TW200930813A - DNA vaccine comprising IL-6-encoding DNA construct and applications thereof - Google Patents

Abstract

The present invention provides a DNA vaccine, which comprises a DNA construct comprising an expression vector which is expressible in a eukaryotic cell, and a nucleotide fragment which comprises an IL-6-encoding sequence and an HPV E7-encoding sequence. In addition, the present invention also provides a pharmaceutical composition and a method of generating said DNA vaccine.

Description

200930813 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD The present invention relates to a DNA vaccine containing a DNA construct. The present invention also relates to a pharmaceutical composition of the aforementioned DNA vaccine and a method for producing the same. [Prior Art] Cervical cancer is one of the leading causes of death in women. High-risk human papillomavirus (HPV) such as HPV subtype i6 (HPV-16) is known to be persistently infected with cervical cancer. The development is directly related to the process. While existing HPV vaccines can be used to prevent cervical cancer', it is not known whether they can be used to treat HPV-associated cervical cancer or to alleviate existing HPV infection or HPV-related disease. Therefore, a safe and effective therapeutic vaccine has been developed to treat HPV-related cervical cancer, other HPV-related genital cancers (such as vagina, vulva, and penis) and other Ηργ-related lesions in the head and neck and gastrointestinal system, and the same treatment. Precancerous lesions at the site are very useful. In recent years, DNA vaccines have been developed and evaluated in a variety of disease models and are considered a prospective therapeutic that can be used to treat a variety of diseases, including use as immunotherapies in certain cancers. 'Therapeutic DNA vaccines are introduced into the antigen-presenting cells (APCs) of the recipient's immune system to express protein antigens, which are derived from major histocompatibility antigen (MHC) molecules of classes j and 11. Treatment and presentation to induce an immune response, such as a helper T cell response, a poisonous tau cell reaction, and a humoral (antibody) response, which directly results in the removal of tumor cells expressing the aforementioned antigen by the immune system. Compared to the “traditional” (protein antigen) vaccine, DNA vaccine 200930813 has several advantages, such as high specificity, safety, stability, cost-effectiveness, and the ability to induce several types of immune responses. Compared with the "live bacteria" and "attenuated" vaccines, DNA vaccines do not pose any risk of infection because only certain sequences in the pathogen DNa are used in the process. In addition, DNA vaccines are not required and are toxic adjuvants for practical use. The DNA vaccine is also simpler to prepare than the subunit vaccine because it does not require protein antigen expression and purification before it is injected into the patient. The turtle's APC cell has a limited life span, so DNA The efficacy of vaccines is also limited by the inability of APC cells to process and present antigens indefinitely. Therefore, several strategies have been used to increase the validity of DNA vaccines, such as targeting molecules by fusion molecules. Enhance antigen treatment (Cheng as α/., 2001; Chen de/α,. 2000); target antigens with rapid intracellular degradation (Rodriguez “α/·, 1997); APC receptor ligand (Boyle 1998) or a pathogen sequence (such as fragment of tetanus toxin C) (King d, 1998) to transfer antigen to APC cells; co-injection with cytokines (Weiss By ^, ❹ 1998 ), and co-administered it with cpG nucleoside acid (Klinman as α/., 1997) ° There have been some merger strategies to enhance the efficacy of DNA vaccines. The development of vaccines and immunotherapy, and the overexpression of anti-apoptotic molecules is a strategy that may overcome the short lifespan of APC cells. For example, administration of a DNA vaccine comprising a coding fragment having an antigen and a dendritic cell (DC) apoptosis inhibitor can prolong the survival of DC cells' to enhance the validity of the DNA vaccine (Kim et al., 2003). Other studies have shown that combined use of tumor antigens and inhibitors of apoptosis (such as Bcl-X bcl-2, XIAP, dominant negative caspase-9 or negative dominant caspase _ 8) It can enhance the specific immunity and anti-tumor effect of antigen 200930813 (Kim eia/., 2003; Kim eia/., 2004; Kim da/., 2005). Therefore, DC-based immunity can be enhanced by a strategy of inhibiting apoptosis in vivo and prolonging the survival time of DC cells exhibiting antigen. However, it is known that certain apoptosis inhibitors (such as Bcl-2 family proteins) may be overexpressed in certain cancers, suggesting that they are involved in cellular immortalization and must be noted. Security issue. © SUMMARY OF THE INVENTION In view of the absence of conventional DNA vaccines, one of the objects of the present invention is to develop a DNA vaccine with improved efficacy comprising a human interleukin-6 (IL-6). The sequence of the gene and a sequence derived from the human papillomavirus E7 gene. The combination of IL-6 and E7 prolongs the lifespan of DC cells, improves the processing and presentation of E7 antigens, and enhances the recipient's immune response. Another object of the present invention is to provide a pharmaceutical composition, I

