Anticancer Activity And Immuno-regulation Based On Recombinant Adeno-associated Virus

With cancer incidence and mortality rates increase year by year, the traditional means of cancer therapy are difficult to meet the clinical needs. Biological treatment of cancer therapy, as the fourth model in a new cancer treatment is unique. Therapeutic cancer vaccines as an important component part, has been shown in clinical optimistic about the prospects.Dendritic cells (DC) treatment is the most representative in the tumor vaccine treatments. The main strategy is to take advantage of DC vaccine therapy in the introduction of exogenous antigen. The ultimate aim is to have highly effective anti-tumor specific immune responses. DC-based cancer vaccines on cancer treatment and prevention studies have been rapidly developed. It has been proved that Clinical substantial expansion is feasible in CD34⁺ monocyte-derived precursors or peripheral blood-derived DC. The safety and immunogenicity of DC immunotherapy have been confirmed. However, researchers still have many clinical puzzles. In these puzzles, how to make optimal DC vaccines, thereby maximize the full potential of immune cells and improve vaccine-induced immune response of tumor gene vaccine, will be the key to clinical application.After years of study, a variety of cytokines such as interleukin-2 (IL-2), interleukin-6 (IL-6), Interfron-γ(IFN-γ) and tumor necrosis factor-α(TNF-α) has been used for tumor immunotherapy. However, they are limited by the systemic toxicity and one-off effects. Cytokine gene transduced tumor cells and immune cells can secret cytokines in the tumor. It does not only minimize the system toxicity, but also can express in the long-term sustainability. Therefore, we could consider the adoption of genetic engineering techniques cytokine gene into tumor cells or modified DC, modification of immune cells. This vaccine can improve the function of DC. But the technical feasibility of its anti-tumor effect is still needed by experiment confirm.IL-12 is one of the most important cytokines for maintaining or enhancing the function of DC. In the process of DC mature, the production of IL-12 lead to Th1 immune response. IL-12 gene is transfected into DC. On one hand, it indirectly promote DC differentiation and maturation through the expression of exogenous IL-12. On the other hand, the expression of the IL-12 by mature DC can promote secretion of endogenous IL-12. The Synergies of them help tumor antigens presenting to T cells and enhance specific anti-tumor immune function. For T cells, IL-2, IL-12 and IL-7 are three different sources. They are similar in function of the T cell stimulatory factor of three to stimulate T cell proliferation in vitro and promote the effect of anti-tumor CTL.Our study choose the unique advantages of recombinant adeno-associated virus (rAAV) as vector, the carcinoembryonic antigen (CEA) gene as the tumor antigen gene to construct rAAV/CEA and rAAV/cytokine (rAAV/IL-2. rAAV/IL-7. rAAV/IL- 12 and rAAV/IFN-γ) plasmids.We transfected the plasmids (rAAV/CEA or rAAV/CEA+rAAV/IL-12) into DC, then transfected rAAV/cytokines (rAAV/IL-2, rAAV/IL-7, rAAV/IL-12 and rAAV/IFN-γ) into T cells. We got the ideal efficiency of expression. The methods of the experiment include CTL-specific surface markers, CTL immune phenotype, intracellular cytokines and cell morphology, etc. We want to know whether these cytokines regulate the immune response against tumors and its mechanism. The aim is to find more effective immunization against the anti-tumor immunity and apply in clinical. Part One The immune regulation of DC transfected by rAAV/IL-12 on CTLMaterials and Methods1. rAAV/CEA plasmid construction, packaging and purification: TRIzol extraction RNA, get out total mRNA by Oligotex mRNA Mini kit, RT-PCR amplification, plasmid construction, Preparation of calcium chloride into Escherichia coli DH5αand analysis. Plasmids were identified by restrict analysis.2. Determination of virus titer: blot hybridization with digoxin-labeled probe.3. The production of DC and rAAV infection: isolation of peripheral mononuclear cells (PBMC) and mononuclear