IL-24 Improves The Therapeutic Effect Of CAR-T Cells In Solid Tumors

BackgroundCancer has been a major threat to human health in recent years,and the incidence and mortality of cancer are also increasing year by year.In clinical therapies,Radiation therapy,chemotherapy,and chemoradiation therapy as well as surgery are the most fundamental treatments for cancer.However,the therapeutic effect is not ideal,the outcomes of patients are not significantly improved.In recent years,immunotherapy has emerged as a major therapeutic modality in oncology.As a new type of immunotherapy,chimeric antigen receptor T cells(CAR-T cells)have emerged as a promising therapy for treating B cell-derived malignancy,especially the CAR-T cells targeting CD 19 in patients with B cell acute lymphoblastic leukemia(B-ALL)have yielded impressive clinical results,the rate of complete remission in patients could be as high as 92%.So far,three CAR T-cell therapies have been approved by the US Food and Drug Administration(FDA)for the treatment of B-ALL and DLB CL patients:Yescarta、Tecartus and Kymriah.Despite advances have been made in the treatment of malignant haematological diseases,the application of CAR-T cells in solid tumors is limited,which is primarily by the complex microenvironment in solid tumors and the off-target effect induced by the absence of sufficiently specific target antigens.A major problem faced during the tumor treatment is the high rate of recurrence,which is the primary factor limiting the effect of CAR-T cells.These shortcomings have led researchers to search for more effective CAR-T cells.Cancer stem cells(CSCs)have been demonstrated to play important roles in tumor recurrence.CSCs have extensively proliferative potential that potentially form metastatic tumors,and CSCs are the critical cause of tumor recurrence.Along with the advancement of immunotherapy in recent years,the innovation of CAR-T cell structure and production have led to a significant increase in efficacy and durability.Especially,the dramatic development of the fourth-generation CAR T cells also provides us more ideas for T cell function optimization.Currently,the combination of cytokine and chemokines like IL-15,IL-2 and IL-23 with CAR-T cells effectively improve the antitumor activity of CAR-T cells in the tumor microenvironment;The application of dual-target CAR-T cells effectively improved the specificity of targeted killing of tumor cells.However,relapse of tumor after CAR-T cell treatment still remains a predominant obstacle,and no solutions have been reported.This study was objected to modifying the structure of CAR based on the tumor recurrence induced by CSCs.IL-24,a cytokine that can effectively inhibit tumor stemness and induce tumor apoptosis,was used to improve the effect of CAR-T cells on CSC.In this study,the function of CAR-T cells combined with IL-24 in tumor recurrence was demonstrated by animal models,tumor cell models and clinical specimens.1.Killing of CAR-T cell leads to the enrichment of cancer stem cellObjectiveTo determine the expression of NKG2DLs and HER2 on tumor tissues and cell lines.To construct CAR-T cells target NKG2DLs and HER2 via molecular biology technology.To detect the target killing ability of CAR-T cells and analyzed the change of tumor cell stemness after Co-culture.Methods(1)Immunohistochemical staining was used to detect the specific expression of NKG2DLs and HER2 in tumor tissues.(2)Lentiviral transfection was used to construct CAR-T cell targeting NKG2DLs and HER2.(3)Flow cytometry and enzyme-linked immunosorbent assay were used to detect the killing effect of CAR-T cells on antigen-positive target cells.(4)Flow cytometry and cell spheroidization methods were used to detect the stemness enrichment of surviving tumor cells after co-culture with CAR-T cells.Results(1)NKG2DLs are highly expressed in lung cancer tissues,and HER2 is highly expressed in esophageal cancer tissues.(2)Lung cancer cell lines H460 and Calu-3 highly express NKG2DLs,while H322 has a low expression NKG2DLs;the esophageal cancer cell line KYSE150 highly expresses HER2,while TE-7 has a low expression of HER2.(3)CAR-T cells can effectively kill target cells.At the same time,CAR-T cells can secrete large amounts of IL-2 and IFN-y.