TWI344989B - Dna vaccine comprising il-6-encoding dna construct and applications thereof - Google Patents

Description

1344989 Date of application: April 29, 100, nin. Illustrated: [Technical Field of the Invention] 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, 16 (HPV-16) persistent infection and cervical cancer are known. The development is directly related to the process. Although 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 lesions. Therefore, 'a safe and effective therapeutic vaccine has been developed to treat HPV-related cervical cancer, other HPV-related genital cancers (such as the vagina, vulva 'penis) and other ΗΡν-related lesions in the head and neck and gastrointestinal system" It is useful to treat precancerous lesions in the same area. — In recent years, DNA has been developed and evaluated in a variety of disease models. It is considered a prospective therapeutic that can be used to treat a variety of diseases, including immunotherapy as a treatment in certain cancers. Therapeutic DNA, which is introduced into the antigen presenting cell (Apc) of the recipient's immune system, is a protein antigen that is treated by a steroidal and πth class of histocompatibility antigen (MHC) molecules. And presented, to read = should be secondary helper U cell reaction 'poisoning type anti ===: will directly lead to the expression of the aforementioned antigen swelling 2 "traditional protein antigen" vaccine compared to DNA vaccine eve Advantages such as high specificity, safety, stability, cost effectiveness 5 1344989 Application date: cost-effectiveness of the Republic of China on April 29, 100, and the ability to induce several types of immune responses. In contrast to the "attenuated" vaccine, the DNA vaccine does not have any risk of infection because it uses only certain sequences in the pathogen DNA. In addition, DNA vaccines do not require 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 prior to injection into the patient. However, 'the lifespan of APC cells is limited', so the validity of sputum 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 antigens by fusion molecules to enhance antigen processing (Cheng as α/., 2001; Chen W α/., 2000); The antigen of the intracellular degradation is targeted (R〇driguez eM/., 1997); it is associated with an APC receptor ligand (B〇yle, 1998) or with a pathogen sequence (eg Fragment of tetanus toxin c) (King eia/., 1998) fusion to transfer antigen to APC cells; co-injection with cytokines (Weiss ei α/., 1998), and cpG oligonucleotides Co-administered to patients (Klinman e/α/., 1997). A number of merger strategies to enhance the efficacy of DNA vaccines have been introduced into the development of cancer vaccines and immunotherapies, and overexpression of anti-apoptotic gastric 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 dendritic cell (DC) apoptosis inhibitor can prolong the survival of DC cells and thereby enhance the validity of the DNA vaccine (Kim at α/., 2003) ). Other studies have shown that 'and tumor antigens and apoptosis inhibitors (such as

Bcl-Xl, Bcl-2, XIΑΡ, d〇minant negative caspase-9 or negative dominant caspase_8 can enhance antigen-specific immunity and anti-tumor effects ( Kim is 0/., 2〇〇3; Kim is 0/., 2004; Kim is α/., 2005). Therefore, it is possible to enhance cell-based immunity by inhibiting apoptosis in vivo and prolonging the date of the 6-recovery date: April 29, 100, in the presence of antigen-bearing DC cell survival time. However, it is known that certain inhibitors of apoptosis (such as Bcl-2 family proteins) may be overexpressed in some cancers, indicating that they must be associated with cell immortalization (cellular imrnortaiizati〇n) Pay attention to this aspect of security. SUMMARY OF THE INVENTION A DNA vaccine having improved efficacies in view of the deletion of a conventional DNA vaccine comprises a sequence derived from the human interleukin-6 (IL-6) gene and a derived from Human mastoid, the sequence of the toxic E7 gene. The combination of IL_6 and E7 will prolong the lifespan of % cells 'improving the treatment and presentation of E7 antigens and enhancing the recipient's immune response. · f Another purpose is to provide a pharmaceutical composition, It further comprises a prodrug S drug acceptable system. Another object of the present invention is to provide a method for producing the aforementioned DNA vaccine. The package provides the above object. The present invention provides a DNA construct, and a representation carrier, Can be expressed in eukaryotic cells. 'It contains -IL_6 coding sequence and -E7 coded from code (4) can be obtained from all subtypes of HPV, especially performance; better: implementation: the expression carrier can be In human cells, the best heart is ==—or in the implementation of the car, the aforementioned IL_6 coding sequence is qing mn〇: 3' and the aforementioned E7 coding sequence is SEQmN^Nahu (1). a DNA vaccine comprising: the DNA construct; and a particle, which is coated with the ship structure. The particles are gold particles; more preferably, the cell exhibits a good sample 2 ' The system can be fine in human ^ PCmJ PCDNA3 ^ PSG5 just as Alex expression vector pCDNA3.

