Antitumor Activity and Mechanisms of DC-targeting DNA Vaccines
|School||Second Military Medical University|
|Keywords||DNA vaccines DC targeting HER2/neu Breast Cancer Cyclophosphamide|
Has great prospects for the application of DNA vaccines in cancer immunotherapy, clinical studies have shown that patients receiving the DNA vaccine capable of generating antigen-specific anti-tumor immune response, however, the DNA vaccine rarely lasting and efficient anti-tumor immune response induced by , suggesting that the DNA vaccine still need to continue to optimize. DNA vaccine to work a very critical part of the expression of the protein antigen need to be antigen-presenting cells (antigen presenting cells, APC) intake, and then processed and presented to antigen-specific T lymphocytes, induced T-cell activation and proliferation. Dendritic cells (dendritic cells, DC) is the strongest known functionality in vivo antigen-presenting cells, which can stimulate the initial (na (?) Ve) T cell proliferation and thus start the immune response, and induce antigen-specific cellular immune response. After subcutaneous or intramuscular injection of plasmid DNA vaccine, most of the plasmid was the site of injection intake of the skin and muscle cells, which encodes an immunogenic antigen in these cells, expression or release. Skin and muscle cells can not effectively presenting the antigen can not be sensitized antigen-specific T cells, DC antigen uptake efficiency is directly related to the immune response induced due to the lack of MHC II and costimulatory molecule expression. Get immune antigen DNA vaccine, DC, there are two main ways that local small DC direct uptake of plasmid DNA and expression of immune antigen, this pathway is relatively minor, after all, the local very low proportion of DC uptake of plasmid DNA directly. Other local DC uptake skin or muscle cells to release the immune antigen or swallowed expression of the death of skin and muscle cells of the immune antigen, which is the main way. Therefore, we assume: DC targeting molecule (DC surface molecule specific antibodies or ligands) the immune antigen directly delivered to the DC will be able to improve the efficiency of the DC uptake of antigen, thus effectively enhance the immune response induced by DNA vaccine. To verify the above assumptions, the present study, we prepared DC targeting and control of non-targeted DNA vaccine scFv of NLDC-145 sup> -HER2/neu and HER2/neu, which scFv NLDC- 145 sup> DEC-205 molecules encoding specific single-chain antibody fragments, DEC-205 DC surface specific expression, phagocytic receptors, effectively mediated antigen endocytosis to enter the delivery of antigen processing pathways; HER2/neu proto-oncogene encoding HER2/neu growth factor receptor overexpression in breast cancer and other tumors, which directly promote tumor growth and development. The experiments show that: DC targeting DNA vaccine encoding immunizing antigen to effective delivery to DC is further processed submissions; in HER2/neu positive transplantation in animal models of breast cancer, compared with the non-targeted HER2/neu DNA vaccine DC targeting vaccine induced significantly enhanced the HER2/neu specific cells and the immune response in the body, can effectively prevent the HER2/neu positive tumor growth in mice, and generates antigen-specific immune memory; is more important is that the DC targeting vaccine combined low-dose cyclophosphamide-mediated regulatory T cells cleared briefly able to eliminate generated HER2-positive to transplant breast tumors and spontaneous mammary tumor growth delay the BALB-neuT transgenic mice. The first part: DNA vaccine Construction and expression of a DNA vaccine construct: gene synthesis of single-chain antibody fragment NLDC-145 (scFv NLDC-145 sup>) by reference to other scholars early post. HER2/neu extracellular region fragments were amplified from SK-BR-3 or TUBO breast cancer cell lines, two extracellular region sequencing with the scFv NLDC-145 sup> constructed by the overlapping PCR method out the scFv NLDC-145 sup>-HER2 scFv NLDC-145 sup>-neu, two PCR products into the pGEM-Teasy vector amplification. With HindIII and XbaI double digestion of the carrier, respectively, we get the scFv NLDC-145 sup>-HER2 and the scFv NLDC-145 sup>-neu fragment, and then loaded by the same digested pcDNA3 .1 () successfully constructed in the expression vector containing the above two fragments. Second, DNA vaccines fusion protein expression: extracted constructed expression plasmid, transiently transfected 293T cells were transfected supernatants were harvested after 72 hours, with the WB method detection plasmid vaccine protein expressed fusion proteins in two targeted vaccine than expected slightly larger, it is presumed fusion protein in eukaryotic expression systems exist for glycosylation. Second part: DNA vaccines in vivo targeting of a construct pcDNA3.1-scFv NLDC-145 sup>-EGFP, the expression vector: EGFP gene was cloned from the plasmid vector pEGFP-N1, and then with EGFP The alternative to the above expression vector scFv gene NLDC-145 sup>-HER2-HER2 fragment constructed scFv NLDC-145 sup>-EGFP expression vector. Fragment scFv NLDC-145 sup> vivo targeting detection: electric shock method of immunizing in BALB / c mice, rear right thigh intramuscularly Fragment scFv NLDC-145 SUP>-EGFP, plasmid gating PE-labeled CD11c-positive cells, EGFP green fluorescent flow cytometry results display scFv NLDC-145 sup> with good DC targeting. Part III: BALB / c mice in vivo anti-tumor effect of a preventive anti-tumor effects: BALB / c mice were randomly divided into five groups, corresponding to each group of mice were used electric