Study On The Mechanism Of IL-21-αHSA Combined With PD-1 Inhibitor In Enhancing Anti-tumor Effect

Tumor immunotherapy is a treatment method to eliminate malignant tumors by activating the own immune system.Immune checkpoint inhibitor therapy(ICI),represented by PD-1,has achieved good clinical results in the treatment of various solid tumors.However,due to the heterogeneity of malignant tumors and presence of a large number of immunosuppressive cells and inhibitory molecules in the tumor microenvironment(TME),the effective response rate of most solid tumor patients to ICI therapy remains low.Therefore,Improving the response rate of tumor patients to immunotherapy has important clinical significance.IL-21 is mainly produced by T helper cells 17(Th17),T follicular helper cells(Tfh),and natural killer T(NKT)cells.IL-21 plays an important role in promoting the maturation of CD8+T and NK cells and enhancing cytotoxicity.During chronic LCMV infection,IL-21 indirectly activates the antiviral activity of CD8+T cells by inhibiting the expansion of Treg cells.Therefore,cytokine-based immunotherapy is expected to further improve the clinical response rate of existing immunotherapies.However,the source of IL-21 in tumor tissue and the mechanism by which IL-21 combined with ICI regulates various immune cells in the TME remain unclear.Furthermore,systemic IL-21 therapy fails to achieve sufficient concentrations in the TME to activate tumor infiltrating lymphocytes(TILs)due to short half-life of cytokine and problems such as peripheral consumption.Therefore,prolonging the half-life of IL-21 and exploring its anti-tumor mechanism in combination with ICI are important scientific issues for improving the clinical therapeutic effect of ICI.Objective:The source of IL-21 in tumor tissue was identified and its cellular characteristics were analyzed.To study the difference in the anti-tumor effect of IL-21 with prolonged half-life(IL-21-αHSA)and rIL-21,and analyze its potential anti-tumor mechanism.To explore the role of IL-21/IL-21 R signaling pathway in PD-1 inhibitor therapy.IL-21-αHSA was further used to combine with PD-1 inhibitor to observe its effect on tumor growth in mice,and analyze the proportion and phenotypic changes of immune cell populations in TME;The differential expression gene of immune cell populations after combination therapy were explored by single-cell transcriptome sequencing to find potential molecular mechanisms.Finally,IL-21-αHSA was combined with PD-1,TIM-3,and LAG-3 inhibitors to seek to solve the tolerance problem in current tumor immunotherapy.Methods:The relationship between the expression of IL-21 and its receptors in different tumor tissues and the survival of patients were analyzed by bioinformatics technology;To further determine the distribution of IL-21 expression in immune cells in colorectal cancer(CRC),non-small cell lung cancer(NSCLC)patients and mouse colon cancer TME;Single-cell transcriptome sequencing and flow cytometry were used to determine the expression distribution of IL-21 R in each subset of TIL in tumor-bearing mice.The CD8+ T cells of spleen in normal mice were enriched by magnetic bead sorting,and IL21-αHSA and rIL-21 were added for in vitro culture,and the proliferation and functional changes of T cells were analyzed by flow cytometry;A mouse MC38 colon cancer xenograft model was constructed,and the effects of IL-21-αHSA and rIL-21 on tumor growth in tumorbearing mice were compared;The proportion and phenotypic changes of mouse TIL population were analyzed by flow cytometry;The tumor-bearing mice were injected intraperitoneally with FTY720 to explore the effect of IL-21 on immune cell function in TME.IL-21 R blocker was used to block IL-21/IL-21R signaling pathway to explore its effect on anti-tumor effect of PD-1 inhibitor.A mouse colon cancer xenograft model was constructed to compare the effects of IL-21-αHSA combined with PD-1 inhibitor on mice tumor growth.The proportion of TIL population and cell phenotype changes in mice were analyzed by flow cytometry;The number of peripheral antigen-specific reactive cells in different treatment groups were