In order to achieve the above object, the present invention provides a DNA construct, which further comprises: a code sequence. An expression vector which can express a nucleotide fragment in a eukaryotic cell, comprising a river: and an IL-6 coding sequence and an E7 coding sequence. The E7 coding sequence is obtained from hpv and is obtained from HPV]6 By. In a preferred embodiment, all of the preceding subtypes, particularly as described, can be expressed in human cells 200930813; more preferably, the aforementioned expression vector is selected from pcDNA3, pSG5 or pCMV; The vector is pcDNA3. In a preferred embodiment, the aforementioned IL_6 coding sequence is SEq ID NO: 3, and the aforementioned E7 coding sequence is seq id NO: ό. The present invention also provides a DNA vaccine comprising: the above DNA construct; and a particle coated with the DNA construct. ❹

In a preferred embodiment, the particles are gold particles; more preferably, the gold particles have a diameter of 1.6 μm. In a preferred embodiment, wherein the aforementioned expression vector is expressed in a human cell; more preferably, the expression vector is selected from pcDNA3, pSG5 or PCMV; and preferably, the expression vector is pcDNA3. In a preferred embodiment, the aforementioned IL-6 coding sequence is SEQ ID NO: 3, and the aforementioned E7 coding sequence is seq ID NO. The present invention further provides a pharmaceutical composition comprising the above DNA vaccine. - In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, and more preferably, the pharmaceutically acceptable carrier is ddH2(R) or PBS (salt buffer solution). In a preferred embodiment, the aforementioned pharmaceutical composition is for treating diseases caused by HPV; more preferably, it is for treating genital cancer (such as cervical cancer, vaginal cancer, vulvar cancer, penile cancer) or cancer. Precancerous lesions (such as precancerous lesions of the cervix, vulva or vagina), head and neck cancer (such as oropharyngeal squamous cell carcinoma (〇r〇pharyngeal called Li (4) _ or \ intestinal cancer (such as esophageal cancer or colorectal cancer) The best one is for the treatment of cervical cancer. 8 200930813 The invention further provides a method for producing the above DNA vaccine, comprising: (1) providing a DNA construct, wherein the DNA construct comprises: a expression vector, It can be expressed in eukaryotic cells; and a nucleotide fragment comprising an IL-6 coding sequence and an E7 coding sequence, and (2) coating the DNA construct on the surface of the particle. In the sample, the aforementioned expression vector is pcDNA3; the aforementioned IL-6 coding sequence is SEQ ID NO: 3, and the aforementioned E7 coding sequence is SEQ ID NO.. 6; more preferably, the aforementioned particles are gold particles; The diameter of the particles is 1.6 μιη The present invention further provides a method for preventing or treating a disease caused by HPV, comprising: administering an effective amount of the above DNA vaccine or effective to a patient suffering from the disease caused by the aforementioned HPV or having a risk of developing the disease; In the preferred embodiment, the disease caused by the aforementioned HPV is a genital cancer or a precancerous lesion, a head and neck cancer or a gastrointestinal cancer; moreover, the genital cancer includes a cervix Cancer, vaginal cancer, vulvar cancer and penile cancer; the genital precancerous lesion includes precancerous lesions of the cervix, vulva and vagina; the aforementioned head and neck cancer comprises oropharyngeal squamous cell tumor; and the aforementioned gastrointestinal cancer includes esophageal cancer , colorectal cancer and anal cancer, and precancerous lesions; preferably, the disease caused by the aforementioned HPV is cervical cancer. In summary, the present invention provides a DNA vaccine comprising: ~DNA constructs' Contains a performance that can be expressed in eukaryotic cells. 