Construction Of CLDN18.2-loaded Dendritic Cell Vaccine And Its Effect On Lung Adenocarcinoma Cells

Objective:The incidence of non-small cell lung cancer(NSCLC)is increasing progressively.The current updating of therapeutic means and new drug development are far from meeting the therapeutic needs of clinical patients.The application of vaccines is the greatest medical achievement of mankind and has played an irreplaceable role in the process of disease eradication.The use of vaccines to intervene in the treatment of malignant tumors date back to the early 19th century.In recent years,with the revelation of tumor-specific antigens,personalized tumor therapy has received increasing attention from researchers.In this study,a recombinant lentiviral(r LV)vector encoding the CLDN18.2 antigen gene was used to prepare a dendritic cell(DC)vaccine loaded with the NSCLC-associated antigen CLDN18.2.And the in vitro activity of cytotoxic T lymphocytes(CTL)induced by DC vaccine against NSCLC was detected.Methods:1.Human CLDN18.2 full-length gene was inserted into the lentiviral expression plasmid p HBLV-CLDN18.2-Zs Green-Puro to construct a recombinant lentivirus encoding human-derived CLDN18.2 gene.2.Mononuclear lymphocytes were isolated from the peripheral blood of HLA-A2+healthy persons,and immature DCs were obtained by differentiation under the stimulation of cytokines GM-CSF and IL-4.The phenotypes of DC cells were identified by flow cytometry and co-stimulatory molecules and major histocompatibility complexes were detected.3.Recombinant lentivirus encoding CLDN18.2 gene was used to infect DC to prepare specific DC vaccine.4.Mature specific DC vaccine was co-cultured with human peripheral blood-derived T lymphocytes in vitro to induce the generation of specific CTL.The phenotype and induction effect of CTL were evaluated by flow cytometry.5.Specific CTL was co-cultured with lung adenocarcinoma cells to detect the killing effect of CTL in vitro and the therapeutic effect of DC vaccine.Results:The recombinant lentivirus r LV-CLDN18.2 constructed in this study showed good biological activity with a titer of about 2x10~8 TU/m L.The recombinant lentivirus could successfully infect human peripheral blood DCs under the infection complex number of MOI=200 and could successfully load tumor antigen CLDN18.2on to DC cells in order to construct DC vaccine.Flow cytometry results showed that the expression of CD83 and CD86 co-stimulatory molecules in DC vaccine cells were significantly increased.DCs could present the tumor antigen CLDN18.2 through MHC class I and MHC class II molecular pathways,inducing the activation and differentiation of initial lymphatic T cell to CD4+T and CD8+T cells.What’s more,when the ratio of effector cells to target cells was 20:1,the DC vaccine could successfully induce the specific CTL,and the CTL had a significant killing effect on lung adenocarcinoma A549-CLDN18.2+tumor cells.Conclusion:A recombinant lentivirus which can stably express the CLDN18.2antigen gene was successfully constructed,and the lentivirus can successfully infect DCs obtained from PBMC in vitro to construct a specific DC vaccine.The DC vaccine was able to stimulate the differentiation and maturation of the initial T cells to generate CTL against CLDN18.2 antigen.The specific CTL was able to kill lung cancer A549-CLDN18.2+tumor cells in a dose-dependent manner.This study provides a new idea and lay theoretical experimental basis for the construction of personalized DC tumor vaccine and immunotherapy of lung adenocarcinoma.