detected by enzyme-linked immunospot assay(Elispot).The tumor-infiltrating CD45+immune cells in different treatment groups were sorted by flow cytometry,the effects of combination therapy on immune cell transcriptomes were assessed using l0×Genomics single-cell transcriptome sequencing.A mouse MC38 colon cancer xenograft model was constructed to explore the effect of IL-21-αHSA combined with PD1/TIM-3/LAG-3 inhibitor on tumor growth in mice.Results:We found that tumor patients with high expression of IL-21 and its receptor had longer survival.In colon cancer,lung cancer patients and mouse colon cancer tumor tissues,IL-21 is derived from CD4+T cells that highly express immune checkpoints and effector molecules.In addition,IL-21 R is widely expressed on immune cells in mouse TME,with an expression ratio of about 40%in CD8+ and CD4+Tconv,and about 54%in B cells.Both IL-21-αHSA and rIL-21 could stimulate the production of GzmB in CD8+T cells.Compared with the control group and rIL-21 treatment group,IL-21-αHSA can more effectively inhibit tumor growth,and the difference is statistically significant(P<0.05).In vivo studies showed that IL-21-αHSA could better inhibit tumor growth.IL-21-αHSA treatment can directly enhance the anti-tumor response of immune cells in the TME and increase the infiltration of immune cells in the TME of tumor-bearing mice,and enhanced CD8+T cell activation and effector function,including the increased expression of PD-1,IFN-y,etc.;decreased the proportion of Tregs cells,and inhibited the expression of TGF-β1,IL-10,and immune checkpoint molecules.PD-1 inhibitor treatment can up-regulate the expression of IL-21 in tumor-infiltrating CD4+T cells,and block IL-21R signaling to reduce the inhibitory effect of PD-1 inhibitor on tumor growth.Compared with the single treatment group,IL-21-αHSA combined with PD-1 inhibitor could significantly inhibit tumor growth(P<0.05)and increase the expression of IL-21 R on CD4+Tconv cells,CD8+T cells and NK cells in TME.The proportions of IFN-γ+ CD8+,GzmB+CD8+,GzmB+CD4+T cells and IFN-γ+NK cells were further increased.Single-cell sequencing data showed that after combined treatment,CD8+T cells underwent clonal expansion,the expression of effector,activation and exhausted molecules was further increased,and CD4+Tconv clonal expansion was increased.Combination therapy resulted in peripheral T cell activation and increased number of antigen-specific cells producing IFN-y.For myeloid cells,IL-21-αHSA combined with PD-1 inhibitor could increases the proportion of cDC1(type Ⅰ conventional dendritic cells),M1(type Ⅰ macrophages)and decreases the proportion of cDC2(type Ⅱ conventional dendritic cells)and M2(type Ⅱ macrophages)in the TME,and the expression of antigen-presenting genes in cDC1 cells and the expression of chemokines that recruited M1 cells into tumor tissues were up-regulated.IL-21-αHSA combined with PD-1 blockade enhanced TME and peripheral antitumor immune responses at late stage.Finally,we combined IL-21-αHSA with PD-1/TIM-3/LAG-3 inhibitors to produce a greater antitumor effect.However,IL-21-αHSA combined with PD-1 inhibitor also increased the ratio of eTregs and MDSCs(myeloid-derived suppressor cells),enhanced the expression of M2 cell suppressor molecules.Conclusions:IL-21 in the tumor microenvironment is derived from CD4+T cells that highly express immune checkpoints and effector molecules.IL-21-αHSA can control tumor growth by enhancing the effector function of CD8+T cells and reducing the suppressive function of Tregs.The antitumor effect mediated by PD-1 inhibitors requires IL-21/IL-21R signaling.IL-21-αHSA combined with PD-1 inhibitor could synergistically inhibit tumor growth by enhancing the immune response mediated by Th1 and CD8+T cells,increasing the ratio of M1 and cDC1,enhancing cDC1 antigen presentation,and reducing the proportion of M2 cells.On the one hand,it can also increase the proportion of MDSCs and eTregs,activate Tregs,and promote the expression of T cell-depleting molecules to maintain the inhibitory TME.Combining IL-21-αHSA with PD-1,TIM-3,and LAG-3 inhibitors further enhances the efficacy of immunotherapy.