shock immune right after thigh intramuscular scFv NLDC-145 sup>-HER2, scFv NLDC-145 sup>-neu, HER2, neu and empty vector pcDNA3.1, each 50μg/50μL. Two weeks after the second re-use the same method immune. BALB / c mice after a week, twice immunization, at the opposite side of the back hypodermic D2F2/E2 the cells (D2F2/E2 is stable expression of HER2 in human breast cancer cell line), after observe and record the growth of tumors. The results show that targeted Fragment scFv NLDC-145 sup>-HER2-group can be effective in preventing tumor growth in this group in the 120 days in 100% of mice survived, while the other group all died at 80 days. It is interesting to scFv NLDC-145 sup>-neu group than the non-targeted HER2-neu and pcDNA3.1 group slightly delayed tumor growth. Second, the therapeutic anti-tumor effects: first verify the DNA vaccine alone the application of anti-tumor effect of BALB / c mice were subcutaneously the inoculation D2F2/E2 cells, when the tumor diameter of approximately 40mm 3 sup>, the above method two sub-immunized mice, tumor growth was observed. The results showed that scFv NLDC-145 sup>-HER2-group than any other group, although a significant inhibitory effect, but to 120 days the group was only 20% of the mice survive, so the scFv NLDC- 145 sup>-HER2 alone a limited role. We then verify that the DNA vaccine combined with cyclophosphamide and anti-tumor effect, BALB / c the mice inoculated D2F2/E2 cells, when the tumor diameter of about 3-4mm, intraperitoneal injection of low-dose cyclophosphamide (100mg/kg), four days after The mice were immunized twice in accordance with the above-described method, after tumor growth was observed. ScFv NLDC-145 sup>-HER2 in combination with cyclophosphamide group 120 can significantly inhibit 80% of tumor growth and tumor volume can be reduced to eliminate other combination group than corresponding to separate applications plasmid immune good antitumor effect. Part IV: the anti-tumor mechanism of DNA vaccines induce HER2-specific T cell responses: the above method twice immunized BALB / c mice, after a week, take the spleen single cells with HER2 or TRP2 protein in vitro stimulation of three days, detected by liquid scintillation counting instrument known Fragment scFv NLDC-145 SUP>-HER2-groups is stimulated by the HER2 protein than TRP2 protein stimulated significant T cell proliferation. Are in the same group stimulated by HER2 protein, results show the scFv NLDC-145 sup>-HER2-group than any other group, significant T-cell proliferation, and scFv NLDC-145 sup >-neu group also proliferation of T cells compared to other non-targeted control group slightly. ELISA detection of each group of the warp HER2 protein stimulates cultured spleen cells in serum Fragment scFv NLDC-145 SUP>-HER2-group of IFN-γ could be detected, the high expression of TNF-α in the same scFv NLDC -145 sup>-Neu IFN-γ, TNF-α Expression of slightly higher than that of other non-targeted control group. The flow detection of HER2 peptide stimulation scFv NLDC-145 sup>-HER2 group CD4 IFN-γ, CD4 TNF-α, CD8 IFN-γ and CD8 TNF-α T cells with high expression. Second, the induction of HER2-specific antibody responses: collecting serum of each of the above groups, ELISA can be detected Fragment scFv NLDC-145 sup>-HER2-group than in the other groups have a high titer of HER2-specific antibody, and mainly IgG2a subtype. Another incubation supernatant D2F2/E2 cell, after with FITC-labeled secondary antibodies binding after incubation flow cytometry Fragment scFv NLDC-145 SUP>-HER2-group D2F2/E2 specific antibodies was significantly higher than the other groups, while The scFv NLDC-145 sup>-neu is also slightly higher than the non-targeted group. Part V: the scFv NLDC-145 sup>-neu the BALB-neuT mice anti-tumor effect of a preventive anti-tumor effects: stably expressing neu antigen mouse breast cancer cell lines TUBO transplanted tumor model; BALB-neuT mice After twice immune week later, at the opposite side of the back hypodermic TUBO cells, tumor growth was observed after. The results show that the targeting of scFv NLDC-145 sup>-neu group can prevent the growth of tumors, this group is 120 days to about 90% of the mice survived, while the other groups in less than 60 days have all died . Second, anti-mouse spontaneous breast cancer effect: BALB-neuT mice expressing neu antigen spontaneous growth of breast cancer in mice, shock method of immunizing mice, 8 weeks and 10 weeks with intramuscular Fragment scFv NLDC-145 sup>-neu CTX, scFv NLDC-145 sup>-neu, neu, pcDNA3.1 and PBS scFv NLDC-145 sup>-neu CTX group the number of spontaneous tumors in the first immune four days before the injection of low-dose cyclophosphamide (100mg/kg), and then intramuscular scFv NLDC-145 sup>-neu, each group of mice was observed after . Of the scFv NLDC-145 sup>-neu combined with cyclophosphamide group can effectively delay spontaneous tumor growth delay of about four weeks of tumor growth than any other group. Cyclophosphamide combined group of mice were alive at 38 weeks, rather than targeting the control group at 26 weeks had been deaths or tumor volume over 1500mm of 3 sup> also scFv that NLDC-145 sup>-neu alone group effect better than other non-targeted group, the group most of the mice also survive at 30 weeks. Conclusion: In summary we built DC targeting DNA vaccine in mice tumor-specific antigens targeted to the DC surface, causing the antigen-specific cellular and humoral immunity, more importantly, if combined with low dose cyclophosphamide briefly to remove regulatory T cells, as a therapeutic vaccine in mice play a better anti-tumor effects, thus providing a potentially viable therapeutic strategy for cancer therapy.