bearing model.(2)The effect of CXCL9-modified CARTmeso-CXCL9 on angiogenesis in vivo was detected in a mouse Matrigel plug assay model.(3)The control of CXCL9-modified CARTmeso-CXCL9 on tumor progression in mouse xenograft model was tested.Results(1)In the mouse xenograft subcutaneous tumor bearing model,CXCL9-modified CARTmeso-CXCL9 cells have a stronger infiltration ability,can recruit endogenous T cells,promote Th1 polarization,and increase the secretion of functional cytokines.(2)In the model of mouse Matrigel plug assay,CXCL9-modified CARTmeso-CXCL9 cells can inhibit angiogenesis.(3)In the mouse xenograft subcutaneous tumor bearing model,CXCL9-modified CARTmeso-CXCL9 significantly inhibited tumor growth.ConclusionIn mouse tumor models,CXCL9-modified CARTmeso-CXCL9 has stronger ability of migration,infiltration,recruitment function,killing function,anti-angiogenesis and anti-tumor efficacy.