SMP30 And GP96 Protein Priming Of Dendritic Cell Vaccination Induces More Potent CTL Response Against Hepatoma

Background:Hepatocellular carcinoma(HCC)is one of the most common malignancies,and its mortality rate ranks second in cancer mortality in China.Surgery,radiotherapy and chemotherapy are currently the three most common methods for the treatment of HCC,but their efficacy is still not satisfactory.Finding more effective and safe therapies has become a new trend in HCC treatment.Throughout the history of cancer therapy research,biological therapy represented by tumor immunotherapy can play an important role in the control of tumor recurrence and metastasis,and dendritic cell(DC)-mediated tumor immunotherapy has become a research hotspots.The core of DC immunotherapy is to give full play to the DC antigen presentation function and to stimulate the production of specific cytotoxic T lymphocyte(CTL).Our group used the SEREX(serome analysis technique of recombinant c DNA expression library)in the previous period to screen a number of clones encoding HCC tumor-associated antigen genes from Guangxi human hepatocellular carcinoma c DNA library.One of the antigens and SMP30 carboxyl were found by DNA sequencing.The terminal 165 amino acids are homologous,and the corresponding antibodies are mainly found in patients with HCC.It is particularly interesting that high positive detection rate of SMP30 antibody is found in AFP-negative HCC patients.We used recombinantly expressed SMP30 protein to sensitize human DCs to find that they can effectively stimulate the proliferation of autologous T lymphocytes,and can induce CTLs with certain cytotoxic activity against hepatocellular carcinoma cell line BEL-7404.Our team earlier showed that SMP30 is highly expressed in paracancerous tissues of HCC but had low levels in HCC tissues.Glycoprotein 96(gp96)belongs to the heat shock protein 90(HSP90)family and is a wellknown adjuvant in immunotherapy.The HSP-antigen complex can be taken up quite efficiently by receptor-mediated means by antigen-presenting cells such as dendritic cells.At the same time,the antigen or HSP in the HSP-antigen peptide complex interacts with dendritic cells,stimulating dendritic cells to express MHC class II molecules,secreting cytokines and chemokines,leading to the maturation of dendritic cells and migration to draining lymph nodes.The antigen is presented to T cells and a CTL response is initiated.Therefore,the HSP formulation vaccine based on its adjuvant effect has attracted more attention in the immunotherapy of cancer or infectious diseases.In addition,HSP can induce memory T cells to produce a more lasting effect.In phase III clinical trials,GP96 has been shown to have a potent effect on CTL cells and has a good anticancer effect.In related research reports at home and abroad,currently used methods of tumor antigen modification mainly include in vitro synthesis of tumor antigen peptides,complete tumor antigen-loaded DC,and tumor antigen gene transfection DC.The immune function of tumor patients is weakened.Whether APC surface receptors,tumor antigens and their chaperones,or MHC-I molecules may not be conducive to the processing and presentation of tumor antigens,thereby affecting the formation of CTL cells.The method of priming DCs with in vitro shocks has the characteristics of direct and rapid action,but it is difficult to induce long-lasting anti-tumor immune responses.The introduction of the tumor antigen gene into the DC will allow the expression of the antigen to be long-lasting and facilitate entry into the MHC-I antigen presentation pathway.Therefore,the construction of a tumor vaccine constructed by transfecting a vector expressing a gene of a tumor antigen peptide has considerable advantages.Objective:At home and abroad have been reported in some studies of liver cancer-associated antigen-loaded DC vaccine,alpha-fetoprotein(AFP)is currently recognized as a marker of liver cancer,although studies have shown that AFP gene transfection of DC can induce specific CTL targeted killing AFP positive liver cancer cells,but its immune response intensity is still far from clinical applications;Japanese researchers transfected DCs with HSP70 m RNA,a phase I clinical trial of 12 liver cancer immunotherapy,but this technique involves the easy degradation of RNA,and single-gene modified DC.The effectiveness of the vaccine is also very limited.Therefore,the current immunotherapy of liver cancer based on DC vaccine is not satisfactory and there is still a lot of work to be done.Based on existing literature reports and our previous research,we proposed the scientific hypothesis that the human liver cancer-associated antigen SMP30 gene gene and GP96 gene can be continuously