3. In the cancer sample, the aforementioned IL-6 coding sequence is SEQ ID NO: and the E7 coding sequence is SEQ mN 〇: 6. V Miaoben is also provided with a pharmaceutical composition comprising the above-mentioned (10) A drug in the sample. The aforementioned pharmaceutical composition further comprises a medical pbs, and more preferably the aforementioned pharmaceutical acceptable carrier * ddH2 or 2 In the preferred embodiment, the aforementioned pharmaceutical system is used for treating HPV bows; the better one is for treating genital cancer (such as uterine cramps, vaginal cancer, vulvar cancer, penile cancer). Or precancerous lesions (such as cervical, sputum, or vaginal precancerous lesions), head and neck cancer (such as oropharyngeal sqUamusus cell carcin〇ma) or gastrointestinal cancer: positive ( Such as esophageal cancer or colorectal cancer); the best, for the treatment of cervical cancer. The present invention further provides a method for producing the above DNA vaccine, comprising: (1) providing a DNA construct, wherein the DNA construct comprises: (2) (2) ___ Auxiliary period: April 29, 1980 a performance vector which can be expressed in eukaryotic cells; and a segment which contains the -l.6 coding sequence and -E7 encoding the above DNA construct on the surface of the particle. IL-42 embodiment of the 'previous expression vector It is PCDNA3; the aforementioned fQiD N〇: 3, and the aforementioned E7 coding sequence is SEQ all temporary adhesion, and more preferably, the particles are gold particles; preferably, the diameter of the H gold particles is L-. 'Description of the method This step provides the prevention or treatment of diseases caused by HPV ϋ ί ί 已 已 已 已 HP HP HP HP HP HP HP = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = In the aspect, the disease caused by the aforementioned Ηρν, precancerous lesions, head and neck cancer or gastrointestinal cancer is reproductive = genital tract cancer includes cervical cancer, vaginal cancer, and external drop, ^ narration of genital cancer The lesion contains the cervix, vulva and penis 2 changes; The head and neck cancer contains the squamous squamous 1 of the oropharynx. The cancer contains esophageal cancer, colorectal cancer and anal cancer, and it is a disease of the stomach. The best, the disease caused by the aforementioned HPV is cancer. [Pre- & cancer] ... ▼ a ... μ low-pressure 々 Wo hip r disease. In addition, the present invention also provides the aforementioned DNA epidemic and its production method. The medical group is described above, the present invention provides A DNA vaccine comprising a construct comprising: j and a nucleotide fragment comprising an IL_6 coding sequence and a steroid; the vaccine has excellent utility, It is used for the treatment of 7 鹄 code. 乩冰,士玫(10)+ ^ . ____ # 1344989 Application date: April 29, 100 [Embodiment] Because HPV's early gene (eariy gene) is in HPV The entire life cycle is manifested, so these early genes can be used as target antigens for therapeutic HP V vaccines. We especially want to make HPV early genes E6 and E7 (because they have the ability to transform host cells, also known as carcinogenic) Gene) for DNA vaccines Treatment of diseases caused by HPV, including cancers of the reproductive tract, head and neck, and gastrointestinal tract, and related precancerous lesions. However, many DNA vaccines do not have sufficient immunogenicity' so they cannot be said to be useful vaccines. Because these DNAs, including vaccines, cannot be amplified or spread in vivo, and the processing and presentation of antigens plays an important role in the manufacture of useful therapeutic DNA vaccines. (IL-6) is a cytokine that is expressed and secreted by normal cells. It plays a heavy role in the expansion and activation of T cells and the differentiation of B cells. It also protects cells from apoptosis by Mcl-1 (myeloid cell leukemia-1). 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 caused by DNA vaccines containing IL-6. The following examples are intended to further illustrate the importance of the invention and are not intended to limit the scope of the invention. "E7" refers to the E7 gene of any subtype of human papillomavirus. 1344989 Application date: April 29, 100 BC DNA construction and preparation of IL-6 lines were amplified using polymerase chain reaction (PCR) using human placental complementary DNA as template and using the following Pair I 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 pcDNA3 vector (Invitrogen, Carlsbad, CA) at the 〇1/£^〇 and I positions 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, -CCCAAGCTTTTGAGAACAGTGGG-3, (SEQ ID NO: 5). The amplified E7 nucleotide sequence (SEQ ID NO: 6) was then cloned into the "ί/ΙΙΙ 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 the Institute of Research, Yang Yanfang, Taiwan Academia Sinica) to produce pcDNA3-Mcl-1/E7. Thereafter, pcDNA3-IL-6/E7 or pcDNA3-Mcl-1/E7 was digested with //ζ·«ί/ΙΙΙ, followed by plastid pcDNA3-E7/GFP (received by Dr. Wu Zibiao from Johns Hopkins Medical Institutes) The GFP fragment obtained by the digestion of five αβΙ/Λ^ΐ was complemented and ligated to produce pcDNA3-IL-6/E7/GFP and pcDNA3-Mcl-1/E7/GFP. All 11 1344989 application period: Republic of China On April 29, 100, DNA constructs were confirmed by DNA sequencing.