200930813 A nucleotide fragment 'which contains - IL_6 coding sequence and an excellent utility' can be used to treat Further, the present invention also provides the aforementioned DNA bacillus composition and a method for producing the same. [Embodiment] Since the early genes of HPV (eadygene) are expressed throughout the entire cycle of Hpv, these early genes can be used as therapeutic © The target antigen of the vaccine. We especially want to treat the HPV early genes E6 and E7 (because they have the ability to transform the host cells, also known as oncogenes) secret DNA vaccine to treat diseases caused by ΗΡν, including the reproductive tract. Cancers in the head and neck and gastrointestinal tract and related precancerous lesions. However, many DNA vaccines do not have sufficient nmnunogenicity, so they cannot be said to be useful because these DNAs containing vaccines cannot be lived = large Dissemination, and the processing and presentation of antigens plays an important role in the manufacture of useful therapeutic DNA vaccines.介 Interleukin-6 (IL_6) is a cytokine that is expressed and secreted by normal cells and plays an important role in the expansion and activation of T cells and the differentiation of B cells. It also protects cells from apoptosis through the Md] (myel〇id cdl leukemia-1) pathway. These properties of il_6 make it a preferred substance that can be used to prolong the lifespan of APc cells, thereby improving the immune response to IL_62 DNA vaccines. The following examples are intended to further illustrate the importance of the invention and are not intended to limit the scope of the invention. Particularly important is "E7" as referred to in the present invention refers to the E7 gene of human breast milk. 200930813 Example DNA Construction and Preparation IL-6 was amplified using polymerase chain reaction (PCR) using human placental complementary DNA (complementary DNA) as a template and using the following primer pair: 5'-CCGCTCGAGAGGAGCCCA GCTATGAACTC-3, (SEQ ID NO: 1) and 5, -CCGGAATTC GACCAGAAGAAGGAATGCCC-3, (SEQ ID NO: 2). The amplified IL-6 nucleotide sequence (SEQ ID NO: 3) was then cloned into the 〇pcDNA3 vector (Invitrogen, Carlsbad, CA) to produce PCDNA3-IL-6. E7 was amplified by PCR using a DNA of a CaSki cell strain (a cell line containing the embedded HPV 16 gene, obtained from ATCC) as a template, and using the following primer pair: 5, -CCGGAAGCT TATGCATGGAGATACACCTAC-3, (SEQ ID NO: 4) and 5, -CCCAAGCTTTTGAGAACAGGG-3, (SEQ ID NO: 5). The amplified E7 nucleotide sequence (SEQ ID NO: 6) was then cloned into the ?ίί/ΠΙ position of pcDNA3 and pcDNA3-IL-6, respectively, to produce pcDNA3-E7 and pcDNA3-IL-6/E7. (See the first figure). The IL-6 and E7 nucleotide sequences contained in the aforementioned pcDNA3-IL-6/E7 are synthesized into a continuous open reading frame, so that the construct expresses a fusion protein comprising IL-6 and E7. In addition, the E7 nucleotide sequence was cloned into PcDNA3-Mcl-1 (derived from Dr. Yang Xingfang, Research Institute of Taiwan Institute of Science and Technology) at the opening > 2ί/ΙΙΙ position to produce pcDNA3_Mcl-1/E7. Thereafter, pcDNA3-IL-6/E7 or pcDNA3-Mcl-1/E7 was digested with milll, and then obtained by plastid pcDNA3-E7/GFP (derived by Dr. Wu Ziwu from Johns Hopkins Medical Institutes by five digestions) GFP fragment fills in, connects, comes

