| Malignant melanoma is derived from melanocytes and mostly occurs in skin and mucosa.Melanoma is highly invasive and prone to local recurrence and distant metastasis.It is the most malignant and lethal type of skin tumors.In the past 10 years,the incidence of melanoma in China has increased year by year,and due to the insufficient early diagnosis of melanoma and its unique subtypes,the population of advanced melanoma requiring systematic treatment is even larger.The therapeutic effect of chemotherapy and targeted therapy is limited for these patients,and it is difficult to maintain treatment after drug resistance.Melanoma has a high tumor mutation burden(TMB),and is characterized by high immunogenicity and immune cell infiltration,so it is suitable for the development and application of immunotherapy.On the other hand,clinical data showed that the objective response rate(ORR)of nivolumab for advanced melanoma was 43.7%in the Caucasian population,but lower at 34.8%in the Asian population.In particular,the ORR of anti-PD-1 was only 16.6%for the high proportion of patients with acromelanomas.The combined treatment effect of anti-PD-1 and anti-CTLA-4 is better than that of single drug,but also leads to a higher rate of adverse reactions.Overall,the immunotherapy of melanoma has made great breakthroughs,but from immune checkpoint inhibitors(ICIs)to multi-mechanism combination regimen,immunotherapy has entered a bottleneck period,and it is urgent to develop new therapeutic strategies.Based on the high TMB of melanoma,which produces a large number of tumor-specific antigen(TSA)and tumor associated antigen(TAA),this project designed a tumor therapeutic vaccine to target and enhance the presentation of tumor antigen by dendritic cells(DC),thus promoting the activation of effector T cells and tumor invasion,so as to fully stimulate the specific immune killing effect.Meanwhile,the synergistic effect of therapeutic vaccine and mainstream immunotherapy was explored.In this study,melanoma cells damaged by hypochlorous acid(HClO)oxidation and ultraviolet B(UVB)irradiation were used.Under high pressure,the two kinds of cells were mixed and extruded through polycarbonate membrane with gradient pore size to prepare recombinant microvesicles(hM-NV).On the basis of hM-NV,CpG ODN 1826(CpG)was loaded as an immune adjuvant to further improve the immune response of the vaccine.Finally,the DC-targeting peptide CBP12 was connected to prepare the recombinant DC-targeting microvesicle vaccine(hMVac-CBP12/CpG).Through the above vaccine design strategies,tumor whole antigen can be targeted to deliver to DC and directly activate DC to enhance its antigen processing and presentation function,increase cytokine secretion,and thus induce anti-tumor specific immune response.In this study,we systematically evaluated the preparation and characterization of hMVac-CBP12/CpG,its immunological effects in vitro and in vivo,its anti-melanoma efficacy,and its synergistic effect with mainstream immunotherapy.This research included three parts.The first part is the construction and characterization of hMVac-CBP12/CpG.This part is mainly divided into three sections.Firstly,the induction and characterization of HClO oxidation and UVB dual-mode damage cells.The second section is the preparation of dual-mode damaged cell recombinant microvesicle vaccine(hMVac-CBP12/CpG).By connecting CpG and CBP12 peptide on the recombinant microvesicle,we successfully prepared DC targeted nanoscale vaccine.Finally,the particle size,PDI and potential of hMVac-CBP12/CpG were determined.The particle size was about209.4±39.8 nm,and the potential was about-20.27±1.74 m V.The spherical and bilame-membrane structures of hM-NV and hMVac-CBP12/CpG were observed by transmission electron microscopy.hMVac-CBP12/CpG can maintain stability at 4℃and-20℃for 30days.Finally,the cells were prestained with fluorescent probes to prepare microvesicles.The fusion of the two damaged cell components in the microvesicles was observed by fluorescence microscopy.The second part is a cytological evaluation of the immunological effect and anti-melanoma effect of recombinant microvesicle vaccine in vitro.Firstly,the toxicity of hMVac-CBP12/CpG to a variety of cells was investigated,and the safety of hMVac-CBP12-CBP12/CPG vaccine at the cellular level was preliminatively evaluated.Secondly,the targeting ability of the vaccine to DC was investigated by cell uptake experiment,and the results showed that the vaccine connected to CpG and CBP12 could significantly increase the uptake of DC cells.It was observed under microscope that part of fluorescently labeled hMVac-CBP12/CpG entered the lysosome after entering the DC,while the rest mainly distributed around the nucleus,and the fluorescence intensity was consistent with that detected by flow cytometry.Finally,the immunological effect of hMVac-CBP12/CpG was verified.The results showed that the vaccine could effectively promote the maturation and antigen presentation of DC,and increased the expression of CD40,CD80,CD86 and MHC Ⅱ on the surface of DC membrane,as well as the secretion of various cytokines.In addition,hMVac-CBP12/CpG sensitized DC had strong activation ability of T lymphocytes,which could induce the amplification and activation of tumor-specific T lymphocytes under the condition of co-culture,and the induced amplification of T lymphocytes had strong tumor killing ability.The third part is to evaluate the lymph node targeting,anti-tumor effect,in vivo immune effect and in vivo safety of hMVac-CBP12/CpG in vivo using mouse tumor-bearing model,and to verify its synergistic effect with PD-1 blocking therapy.The rapid accumulation of hMVac-CBP12/CpG in lymph nodes after subcutaneous injection suggested that HMVAC-CBP12/CPG had DC targeting characteristics.hMVac-CBP12/CpG significantly inhibited the growth of transplanted melanoma in mouse melanoma models,and had synergistic effect with anti-PD-1 monoclonal antibody.hMVac-CBP12/CpG played an anti-tumor role by increasing the level of anti-tumor cytokines,increasing the CD8~+/CD4~+ratio of T cells,and increasing the infiltration of CD8~+T cells in cancer tissues.In the tumor prevention model,hMVac-CBP12/CpG could also delay tumor recurrence and progression.Pharmacodynamic results indicated that CpG could be used as an excellent immune adjuvant,acting together with CBP12 targeted peptide to increase the immune activation effect of recombinant microvesicle vaccine.At the same time,there were no abnormalities in body weight,blood biochemistry and histological indexes of organs after hMVac-CBP12/CpG treatment,which proved that HMVAC-CBP12/CPG had good biological safety.In summary,we constructed a nanoscale cell recombinant microvesicle vaccine hMVac-CBP12/CpG.On the one hand,the vaccine contains tumor cell antigens and multiple damage signaling molecules through dual-mode cell recombination;on the other hand,CBP12 and CpG can further increase the DC targeting and activation of the vaccine.Ultimately,strong anti-tumor specific immunity is induced.This project is expected to provide new ideas and research methods for the design of tumor vaccine,and at the same time provide a new strategy for the combined immunotherapy of melanoma. |
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