transferred into DC to continuously sensitize and enhance DC antigen presentation function,becoming more effective and specific.Anti-hepatoma vaccine.If our study confirms that loading SMP30 and GP96 DC can induce specific liver cancer antigen CTL toxicity and enhance anti-hepatoma immune effects,plus our previous research results,we will further understand the role of SMP30 in HCC immunotherapy,but also for the development of the study of SMP30 as an intervention target provides experimental evidence.Method: We used bioinformatics technology to search for gene sequences,and introduced SMP30 and GP96 gene fragments into lentiviral vectors to construct eukaryotic recombinants,and then transfected into healthy volunteer DCs.The experimental components were smp30-gp96 group,GP96 group,and SMP30 group,DC group,empty vector group,liver tissue extracts were incubated in DC group.Flow cytometry was used to evaluate CD80,CD86,and CCR7 changes in DC vaccine before and after transfection.Using DC vaccine and T cell coculture,relevant CTLs were obtained and TNF and cytokines such as interferon(IFN)were detected by ELISA.And mixed with the target cell human hepatoma cell line SMMC7721,flow cytometry CTL killing effect.5-6 week old female BALB/c nude mice(special pathogen-free,SPF)were injected subcutaneously(s.c.)into SMMC-7721 cells(1 x 107)into the right flank of the mouse.After injection every four days and two injections of tumors,the mice were randomly assigned on the 9th day to the experimental group and the control group,with 4 mice in each group.In the experimental group tumors were injected with gp96-smp30-modified DC-induced CTL(1×108),and CTLs were injected once every other day for a total of seven times.This experiment was repeated 3 times.The control group was injected with PBS.Animals were euthanized after 16 days and transplanted tumors were weighed and harvested for histological analysis.The results : the SMP30 and GP96 gene fragments can be introduced into the same lentiviral vector and transfected with DCs to express more surface molecules such as CCR7,CD86 and CD80,and secrete more IL-1 and IFN-?;smp30-gp96 group and SMP30 The cytokines(INF-?,INF-?,TNF-?,TNF-?, IL-12,etc.)secreted by DCs induced by DCs in the group and the liver cancer tissue lysate group were more than those in the empty group and the blank group.There was a significant difference(P<0.05/0.01);however,there was no significant difference between the three groups.The induced CTL has a stronger killing effect on human hepatoma cell line SMMC-7721 cells.In vivo experiments with human tumor-bearing mice showed that DCs induced by gp96-smp30 transfection of DCs were also effective in reducing mouse liver tumors,and that more INF-gamma and IL-6 were detected in serum.Increased apoptosis.Conclusion: We successfully introduced SMP30 and GP96 gene fragments into lentiviral vectors to construct bispecific gene eukaryotic recombinants;SMP30 and GP96 dual-purpose genomes(mixed group),SMP30 group and lysate group compared with empty and blank groups.,Promote DC maturation;Strengthen antigen presentation ability.DCs sensitized with the mixed group,SMP30 group and lysate group all significantly promoted T cell proliferation;DC induced CTLs in the mixed group,SMP30 group and lysate group compared with the empty group and blank group,compared with the human hepatoma cell line.SMMC-7721 cells had a stronger killing effect;in vitro experiments showed that compared with the SMP30 alone group,the sensitized DCs(the production of surface molecules CCR7,CD86 and CD80 and secrete IFN-? and IL-1),and promote T Cell proliferation,induced CTL and secreted cytokines(INF-?,INF-?,TNF-?,TNF-? and IL-12)and killing effect on human hepatocellular carcinoma cell line SMMC-7721 cells were basically insignificant Sex differences;but compared with the gp96 group showed a significant increase in the trend;comprehensive preliminary results of this study showed that: SMP30 and GP96 dual purpose genome(mixed group)and lysate group have the same anti-HCC effect.However,the specificity and side effects of the two groups need to be further compared;the effect of SMP30 alone is better than that of gp96 alone;the anti-HCC effect of the mixed group is better than that of in vitro experiments,both SMP30 and GP96 can mediate lentivirus-promoted DC Mature,and then induced CTL,and the two may have a synergistic effect.This study shows that GP96 and SMP30 mediate Lentivirus co-transfection of DC,respectively,can synergistically stimulate DCs to promote its maturation,enhance antigen presentation capacity,and then induce CTL and produce more IFN-?,IFN-?,TNF-?,TNF-? and IL-12,better effect of anti-human hepatoma cell line SMMC-7721 cells in vitro and in vivo.