cell; rAAV/CEA and rAAV/IL-12 infection.4. Chromosomal integration, expression and efficiency: transcription and expression of CEA, PCR/Southern blot analysis, rAAV/CEA DNA chromosomal integration, the efficiency of rAAV/CEA infection.5. Mixed lymphocyte reaction(MLR)6. CTL killing assay in vitro: ⁵¹Chromium (⁵¹Cr) release assay.7. Cell proliferation: magnetic bead Separation of total T lymphocytes, ³H incorporation assay.8. Flow cytometry(FACS) analysis of cell surface markers: surface markers of mature DC(CD14, CD80, CD86, CD83, HLA-DR and CD40).9. Determinate immunophenotype(CD8/CD4 and CD8/CD56) of activated CTL: FACS double staining method.10. FACS detection of the expression of cytokines: expression of IL-12 and IL-10 of mature DC, expression of IFN-γof Activated CTL.11. Determinate immunophenotype(CD8/CD69 and CD25/CD4) of activated CTL: Separating CD4⁺ or CD8⁺ T cells by magnetic bead Separation; detected by FACS.12. Enzyme-linked immunosorbent assay (ELISA) detect the expression of cytokines.Results 1. The successful construction of high titer of rAAV/CEA and rAAV/IL-12 plasmids.2. rAAV/CEA and rAAV/IL-12 integrated into the chromosome DC, CEA and p40 subunit of IL-12 express in DC.3. DC infected by rAAV/CEA active CTL. Its activity is target-antigen-specific and MHC classâ… restrictive. The plasmid of rAAV/IL-12 can increase the activity.4. DC infected by rAAV/CEA, can significantly increase the expression of CD40, CD80, CD83 and CD86 molecules, reduced the expression of CD14. Thereby enhance the biological activity of DC function. If DC were infected by rAAV/IL-12 at the same time, the endogenous expression of IL-12 will increase, the expression level of CD40,CD80 and CD83 will significantly increase, the expression of CD14 will decrease. DC function will be enhanced further.5. rAAV/CEA infection increase the proliferation of lymphocytes. rAAV/CEA+rAAV/IL-12 infection can further increase this proliferation.6. DC infected by rAAV/CEA can significantly increase the secretion of IL-12 and reduced the secretion of IL-10. rAAV/CEA+rAAV/IL-12 infection can strengthen this effect.7. Immunophenotype of DC-activated T cells:â‘ Analysis of CD8/CD4 T cell subsets: no significant difference in the level of CD4⁺ T cells. DC infected by rAAV/IL-12+rAAV/CEA can significantly up-regulate the levels of CD8⁺ T cell subsets.â‘¡CD8/CD56 ratio: DC infected by rAAV/CEA can selectively stimulate CTL. Moreover, if the DC is infected by rAAV/IL-12 at the same time, the effect of stimulating CTL will increase, the activation of NK cells will reduce further.â‘¢Analysis of CD69⁺ cell in CD8⁺ cells: The number of CD69⁺CD8⁺ T cell can significantly increase in the group of rAAV/CEA infection. If the DC was infected by rAAV/IL-12 at the same time, the number will further increase.â‘£Analysis of CD25⁺ cells in CD4⁺ cell: The number of CD25⁺CD4⁺ T cells can significantly reduce in the group of rAAV/CEA+rAAV/IL-12 infection. 8. DC infected by rAAV/CEA can significantly increase the expression of IFN-γ; if infected by AAV/IL-12 at the same time, the expression of IFN-γand the activity of CTL will further increase.Conclusions1. rAAV/CEA can transfected DC and induce CEA-specific CTL for the treatment of CEA-positive cancer.2. rAAV/IL-12 can enhance the immune regulation of the specific CTL induced by rAAV/CEA.Part Two The immune regulation of cytokine gene transfection in PBL on CTL activityMaterials and Methods1. rAAV/cytokines plasmids(rAAV/IL-2, rAAV/IL-7, rAAV/IL-12, rAAV/IFN-γ) construction, packaging and purification: TRIzol extraction RNA, get out total mRNA by Oligotex mRNA Mini kit, RT-PCR amplification, plasmid construction, Preparation of calcium chloride into Escherichia coli DH5αand analysis. Plasmids were identified by restrict analysis.2. Determination of virus titer: blot hybridization with digoxin-labeled probe.3. The production of DC and rAAV infection: isolation of peripheral mononuclear cells (PBMC) and mononuclear cell. rAAV/CEA infected into DC and rAAV/cytokines infected into PBL.4. Chromosomal integration, expression and efficiency: transcription and expression of cytokines, PCR/Southern blot