II 200930813 Production of pcDNA3-IL_6/E7/GFP and pcDNA3-Mcl-1/E7/GFP. All DNA constructs were confirmed by DNA sequencing. Mice The studies described below were performed in groups of six using six to eight week old female C57BL/6J mice. All animal experiments were performed at the National Taiwan University Medical Center Animal Center in accordance with approved procedures and in accordance with the appropriate use and care recommendations for laboratory animals. DNA immunization to prepare DNA-coated gold particles, and using helium gene robbing to carry out DNA immunity mediated by particles, which is based on low-pressure accelerated gene gun (Bio-Gal Technology Co., Ltd., Taipei, Taiwan) get on. Gold particles (Bio-Rad 1652263) were weighed and suspended in 70% ethanol. After the suspension was shaken vigorously, it was centrifuged and the particles were collected. After washing three times with distilled water, 0.025 pg of the collected gold particles were placed in an Eppendorf tube and mixed with 1 〇〇5 μspermidine, and the mixture was ultrasonicated. Process for 10 to 20 seconds. Next, 25 pg of DNA dissolved in 25 mL of ddH20 was added, and the mixture was shaken. 1 μ CaCl 2 of loo was added, and the final mixture was shaken and cultured on ice for 1 minute, and the DNA was coated on the gold particles by spermidine. Finally, the coated particles were washed three times with 100% ethanol and resuspended in 2 to 250 pL of 100% ethanol. These DNA-coated gold particle suspensions are used as bullets for gene guns. Naked DNA (naked DNA) dissolved in ddHbO or pBS can also be used as a bullet. Dna-coated gold particles 12 200930813 pellets were delivered from the bristled abdomen to mice using a low-pressure accelerated gene gun with a helium gas pressure of 50 psi. Intracellular cytokine staining and flow cytometry analysis Each group of mice was immunized with one of the following DNA vaccines: pcDNA3 (no insert), pcDNA3-E7, pcDNA3-IL-6, pcDNA3-E7 + pcDNA3- IL_6, pcDNA3-Mcl-1/E7 and pcDNA3-IL-6/E7 (2 pg DNA construct/2 pg gold particles/mouse); all mice received boost immunity after one week. The naive group in which Ο mice were not immunized was used as a negative control group. After one week of rejuvenation, the mice were sacrificed and their spleen cells were harvested with a short E7 peptide RAHYNIVTF (aa 49-57, SEQ ID NO: 7) of 1 pg/ml or a long E7 peptide DSSEEEDEIDGP AGQAEPDRAHYNIVTFCCKCDSTLRL of 10 pg/ml ( Aa 30-67 » SEQ ID NO: 8) Co-cultivation. In general, short E7 peptides can be directly presented; while long E7 peptides need to be taken up by APC cells before they are processed and presented. The cells are mixed with the short or long E7 peptide described above for 16 to 20 hours. Thereafter, 1 pL/mL Golgistop (PharMigen, _ SanDiego, CA) was added to prevent secretion of cytokines (e.g., or il-4). After six hours, the cells were harvested, transferred to a tube, and then centrifuged at 40 °C for 5 minutes at L200- 1,600 rpm. Thereafter, the cells were washed with 500 pL of FACScan buffer (Shibuya in 0.5% BSA of PBS), and centrifuged again at 4 ° C for 5 minutes. The cells were resuspended in anti-CD4 or anti-CD8 antibody (PharMingen) conjugated with PE at a dilution of 1 ML (0.5 飓) diluted with 50 pL of FACScan buffer, and the cells were cultured in the dark for 3 minutes. The cells were then washed twice with FACScan buffer and centrifuged. These cells were resuspended in 500 pL of fixation buffer and protected from light for 2 minutes on ice; then, the cells were again centrifuged and 500 pL Perm Wash Buffer

