The Study Of Sequential Administration Of Anti-PD-1 And Anti-Tim-3 Recovered T Cell Apoptosis Of GM-CSF-anchored Vaccine For Metastatic Bladder Cancer

Background and ObjectivesBladder cancer is the most common malignant tumor in the urinary system which threatens human health,and muscle invasive bladder cancer is particularly harmful.The possible reasons for the high relapse rate and malignant progression of bladder cancer are that the lack of tumor infiltrating lymphocytes(TILs)in the tumor microenvironment(TME)and tumor cells escape the killing of immune cells through the immune escape mechanism and leading to the continued growth of the tumor.Tumor vaccine can activate immune response by inoculating tumor antigens into the host in a variety of ways.In our previous researches,based on the unique property of Streptavidin(SA)to bind rapidly and almost irreversibly to any biotin linked molecule and the outstanding ability of biotin to be incorporated easily into proteins on the cell surface,we had developed a protein anchor platform to immobilize SA tagged bioactive molecules on the surface of biotinylated MB49 bladder cancer cells,and confirmed that cytokine-modifined MB49 bladder cancer cell vaccines could effectively induce a specific antitumor immunity which inhibited the growth of bladder cancer in mice model.However,we also found that the majority of the vaccine-treated tumors experienced slowly regrew through long-term observation.PD-1/PD-L1 is an important costimulatory molecule.PD-1/PD-L1 has been shown to negatively regulate immune responses by inhibiting T cell activation and proliferation.T-cell immunoglobulin and adhesion protein-3(Tim-3)is a member of the Tim family,when combined with its natural ligand galectin-9 can cause helper T-cell 1 programmed death,thereby negatively regulating the anti-tumor ability.Combined with our previous research work and present research progress,we intend to perform the following studies:established the subcutaneous models of mice with metastatic bladder cancer to be tumorigenic for subsequent animal studies.The use of SA-GM-CSF membrane modification bladder cancer cell vaccine therapy model in mice and assessed the therapeutic effect,and to verify whether the PD-1,Tirn-3 expression-related immune escape could be happened in TME of the vaccine treated mice.According to detect the funcuons of different subsets of CD8+ TILs,we could understand the effect of PD-1 and Tim-3 expression on the functions of CD8+TILs.Finally,we used anti-PD-1 and anti-Tim-3 antibody sequential blocking combined with the vaccine treatment program to assess the effect,and explored the relevant immunological mechanisms.MethodsProtein-modified cell surface technology was used to prepared SA-GM-CSF membrane-modified bladder tumor cell vaccine.Through the comparison of tumor size,and some detection methods of CTL,flow cytometry,immunohistochemistry,ELISA,we assessed the vaccine or vaccine combination of targeting costimulatory molecules in the treatment of metastatic bladder tumor effect and explored the relevant immunological mechanisms.Results1.In this researches,based on the unique property of SA to bind rapidly and almost irreversibly to any biotin linked molecule and the outstanding ability of biotin to be incorporated easily into proteins on the cell surface,we developed a protein anchor platform to immobilize SA tagged bioactive molecules on the surface of biotinylated MB49 bladder cancer cells,and confirmed that cytokine-modifined MB49 bladder cancer cell vaccines could induce DCs activation,enhance effector T cell infiltration into TME and effectively improve the specific cytotoxic activity,but insufficient to inhibit tumor growth in long-established subcutaneous models of bladder cancer.2.SA-GM-CSF-anchored vaccine Ccould upregulate PD-1 and Tim-3 expression on CD8?TILs,and could also upregulate PD-L1 expression in TME depending on IFN?.3.The combination therapy with PD-1 blockade and SA-GM-CSF-anchored vaccine could synergistically induce anti-tumor immune responses,which greatly inhibit the tumor growth,even regress the established tumor.But a part of mice still undergone tumor progressed eventually,the tumor regression rate was too low(20%regression).4.Anti-PD-1 antibody could also upregulate Tim-3 expression on CD8+ TILs.5.PD-1+Tim-3+ CD8+ TILs were the most dysfunctional in terms of Thl cytokine production and proliferation,and PD-1-Tim-3+ CD8+ TILs were highly proliferative but defective in cytokine production.So we could considered that PD-1 expression was intensely associated with CD8+ TILs exhausted,and Tim-3 expression was closely correlated with secretion function but not proliferation of CD8+ TILs.6.We combined SA-GM-CSF-anchored vaccine with blockade of PD-1 and Tim-3 in long-established subcutaneous bladder cancer models treatment and found that this combination therapy could significantly suppressed tumor growth,and tumor regression was increased in over 50%.The observation of improvement the specific cytotoxic activity,decrease in apoptosis of CD8+ TILs,together with a reduction of tumour-promoting cytokines,indicated that sequential administration of anti-PD-1 and anti-Tim-3 in treatment with SA-GM-CSF-anchored vaccine could induced a more robust anti-tumor immune response.ConclusionSequential administration of anti-PD-1 and anti-Tim-3 in treatment with SA-GM-CSF-anchored vaccine could effectively inhibit the growth of advanced bladder cancer.