analysis, rAAV/cytokines DNA chromosomal integration, the efficiency of rAAV/cytokines infection.5. Mixed lymphocyte reaction (MLR)6. CTL killing assay in vitro: ⁵¹Chromium (⁵¹Cr) release assay.7. The effect of rAAV/cytokines infection on the cell proliferation: magnetic bead Separation of total T lymphocytes, ³H incorporation assay.8. Flow cytometry (FACS) analysis of cell surface markers: surface markers of mature DC (CD14, CD80, CD86, CD83, HLA-DR and CD40).9. Determinate immunophenotype (CD8/CD4 and CD8/CD56) of activated CTL: FACS double staining method.10. FACS detection of the expression of cytokines: expression of IL-12 and IL-10 of mature DC, expression of IFN-γof Activated CTL.11. Determinate immunophenotype (CD8/CD69 and CD25/CD4) of activated CTL: Separating CD4⁺ or CD8⁺ T cells by magnetic bead Separation; detected by FACS.12. Enzyme-linked immunosorbent assay (ELISA) detect the expression of cytokines.Results1. Successfully construct high titer rAAV/IL-2, rAAV/IL-7, rAAV/IL-12 and rAAV/IFN-γplasmids.2. The high efficiency of protein expression was detected in rAAV/IL-2, rAAV/IL-7, rAAV/IL-12 and rAAV/IFN-γtransfecting PBL.3. rAAV/IL-7, rAAV/IL-12 and rAAV/IFN-γtransfecting PBL strengthen rAAV/CEA CTL response. The D3 rAAV/IL-12 infection, D3, D5 rAAV/IFN-γand rAAV/IL-7 infection show the best results. But rAAV/IL-2 does not work.4. Promotion of the proliferation was showed in D3 rAAV/IL-12, D3 rAAV/IL-2 and D5 rAAV/IL-7 infection groups. Inhibition of the proliferation was observed in D3 rAAV/IFN-γinfection group.5. Immunophenotype of T lymphocytes activated by DCâ‘ CD8/CD4: rAAV/IL-2 probably inhibit the proliferation of CD8⁺ T cells. CD8⁺ T cell proliferation can be significantly enhanced by rAAV/IL-7 infection in the fifth day of PBL culture, and CD4⁺ T cell's can be promoted in the third day. The target cells of rAAV/IL-12 are CD8⁺ T-lymphocytes, rather than CD4⁺ T lymphocytes. Meanwhile, in third day of PBL culture, proliferation of CD8⁺ T cells induced by rAAV/CEA infected DC can be significantly enhanced, but no effect on CD4⁺ T cells. rAAV/IL-7 infection in fifth day of PBL culture can significantly increase the number of CD8⁺ T cell and reduce CD4⁺ T cell's proliferation. No similar effect was observed in early infection.â‘¡CD8/CD56: rAAV/Cytokines can not promote the proliferation of CD56⁺ cells and NK cells, rAAV/IL-12 or rAAV/IFN-γinfection in fifth day of PBL culture may selectively promote the proliferation responses of CTL and inhibit the proliferation and the expression of activity of NK cell.â‘¢CD8/CD69: In this experiment, all viruses except rAAV/IL-2, can increase the number of CD69+/CD8⁺. AAV/IL-12 infection can significantly increase the number of CD69⁺/CD8⁺ T cells in third day of PBL culture, rAAV/IL-7 or rAAV/IFN-γinfection can do the same in fifth day of PBL culture.â‘£CD25/CD4: Except rAAV/IL-2 infection group, all groups did not significantly increase the number of CD25⁺/CD4⁺ T-lymphocytes. This mean did not enhance the effect of Ts cells.6. Cytokine expression of activated CTL: The level of IFN-γin rAAV/IL-2 infection group is lower than those in the control group. The level of IFN-γincreases significantly in D3 rAAV/IL-12 infection group, D5 AAV/IL-7 infection and D5 rAAV/IFN-γgroups.ConclusionsrAAV/IL-7, rAAV/IL-12 and rAAV/IFN-γcan effectively enhance the anti-tumor response to CTL, the effect of various cytokine gene differ according to the different infection time of PBL. Although rAAV/IL-2 does not enhance the anti-tumor response, it may still be considered as the substitution of exogenous IL-2.Summary1. It has been confirmed that rAAV/CEA transfected dendritic cell vaccine, which can induce anti-tumor activity.2. rAAV/IL-12 virus can enhance the anti-tumor activity of DC vaccine, which loading antigen gene. It is worthy of further study.3. Compared with exogenous cytokines, rAAV/IL-7, rAAV/IL-12 and rAAV/IFN-γviruses can effectively enhance the anti-tumor CTL responses. Different cytokine genes play inconsistent roles at different times.4. Although the effect of rAAV/IL-2 in enhancing the anti-tumor response is not satisfied. However, it is worthy of future research.