(BioLegendBiotech) cleaning. Dilute 1 (0.5 μδ) of anti-IFN-γ antibody (PharMingen) or anti-IL-4 antibody (Biolegend, San Diego, CA) co-depleted with FITC 13 200930813 into 50 pL Perm Wash buffer and add it. The aforementioned cells were cultured on ice for 3 minutes in the dark. The cells were centrifuged and washed twice with 500 pL Perm Wash buffer. The cells were then resuspended in 300 to 500 pL of FACScan buffer' and analyzed by flow cytometry. All double-stained cells were analyzed in a standard procedure using a FACScan or FACSCalibur equipped with CELLQuest software (Becton Dickinson Immuno- cytometry System, Mountain View, Calif., USA). The result ❹ is shown in the second figure. Figure 2a shows the flow cytometric analysis of E7-specific lFN-γ secreting CD4+ T lymphocytes, while the bar graphs of the second and second graphs c depict the spleen per 3.5xl〇5 The number of E7-specific IFNs in the cells is the number of secretory CD4+ T lymphocytes and E7-specific IL-4 secreting CD4+ T lymphocytes. These data show that mice immunized with IL_6/F7 produced more E7-specific IFN secreted CD4+ T lymphocytes and E7-specific IL-4 secreting CD4+ T lymphocytes than the other groups. ball. It is easy to say that IL_6/E7 activates the Th1 path (Fig. 2b) and the Th2 path (Fig. 2c). Figure 2d shows flow cytometric analysis of E7-specific π?Ν-γ secreting CD8+ T lymphocytes, while the bar graph of second figure e depicts E7-specific iFN-γ secretory CD8+ τ Number of lymphocytes: il_6/E7 activates the poisonous T lymphocytes (Fig. e). None of the E7 peptides we used in these experiments, compared with mice immunized with E7 alone, CD4+ IFN-γ secretion and CD8+ IFN_Y secretion in mice immunized with IL_6/E7 The number of E7-specific sputum lymphocytes is significantly higher. 200930813 Anti-E7 antibody-enzyme-free residue-free adsorption assay (ELISA) All mice were sera taken 14 days after the last immunization and used to detect HPV by the following direct enzyme-labeled immunosorbent assay. -16 E7 specific antibody. '100 pL of HPV-16 E7 protein (0.5 pg/ml) from bacteria was coated on a 96-microplate' and incubated overnight at 4 °C. These wells were then blocked with pbS containing 20% fetal bovine serum at 37 °C for 2 hours' followed by the addition of 100 pL of serum diluted 1:100, 1:500 or 1:1,000 in PBS. The wells were incubated at 370 C for 2 hours. These wells were then washed with PBS containing 0.0% Tween 20 and incubated with rabbit anti-mouse IgG antibody (Zymed, San Francisco, CA) 1 : 2,000 dilutions conjugated with peroxidase for 1 hour at room temperature. . The wells were washed, developed with l_StepTurboTM B-ELISA (Pierce, Rockford, IL), then stopped at 1 MH 2 SO 4 and read the ELISA plate at 450 nm using a standard ELISA analyzer. Figure 5f shows E7-specific antibodies in mice immunized with various DNA vaccines. Mice that were immunized with the IL_6/E7 DNA vaccine had higher titers than the other groups. In vivo tumor preservation 眚 To determine whether we observed an enhanced E7-specific T cell immunity can be converted to a distinct E7-specific protective anti-tumor effect, the following in vivo tumor protection experiments were performed. Each group of mice was immunized with pcDNA3 (no insert), pcDNA3-E7, pcDNA3-IL-6, pcDNA3-E7 + pcDNA3_IL_6, pcDNA3 Mcl l/E7 or pcDNA3_I^6/E7 (2 mice), and all The mice received double reinforcement after one week. One week after the reinforcement, the mice were subcutaneously injected with 5χ1〇4 15 200930813, TC-1 cells/mouse for challenge (chaiienge). These mice will be monitored until 60 days after TC-1 challenge. The mouse was not subjected to the immunologically intact group as a negative control group. 〇TC-1 cell line supplemented with 10% (v〇l/v〇l) fetal bovine serum, 50 units/mL penicillin/streptomycin, 2 mM Z-glutamic acid, 1 mM sodium citrate, 2 mM non-essential amino acid (Gibco company) and 0.4 mg/mL G418 in RPMI-1640 were grown at 37 ° C and 5% CO 2 . In the case of tumor challenge, TC-1 was finely diluted by trypsinization, collected, washed twice with lxPBS, and finally resuspended in TxHanks buffered saline solution for TCM ® challenge. After challenge with TC-1 tumor cells, 100% of mice receiving the il_6/E7 DNA vaccine remained tumor-free on day 6 of the day. Only 40°/. Mice receiving the Mcl-1/E7 DNA vaccine remained tumor-free and all mice in the other groups (including the E7 group) developed tumors within 14 days after TC-1 challenge. The results are shown in the third graph a. Antibody depletion sinus test® In order to determine whether the lymphoid subpopulations (CD4+ T lymphocytes, CD8+ T lymphocytes and axillary lymphocytes) play an important role in antitumor effects, we performed a living Endogenous antibody depletion experiments. The mice were immunized with IL-6/E7 DNA vaccine, reinforced one week later, and then subjected to depletion of lymphocyte subpopulations, in which monoclonal antibody GK1.5 was used for CD4 depletion (Berkeley Antibody Company), single plant Antibody 2.43 was used for CD8 depletion (Berkeley Antibody Company) and monoclonal antibody PK136 was used for Natural Killer 1.1 depletion (Berkeley Antibody Company). These early strain antibodies were administered to mice by intraperitoneal injection. Consumption 16 200930813 After one week, all mice in the group were challenged with 5X1 〇 4 TC-1 cells/mouse. Flow cytometric analysis showed that 99% of these specific lymphocyte subpopulations were exhausted, while other subpopulations remained at normal numbers (data not shown). All mice were terminated on day 4 after tumor challenge.

