Fabricating Effective Autologous Tumor Vaccine Combined With PPAR-α Agonist And PD-1 Blockade Triple-enhanced T Cell Immunity Against Postoperative Melanoma

Immunotherapy continues to be the focus in current tumor treatment,which mobilizing the body’s immune system to recognize and eliminate tumors.T cells are the core effector cells in immunotherapy,as they can directly and specifically kill tumor cells.Based on this,Immune checkpoint blockade therapy has achieved good therapeutic effects in clinical anti-tumor therapy.Moreover,by directly editing T cells,CAR-T cell therapy showed almost 100%tumor eliminating rate in several blood tumors treatment.Despite these promises in T cell based therapy,the low response rate and insufficient immune response in solid tumor still limited its clinical applications.Current studies have demonstrated that the immune system is a complex network with a variety of regulatory mechanisms.To successfully eliminate tumors,the body need to firstly generate sufficient tumor-specific cytotoxic T cells(CTLs),which is often suppressed by low immunogenicity of tumors.The low immunogenicity of tumors often cannot stimulate the body to activate a sufficient number of CTL,and after the activated T cells reach the tumor tissue,their effector functions are influenced by metabolism.Moreover,tumor cells disguised themselves as normal cells by expressing immune checkpoints,so as to escape the killing effect of CTLs.Therefore,based on the need for effective CTL therapy,we propose a triple promotion strategy of increasing the number of CTL,promoting CTL efficacy,and relieving tumor immunosuppressive CTL,aiming to provide a new solution for generating effective T cell therapy.To generate sufficient tumor-specific CTLs,personalized tumor vaccines received great attention cause they can initiate immune response by stimulate antigen presentation.By using resected tumors as antigens,the vaccinated patients can elicit high-specific multivalent anti-tumor T-cell immunity to against autologous tumors.However,the low immunogenicity highly limited its immune effect and clinical application.Here,we aimed to design a pre-vaccine reagent,which can simply fuse with resected tumors to prepare a high-effective tumor autologous vaccine.To making the fusioned tumor autologous vaccine high-effective,the adjuvant combination strategy was used.Among adjuvant-related pathways,STING,an intracellular pattern recognition receptor,holds great promise.Activation of the STING pathway triggers I IFN response that promotes DC maturation and antigen cross-presentation.Meanwhile,the TLR9 pathway plays an important role in immunity.activation of TLR9 pathway it can induce the secretion of various cytokines such as TNF-α,IL-6,IL-12,etc,and regulates the immune response of the body.The combination of TLR9 and STING pathway activation can synergistically promote the secretion of inflammatory cytokines such as IFN-I and TNF-α,and enhance antigen-specific adaptive immune response.DOPE,which can undergo a phase transition in an acidic intracellular environment,was incorporated to induce the lysosomal escape of antigens,thus promotion of CTL differentiation through antigen cross-presentation.Therefore,adding DOPE and mannose modification in vaccine preparation can deliver tumor antigens and adjuvants to DCs,achieve antigen cross-presentation,and effectively activate T cells.In this paper,the lipid DOPE and DOTAP were used as carrier materials,and DSPE-PEG-Man was used as the target to prepare a co-loaded CpG and cGAMP vaccine pre-liposome preparation.Then it was fused with the tumor cell membrane extracted from the tumor to obtain the high-efficiency autologous tumor vaccine C/G-HL-Man.The activity of CTLs also affects antitumor immunity.The activity of CTLs in tumor tissue is impaired by lack of energy.In order to survive in harsh environments,immune cells undergo metabolic reprogramming and use fatty acids as a "second supply station" for energy.Fatty acid synthesis and uptake are key features of effector T cells,and fatty acid catabolism may improve CTL cell function through an alternative pathway.Therefore,reprogramming the lipid metabolism of T cells could improve immunotherapy.fenofibrate can induce the expression of peroxisome proliferator-activated receptor PPAR-α and promote fatty acid metabolism,alleviate the metabolic stress caused by hypoglycemia in the tumor microenvironment.Therefore,this paper uses fenofibrate to improve the metabolic process of CTL cells and protect the effector function of CTL cells.Immunosuppressive signaling also affects the killing effect of CTLs.PD-1 is an immune checkpoint receptor on T cells.PD-1 binds to PD-L1,transmits inhibitory signals,and promotes immune evasion of tumor cells.Immune checkpoint blockade therapy is an effective means to alleviate T cell inhibitory signals,PD-1 mAb blockade effectively restored T cell function.Since immune checkpoint therapy relies on effective T-cell activation,it can be used in combination with tumor vaccines to exert an effective synergistic immunotherapeutic effect.we propose an immunotherapeutic scheme to enhance the effect of CTL in the process of anti-tumor immunity,establish a triple postoperative dosing