The Effects Of Anti-tumor By Active Immunization Against OX40

Background(s):As we all know,CD8 positive T cells show its potential role in anti-tumor cellular immunity.They can recognize specific tumor antigens and Secrete IFN-y,granzyme,perforin and so on.Tregs can inhibit anti-tumor effector cells by interfering with Antigen presentation,secreting inhibitory molecules,producing granzyme,metabolic regulation such as depletion of IL-2,etc.to help the tumor cells escape.The interaction between effector cells and immunosuppressive cells determines the direction of tumor development.Tumor infiltrating effector T cells are often inhibited by tumor and immunosuppressive cells,which can not effectively recognize and kill tumor cells.Immune checkpoint inhibitor therapy has been widely applied in clinical for anti-tumor immunotherapy,which has acquired favorable curative effect,regulated immunosuppress molecular network in the tumor microenvironment,and achieved "immune normalization".More and more studies have identified the drug-resistance in the first generation checkpoint immunotherapy,thus,new and effectively targets are urgently needed.OX40 also named CD134/TNFRSF4 has been identified as one of the most representative targets in the second generation checkpoints,which is widely expressed in immune cells in inflammation and tumor sites.The activation of the OX40-OX40XL-axis promotes the proliferation and survival of T cells,and at the same time contributes to the generation of memory T cells.The inhibition of Treg cells and the stimulation of effector t cells by OX40 agonist antibody contribute to the anti-tumor effect of immune system.Objective(s):To evaluate the anti-tumor immune response by active immunization with OX40,we prepare the HBcAg Virus-like particles(VLP)to present the predicted epitopes of the mouse OX40 extracellular domain(P1-7).To better explore the VLP vaccine effects in vivo,we establish the 4T1-based orthotopic breast cancer model and TC-I-grafted mouse HPV model.VLP vaccines are employed in the 4T1 modeland TC-1 model.After active immunization,effective vaccine candidates are obtained and identified for further therapeutic strategy evaluation and the influence on HPV therapeutic vaccine,revealing the effects of active immunization strategies targeting OX40 on tumor growth,anti-tumor immune response in mice and the synergy with tumor vaccines,providing a new strategy for tumor immunotherapy.Methods:The prediction of potential OX40 linear B cell epitopes based on the frequency of occurrence of amino acids in the antigen peptide database.The epitopes are inserted into HBcAg-VLPs for antigen presentation,which is produced by the Escherichia coil expression system.The electron microscope(TEM)are used for chimeric VLPs morphological observation.For prevention strategies,six-to eight-weeks old female BALB/c and C57BL/6 mice are immunized with purified chimeric VLPs(P1-7)for four times.VLPs are applied in 4T1 model and TC-1 model.Tumor cells were injected two weeks after the last immunization and continuously monitoring tumor growth.At the endpoint of experiments,mice are sacrificed,spleen and tumor tissues were stripped for immune assay.For therapeutic strategy,TC-1 and B16-F10 tumor cells are first subcutaneously injected to establish the tumor model.Then,50 ug P6-VLPs are administrated for three times when tumor volumes reach 2-3 mm in diameter,mice tumor growth curve and survival rates were monitored.To evaluated the combination effects,TC-1-grafted tumor are established and immunized with P6-VLPs twice,and then immediately after administrated HPV peptides vaccine for three times,mice tumor growth curve and survival rates were monitoredResults:In this study,seven potential epitopes are obtained from the OX40 extracellular domain for further assay.All chimeric VLPs(P1-7)is successfully expressed in E coil expression system,the complete VLPs could be observed under the electron microscope(TEM)with all P1-P7-VLPs.OX40 specific-IgG titers occur after immunization in serum samples.In the 4T1 model,we found that P6-VLP demonstrates the robust anti-tumor response in suppressing tumor progression and metastasis,while P3-and P7-VLP does not work.Additionally,CD8 positive T cell numbers were increased in both splenic-and tumor-infiltrating lymphocytes,along with the increasing CTL,TH1,and decreasing Treg,Th2,and MDSCs.In the TC-1 model,P4-and P5-VLP significantly suppress tumor growth in vivo.We also found that P6-VLP also suppresses TC-1 and B16-F10-grafted tumor growth and improve the survival rate of tumor-bearing mice in therapeutic strategy.In addition,P6-VLP combination with PLGA-E7 was effective in tumor suppression,however,the P6-VLP combination showed better therapeutic efficacy.Conclusion(s):This study successfully constructed,prepared,and purified the recombinant OX40-VLPs,and then evaluated the anti-tumor effects in different mouse tumor models,demonstrating that active immunomodulation against OX40 can effectively suppress tumor progression,prolong the survival time of tumor-bearing mice,enhance cellular response on anti-tumor immune effects.In addition,the synergistic tumor antigen therapeutic vaccine with OX40-VLP showed a robust anti-tumor response better than monotherapy.Taken together,our finding provides a novel,potential strategy for tumor immunotherapy.