All mice in the normal group and all CD8+ τ cell depleted mice developed tumors within 14 days after TC-1 challenge. In addition, 2%% CD4+ T cell depleted mice and 40% natural killer cell hl' cell depleted small ^ will develop tumors within 60 days after T (^-l challenge). The results are shown in the second figure. b, this result indicates that CD8+ gamma cells, CD4+ sputum cells, and sputum NK cells are important for the anti-tumor immunity produced by the IL-6/E7 0 vaccine. In vivo tumor treatment age due to lung blood The model of lung hemat〇genous spread is similar to cancer metastasis, so it is evaluated by this model.

IL 6/E7 embedded a-type DNA vaccine (Caf de Yejiang _6/£7 DNA ❿

The therapeutic efficacy of VaCCine) (see Chengeia/., 2005a). In this test, 5χ104 Τ(Μ cells were injected into the mouse from the tail vein. Two days later, each group of mice was pcDNA3 (

Pc brain-E7, pc cryptic IL_6, p__ = H PCDNA3_MC1_1/E7 or _ il 6/E7 days - reinforced immunity. The tcm S group was used to estimate and calculate the lungs of each group of mice ... that is, the original group that was neither immunized nor immunized as a negative control group. The representative lungs of each of the mice immunized with each other, and the number of lung nodules in the lungs were not shown in Fig. 4b and Fig. 4C, respectively. Pan 17 200930813 In comparison with other groups of mice, mice immunized with IL-6/E7 had much lower lung weights, and their lung tumor nodules were significantly more common than all other groups of mice. I have to come less. ' For direct comparison of clonal DNA vaccines, mice were grouped and immunized using two different vaccine combinations: IL-6/E7 DNA only, ETA (dII)/E7 DNA only (Hung d α/., 2001 ) or a combination of IL-6/E7 and ETA(dII)/E7 DNA, and then reinforce every 7 days. These mice were sacrificed 28 days after TC-1 challenge and lung tumor nodules were estimated as previously described. The results are shown in Figure 5a and Figure 5b. In the three groups of mice, mice immunized with IL-6/E7 in combination with ETA(dII)/E7 had the lowest lung weight and the least tumor nodules in the lungs. The results are shown in the fifth graph. Inguinal lymph node preparation of CDJC+ condylar cells of mice with JjA vaccination was performed in the abdomen of mice with pCDNA3-GFP, pcDNA3-E7/GFP, pcDNA3-Mcl-1/E7/GFP or pcDNA3-IL-6/E7 Intradermal injection of /GFP. After 1 or 5 days of immunization, 小鼠 all the inguinal lymph nodes of the mice were collected. Dendritic cells with CDllc+ expression on the cell surface were enriched from the lymph nodes using CDllc (N418) microparticles (Miltenyi Biotec, Auburn, CA). Detection of apoptotic cells in GFP+ CD1 lc+ cells using Annexin V-PE cells> annexjn v_pe. apoptosis detection kit (BD Bioscience, San Diego, CA) Method to calculate the percentage of apoptotic cells. The intact group in which the mice were not immunized served as a negative control group. In addition, 2χ1〇4 of the above enriched CDllc+ dendritic cells were co-cultured with 2xl〇6 Ε7-specific CD8+ T cell lines (see Kim as α/·, 2003). These cells were then stained for both surface CD8 and intracellular IFN-γ, and analyzed by flow cytometry 18 200930813 as described above. ❸