(4)After co-culture with CAR-T cells,some tumor cells still survived and the stemness of the surviving tumor cells was significantly enriched.ConclusionThe existing classic second-generation CAR-T cells can effectively kill target cells,but they will lead to the enrichment of surviving tumor stem cells.2.IL-24 inhibits tumor cell stemness and promotes tumor cell apoptosisObjectiveTo detect the effect of IL-24 on tumor cell stemness and apoptosis.Methods(1)RT-PCR experiment was used to detect the expression of IL-24 in tumor tissues and analyzes the correlation with the patient’s prognosis.(2)RT-PCR experiment was used to detect the correlation between IL-24 expression and tumor stemness genes.(3)Cell fluorescence and flow cytometry were used to detect the effect of IL-24 on tumor cell apoptosis.(4)TR-PCR,flow cytometry,cell immunofluorescence,immunoblotting and spheroidization experiments were used to detect the effect of IL-24 on tumor cell stemness.Results(1)Analysis of clinical specimens showed that the expression of IL-24 was lower in tumor tissues than normal tissues and was positively correlated with patients’prognosis.(2)Analysis of clinical specimens showed that the expression of IL-24 in tumor tissues was negatively correlated with the expression of multiple tumor stem genes.(3)IL-24 induced tumor cell apoptosis.(4)IL-24 inhibited tumor cell stemness.ConclusionIL-24 was low expressed in tumor tissues and was positively correlated with patient prognosis.IL-24 induced tumor cell apoptosis and inhibited tumor cell stemness.3.IL-24 can promote T cell proliferation and activationObjectiveTo analyze the relationship between IL-24 expression in tumor tissues and T cells,and detect the effect of IL-24 on T cell activation,proliferation,differentiation and apoptosis.Methods(1)RT-PCR experiment was used to detect the correlation between IL-24 expression in tumor tissues and T cell-related genes.(2)TCGA data was used to detect the correlation between IL-24 expression in tumor tissues and T cell-related genes.(3)RT-PCR experiment was used to detect the effect of IL-24 treatment on the expression of T cell-related genes.(4)Flow cytometry experiments were used to detect the effects of IL-24 treatment on T cell activation,proliferation,differentiation and apoptosis.Results(1)Analysis of clinical specimens and TCGA data showed that IL-24 expression in tumor tissues was positively correlated with the genes that were related T cell infiltration,activation and functional.(2)IL-24 treatment promoted T cell activation and proliferation,and promoted T cell differentiation to central memory subtypes;(3)IL-24 treatment did not affect T cell apoptosis.ConclusionThe expression of IL-24 in tumor tissues was positively related to the genes that were related with T cell infiltration,activation and function.IL-24 treatment promoted the activation and proliferation of T cells,and promoted the differentiation of T cells to the central memory subtype,but did not affect T cell apoptosis.4.IL-24 secreting CAR-T cells better control tumor progressionObjectiveTo construct IL-24 secreting CAR-T cells and to verify their anti-tumor activity.Methods(1)The co-culture experiment was used to detect the killing activity of IL-24 secreting CAR-T cells on tumor cells and the inhibition of tumor cell stemness;(2)Lung metastasis model and subcutaneous tumor-bearing model were used to detect the effect of IL-24 secreting CAR-T cells on inhibiting tumor progression;(3)The late-stage treatment model was used to detect the effect of IL-24 secreting CAR-T cells on controlling the progression of advanced tumors;(4)The recurrence model was used to detect the effect of IL-24 secreting CAR-T cells on inhibiting tumor recurrence.Results(1)IL-24 secreting CAR-T cells had stronger anti-tumor activity and can inhibit tumor cell stemness.(2)IL-24 secreting CAR-T cells could better control tumor progression in mouse mode.(3)IL-24 secreting CAR-T cells could better control the progression of advanced tumors in mouse mode.(4)IL-24 secreting CAR-T cells could better control the tumor recurrence in mouse mode.ConclusionIL-24 secreting CAR-T cells had stronger anti-tumor activity and could inhibit tumor cell stemness.IL-24 secreting CAR-T cells could better control tumor progression and recurrence in mouse mode.