Improvement Of Nanoscale Artificial Antigen-presenting Cells And Combination With Nano Tumor Vaccine

Background:In tumor immunotherapy,antigen-presenting cells(APCs)are effective tools to expand antigen-specific T cells in vitro and in vivo,but still face time-consuming,costly,difficult amplification and biosafety issues.With the rapid development of acellular artificial antigen-presenting cells,nanosacle artificial antigen-presenting cells(aAPC)have been developed in recent years,which have better biodistribution and less embolism than cell-sized aAPC,but there are still shortcomings:compared with cell-sized aAPC,it is more easily uptaken by macrophages and has a smaller surface area for contact with T cells,so the ability to amplify T cells is relatively weak.In addition,only few T cell clones can be amplified by aAPC,not broad enough.Objective:The nanoscale aAPC is optimized to improve its ability to resist phagocytosis and amplify T cells,and further combined with other therapy(tumor vaccine nanoparticles)to induce a better immune response.Methods and results:(1)Preparation and verification:firstly,spherical PLGA nanoparticles were prepared by double emulsion method using PLGA(polylactic-co-glycolic acid)as raw materials,then stretched and PEGylated,and finally,surface-modified with H-2Kb/TRP2180-188-Ig dimer(signal 1),anti-CD28(signal 2)and/or CD47-Fc,thereby obtaining corresponding a APCs.The results show that we successfully generated spherical aAPCs(SaAPCs)with a diameter of about 200 nm,ellipsoidal aAPCs(EaAPC),PEG-covered ellipsoidal aAPCs(EaAPCPEG)and EaAPCPEG surface-modified with anti-phagocytosis molecule CD47-Fc(EaAPCPEG/D47);(2)Evaluation of anti-phagocytic ability in vitro and in vivo:PE-labeled aAPCs were incubated with macrophage RAW264.7 for 6 hours,and then confocal microscopy and flow cytometry were used to investigate the engulfment of aAPCs by RAW264.7 cells The results show that compared with the EaAPCPEG,EaAPC,SaAPC and Blank-NPs group,the EaAPCPEG/CD47 group was uptaken with the least amount in vitro;aAPCs were injected into tumor-bearing mice to detect contacts with immune cells in spleen,the EaAPCPEG/CD47 showed most contacts to CD8+T cells,and the least co-localizations with macrophages and dendric cells;(3)Anti-tumor effects and tumor-specific T cells proliferation:on the 7th,9th and 11th day after inoculation of melanoma cells in C57BL/6 mice,aAPCs were injected into the tumor-bearing mice through the tail vein,and then the tumor growth was measured every three days.On the 28th day,the mice were sacrificed to evaluate the anti-tumor effects of aAPCs.Flow cytometry was used to detect the frequencies of TRP2180-188 specific CD8+T cells and IFN-?+/CD8+T cells in peripheral blood,spleen and tumor tissue samples;mouse spleen lymphocytes were pre-stained with CSFE and then co-cultured with B16 cells to detect their cytotoxicity to tumor cells.The results show that among the groups,the EaAPCPEG/CD47 group had the strongest effects in inhibiting tumor growth and prolonging mouse survival time,with the highest frequencies of specific CD8+T cells and IFN-?+/CD8+T cells,and spleen lymphocytes presented the strongest ability to kill B16 cells;(4)Combination therapy and mechanism exploration:tumor cell lysate(TCL)was encapsulated in spherical PLGA nanoparticles followed by surface modification with PEI to obtain nanoscale tumor vaccine TPP(TCL-PLGA-PEI).Then the TPP was combined with aAPC for anti-tumor immunotherapy.The results show that the nanoscale TPP was successfully prepared and could be uptaken by maocrophage RAW264.7 and dendritic cells DC2.4 in vitro.The combination therapy achieved better anti-tumor effects than the monotherapy.Compared with TPP treatment group and aAPC treatment group,the combination treatment group could more effectively inhibit tumor growth and prolong mice survival;increase the frequencies of CD4+T,CD8+T and TRP2180-188 specific CD8+T cells;enhance the activation,degranulation,cytotoxicity and proliferation activity of CD8+T cells;inhibit regulatory T cells proliferation;promote inflammatory cytokines secretion while reduce inhibitory cytokines production;in addition,CD4+T and CD8+T cells were more invasively infiltrated into the tumor tissues in the combination treatment group.Conclusion:The superimposed or synergistic effects of ellipsoidal stretch,PEGylation,and CD47-Fc conjugation minimized cellular uptake of the nanoscale aAPC by phagocytic cells and induces stronger antigen-specific T cell responses and tumor growth inhibition.In addition,combination therapy of tumor vaccine nanopaarticles and optimized aAPCs can induce a broader immune response and better anti-tumor effects than monotherapy.Therefore,this study provides an optimization solution for tumor-specific active immunotherapy.