The results of the flow cytometry analysis are shown in Figure 6 a' and the results of the Cell > Death Detection Kit are shown in Figure 6 b-d. As shown in Figure 6b, + was in progress, and there was no significant difference in the number of GFP+CDllc+ cells in the inguinal lymph nodes of each group on the first day after the episode. However, on day 5, we found that the immunization with Mcl-1/E7/GFP and IL-6/E7/GEP was compared with the group immunized with E7/GFP and only GFP. The percentage of GFP+CDllc+ cells in the lymph nodes collected by the rats will be higher. We further determined the apoptosis of GFP+ CDllc+ cells obtained from the inguinal lymph nodes of each group of mice. The percentage of apoptotic cells in mice immunized with DNA with IL-6/E7/GFP or Mcl-1/E7/GFP was significantly lower than in mice immunized with GFP or E7/GFP. As shown in the sixth figure c. In other words, apoptosis of dendritic cells is inhibited by IL-6/E7 or Mcl-1/E7 DNA immunization. We also estimated the ability of these enriched CDllc+ dendritic cells to stimulate IFN-γ secretion. As shown in Figure 6d, we compared enriched CDllc+ dendritic cells at 1 and 5 days after immunization with the gene grab. CDllc+ dendritic cells isolated from mice immunized with IL-6/E7/GFP or Mcl-1/E7/GFP were activated in E7 specificity compared to mice immunized with GFP and E7/GFP. The CD8+ T cell strain is more effective in secreting IFN-γ. Peripheral blood single 19 200930813 nucleated cells were obtained from HLA-A2-positive human volunteers using CaSki cells as the target cell's toxic T lymphocytosis assay and exposed to granules _ macrophage colony stimulating factor (GM- CSF) (800 U/ml) and Jiang_4 (500 U/ml) were induced to differentiate into dendritic cells (DC) for 6 days. The differentiated DC cells were grouped' and each group was transfected with pCDNA3 (no insert), pcDNA3-E7, pcDNA3-IL-6 or pcDNA3-IL-6/E7 DNA constructs overnight using 50 mmol/l. The lysate of 293 DbKb cells was pulsed' after each group of shocked dc cells were co-cultured with fresh peripheral gold mononuclear cells from the same human volunteers to produce four E7-specific CD8+T cell. Thereafter, an assay was carried out in which: various E7-specific CD8+ T cells were used as effector cells, and CaSki cells were used as target cells. Toxic T cell activity values (CTL levels) are measured using standard lactate dehydrogenase (LDH) release assays (see cheng ei «/., 2005b). Effector cells and target cells (<1>4 target cells per well) were mixed together at various ratios (1:1, 5:1, 15:1, and 45:1) and the final volume was 200 pL. These cell mixtures were incubated at 37 ° C for 5 hours, after which 50 juL of medium was collected to estimate the LDH in the medium and the cell lysis was calculated according to the instructions of the CytoTox assay kit (Promega, Madison, WI). Percentage ® ratio. All experiments were three replicate experiments. As shown in the seventh panel, compared with the E7-specific toxic T cells stimulated by E7 protein, the percentage of CaSki cell-specific lysis caused by IL-6/E7 protein-impacted tau cells was significantly higher. high. Statistical Analysis All data expressed as mean ± SEM represent at least two different experiments. Intracellular cytokine staining data and tumor treatment experimental data obtained by flow cytometry were estimated by ANOVA 20 200930813. [Simple description of the diagram] The first figure is a schematic diagram of the DNA construct pCDNA3-IL-6/E7. Figure 1 shows the E7-specific immune status (immunological pr0file) of immunized mice. (4) A flow cytometric analysis representative of E7-specific IFN-γ secreting CD4+ T lymphocytes in each group. (b) The histogram depicts the number of E7-specific IFN-γ secreting CD4+ T lymphocytes per 3.5 x 1 5 spleen cells (mean ± SEM, corpse < 0.05 'single factor variation analysis (one_way) ANOVA)). (c) The histogram depicts the number of E7-specific il-4 specific for secretive CD4 T lymphocytes per 3.5 x 105 spleen cells (mean SEM, /^<0.01, single factor variance analysis) . (d) Flow cytometric analysis representative maps derived from E7-specific IFN-γ secretory CD8+ T lymphocytes in each group. (The histogram depicts the number of E7-specific IFN-γ secreting CD8+ T lymphocytes per 3.5 x 105 spleen cells (mean SEM, Ρ <0·01 'single factor variance analysis). (7) The histogram shows E7-specific antibodies in mice immunized with various DNA vaccines (mean SEM, corpse <〇.〇1, single factor analysis). The third panel shows the living body of mice. Results of in vivo tumor protection assays and in vivo antibody depletion experiments. (a) In vivo tumor protection experiments in mice immunized with various DNA vaccines. (b) Mice immunized with IL-6/E7 DNA vaccine In vivo antibody depletion experiments. The fourth panel shows the results of in vivo treatment with high therapeutic doses in mice. (a) Representative lung tumor nodules of immunized mice in each group. Naive ), 2 : no insertion sequence, 3 : E7, 4 : IL-6, 5 : E7 + IL-6 ' 6 : Mcl-1/E7 ' 7 : IL-6/E7. (b) Immunization of each group The lung weight of the mouse (mean ± 8 £) ^ 1, corpse < 0.001, 21 200930813 i, due to: ϊ f jin (8) each group of immunized mice The number of lungs in the lungs (should be job-like, ?<_, single-factor variation analysis) the result of the tumor (four) treatment, which is the anti-senior SE"l' in mice treated with the να vaccine /2001 'Single-factor variation analysis'. (8) Number of lung tumor nodules in mice of each group (mean ± SEM).