regimen combining tumor autologous high-efficiency vaccine,immune checkpoint blockade therapy and lipid metabolism regulation.At the same time,it acts on the production of CTL,lipid metabolism and relieves the inhibitory signals of CTL,so as to effectively increase the proportion and activity of activated CTL,and improve the tumor treatment effect.A personalized patient autologous tumor vaccine was designed and prepared,and the tumor burden was reduced by surgical resection while obtaining the patient’s autoantigen.It is fused with liposomes loaded with immune adjuvants CpG and cGAMP,and modified with mannose to obtain a potent autologous vaccine(C/G-HL-Man),which can effectively target DC,regulate antigen cross-presentation and DC maturation,subcutaneous injection of C/G-HL-Man triggered robust T-cell immunity,includes potent CD8+T cells,CTL,and cytokine secretion.In mouse melanoma models,the induced immune response significantly suppressed tumor development and recurrence.Combining C/G-HL-Man with PD-1 antibody and/or lipid-lowering drug fenofibrate significantly inhibited the growth of recurrent tumors and prolonged survival,showing a promising clinical application prospect.The main contents and results in this study are listed as follows:1.Establishment of cGAMP assay methodologyThe method for the determination of cGAMP was successfully established by HPLC.2.Preparation and characterization of autologous tumor vaccinesIn order to realize the DC targeting of the vaccine,the target DSPE-PEG2000-Man was synthesized.The cGAMP-loaded cationic liposomes were prepared by thin-film hydration method,and then incubated with CpG to obtain a system co-loaded with cGAMP and CpG.The individualized targeted vaccine system C/G-HL-Man was obtained by extrusion fusion with tumor cell membrane extracted from surgical cuttings.The physicochemical properties of C/G-HL-Man showed that the particle size was 167.53±6.9 nm;the potential was-8.677±1.21 mV;The nano vaccine system completely retains the protein components on the tumor cell membrane.And the vaccine system has pH-responsive release properties.3.In vitro evaluation of C/G-HL-ManCells were extracted from mouse bone marrow and induced to investigate the effect of the vaccine system on BMDC.In the cellular uptake assay,C/G-HL-Man could target BMDCs mediated by mannose,promoting uptake of vaccines and lysosomal escape of antigens.In in vitro stimulation experiments,Compared with Membrane and adjuvant single-loaded nanovaccine groups C-HL and G-HL,C/G-HL-Man significantly promoted DC maturation and cytokine secretion.The in vitro imaging results of lymph node aggregation experiments showed that nanocarrier delivery could increase the accumulation in lymph nodes,and mannose modification promotes vaccine accumulation in lymph nodes.These results suggest that C/G-HL-Man has distinct DC targeting and lysosomal escape capabilities,which can promote DC maturation and antigen presentation.4.Evaluation of the effect of high-efficiency autologous tumor vaccine combined with immunotherapy such as lipid metabolism regulation and immune checkpoint blockade in inhibiting tumor recurrenceThe effects of the vaccine system and combination therapy on the occurrence and development of tumors were investigated through preventive experiments and inhibition experiments on recurrent tumors.The results show that C/G-HL-Man treatment can induce immune memory and has a good effect on preventing the occurrence and development of tumors.In vivo immunological evaluation showed that C/G-HL-Man induced specific anti-tumor immune responses by effectively activating T cells.It can promote the infiltration of CD4+and CD8+T cells in recurrent tumors,reduce the level of regulatory T cells(Treg),promote DC maturation in lymph nodes,and increase the proportion of memory T cells and antigen-specific CTLs.Increase the level of cytokines that promote anti-tumor immunity and reduce the level of immunosuppressive cytokines,improve the tumor microenvironment,and promote anti-tumor immunity.It can effectively inhibit the development of tumor in a mouse model of tumor recurrence,resulting in anti-tumor efficacy.C/G-HL-Man,lipid metabolism and immune checkpoint blockade combined for postoperative anti-relapse therapy in mice.The results show that they can respectively promote T cell activation,improve T cell efficacy and relieve immunosuppression in the tumor microenvironment,produce obvious anti-tumor effect.In conclusion,based on the concept of promoting the immune effect of CTL in the anti-tumor process,this paper established a triple postoperative dosing regimen combining highly effective autologous tumor vaccine,lipid metabolism regulation and immune checkpoint blockade therapy.The autologous tumor vaccine system is obtained by fusion of tumor cell membrane and pro-vaccine system.At the same time,PD-1 antibody and fenofibrate were given to the tumor-removed mice.The three acted on the stage of CTL production,improvement of CTL lipid metabolism and release of CTL inhibitory signal,respectively.It can effectively increase the proportion and activity of CTL,improve the tumor treatment effect,and provide an effective strategy for personalized combined therapy after tumor surgery.