Figure 6 shows flow cytometric analysis of dendritic cells transfected with inguinal lymph nodes DN A in immunized mice, and E7-specific CD8+ T cell activation from isolated petile inguinal lymph nodes. (a) Representative flow cytometry data for the fraction of CDllc+ cells transfected with GFP in gated monocytes. (b) The histogram depicts the percentage of cDllc+ GFp+ mononuclear spheres in the mononuclear sphere population (mean SEM, ρ < 〇.〇ι, single factor variance analysis) ° (c) the histogram The percentage of peripheral cells in the CDllc+ GFP+ cells is depicted (mean soil SEM, ρ < 〇.〇ι, single factor variance analysis). (d) The histogram depicts the percentage of IFN7-specific IFN-γ secreting CD8 Τ cells per χ1〇5 cells (mean SEM', Ρ<0·001, single factor variance analysis) . Figure 7 shows CTL of Ε7, IL-6 and IL-6/E7. 'Results' Ε7-specific CD8+ Τ cell line impaired by Ε7, IL-6 or IL-6/E7 protein was used as an effector cell. , while caSki cells are used as target cells. E: The ratio of T is the ratio of effector cells to target cells, and IL_6 is interleukin-6. [Main component symbol description] None 22 200930813 References

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Claims (1)

200930813 X. Patent application: A DNA construct comprising: a two-representation vector which can be expressed in eukaryotic cells; and a nucleotide fragment comprising an IL 6 coding sequence-E7 sequence. 2. The DNA construct of claim 1, wherein the expression vector is expressed in a human cell. 3' is the DNA construct according to item 2 of the full-time application, wherein the pre-expressing expression vector is selected from the group consisting of pcDNA3, pSG5 or PCMV. 4. The DNA construct of claim 3, wherein the expression vector is pcDNA3. 5. The DNA construct of claim 1, wherein the aforementioned 1L-6 coding sequence is SEQ ID NO: 3. 6. The DNA construct of claim 1, wherein the E7 coding sequence is SEQ ID NO: 6. A DNA vaccine comprising: (4) a DNA construct according to item i of the patent garden; and a particle 'coated with the DNA construct. 8. The DNA vaccine according to claim 7, wherein the aforementioned particles are gold particles. 〃 9' For the dNa vaccine as described in item 8 of the patent application park, the direct control of the aforementioned gold particles is 1.6. Ίο. The DNA vaccine of claim 7, wherein the aforementioned expression vector is expressed in human cells. 11. The DNA vaccine of claim 10, wherein the aforementioned expression vector is selected from the group consisting of pCDNA3, pSG5 or pCMV. 12. The DNA vaccine of claim n, wherein the aforementioned 24 200930813 expression vector is pcDNA3. 13. The DNA vaccine according to claim 7, wherein the 妒IL-6 coding sequence is seq ID Ν〇·3. 8. The DNA vaccine according to claim 7, wherein the coding sequence of 驴7 is SEQ ID NO: 6. Do not κ 15. A pharmaceutical composition comprising a DNA vaccine as claimed in the patent application. The pharmaceutical composition as described in claim 15 of the patent application, wherein - φ comprises a pharmaceutically acceptable carrier. VIII. 17. The pharmaceutical composition of claim 15 for use in the treatment of a disease caused by HPV. 18. The pharmaceutical composition according to claim 17, which is for the treatment of cervical cancer, vaginal cancer, vulvar cancer or penile cancer. 19. The pharmaceutical composition of claim 18, which is for use in the treatment of cervical cancer. 20. A pharmaceutical composition as described in Section π of the Patent Application, which is used to treat precancerous lesions of the cervix, vulva or vagina. ❹ 21. A method for producing a DNA vaccine according to claim 7, wherein: (1) providing a DNA construct, wherein the DNA construct comprises: a expression vector, which is in a eukaryotic cell And a nucleotide fragment comprising an IL-6 coding sequence and an E7 coding sequence; and (2) coating the DNA construct on the surface of the particle. 22. The method of claim 21, wherein the aforementioned expression vector is pcDNA3. 23. The method of claim 21, wherein the aforementioned IL_6 25 200930813 coding sequence is SEQ ID NO: 3. 24. As claimed in claim 21, the sequence of A Λ — , 剐 剐 7 is SEQ ID NO: 6. 25. The method of claim 21, wherein the particles are gold particles. The aforementioned gold particles are as described in claim 25, and the direct control is 1.6 μm. 27. A method for preventing or treating a disease caused by hpv, which comprises administering an effective amount of DNA as described in the scope of the patent application to a patient suffering from or suffering from a disease caused by the aforementioned HPV. A vaccine, or an effective amount of the pharmaceutical composition as described in claim 15 of the patent application. 28. The method of claim 27, wherein the disease caused by the aforementioned Ηρν is cervical cancer, vaginal cancer, vulvar cancer or penile cancer. 29. The method of claim 28, wherein the disease caused by the pre & HP°V° is cervical cancer. The method of claim 27, wherein the disease caused by the foregoing is a precancerous lesion of the cervix, vulva or pubic lice. 26