The study interspersed below was performed in groups of six by eight to eight week old female C57BL/6J mice. All animals 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. PNA immunization, > preparation of DNA-coated gold particles, and using the helium gene to steal DNA mediated by the particle, which is based on low-pressure accelerated gene gun (Bio-Gal Technology Co., Ltd. , Taipei, Taiwan) to come. Gold particles (Bi〇-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 twice with distilled water, 0.025 pg of the collected gold particles were placed in an Eppendorf tube 'and mixed with 丨00 〇.05 μ spermidine (spermidine) and the mixture was ultrasonically processed 1 〇 to 20 seconds. Next, 25 pg of DNA dissolved in 25 pL ddHzO was added, and the mixture was added to the shock. 100 μL of 1 M CaCI was added and the final mixture was shaken, incubated on ice for 10 minutes, and DNA was coated on the gold particles by spermidine. Finally, the coated particles were washed three times with 100% ethanol and resuspended in 200 to 250 μί of 100% ethanol. These DNA-coated gold particle suspensions serve as bullets for the gene gun. Naked DNA (near DNA) dissolved in ddH2〇 or PBS can also be used as a bullet. The DNA-coated gold particles were sent from the shaved abdomen to the mice using a low pressure accelerated gene gun with a helium gas pressure of 50 psi. 12 1344989 Date of application: cytokine staining and flow-type fine-face analysis in cells of the Republic of China on April 29, 100 Each group of mice was immunized with one of the following DNA vaccines: pcDNA3 (no insertion), pcDNA3- E7, pcDNA3-IL-6, pcDNA3-E7 + pcDNA3-IL-6, pcDNA3-Mcl-1/E7 and pcDNA3-IL-6/E7 (2 pg DNA construct/2 gold particles/mouse); The mice received boost immunity after one week. The naiive group in which the mice were not immunized was used as a negative control group. Reinforce one week later, sacrifice the mice' to collect their spleen cells and mix them with 1 gg/ml

Short E7 peptide RAHYNIVTF (aa 49-57, SEQ ID NO: 7) or 10 gg/ml long E7 peptide DSSEEEDEIDGP

AGQAEPDRAHYNIVTFCCKCDSTLRL (aa 30-67, SEQ ID NO: 8) was co-cultured. In general, short E7 peptides can be presented directly; 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 gL/mL Golgistop (PharMigen, San Diego, CA) was added to prevent secretion of cytokines such as IFN-γ or IL-4. Six hours later, the cells were harvested, transferred to a tube, and then centrifuged at 1,200-l, 600 rpm for 5 minutes at 40C. Thereafter, the cells were washed with 500 pL of FACScan buffer (0.5% BSA in PBS), and further centrifuged at 4 ° C for 5 minutes. The cells were resuspended in 1 pL (〇.5 pg) diluted with 50 g of LFACScan buffer and conjugated with PE anti-CD4 or anti-CD8 antibody (PharMingen), and the cells were incubated for 30 minutes in the dark. The cells were then washed twice with FACScan buffer and centrifuged. The cells were resuspended in 500 μL of fixing buffer and protected from light for 20 minutes on ice; next, the cells were again centrifuged and washed with 5 μM pL Perm Wash buffer (BioLegend Biotech). Dilute 1 pL (0.5 pg) of anti-IFN-γ antibody (PharMingen) or anti-IL-4 antibody (Biolegend, San Diego, CA) conjugated to FITC to 50 μί

In Perm Wash buffer, the cells were added to the above cells and incubated on ice for 30 minutes in the dark. The cells were centrifuged and reconstituted with 500 pL of Perm Wash buffer 13 Date: April 29, 100, washed twice. The cells were then resuspended in 300 to 500 μί 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 results are shown in the second figure. Figure 2a shows the flow cytometric analysis of E7-specific IFN-γ secreting CD4+ T lymphocytes, while the bar graphs of the second panel b and the second panel c depict each spleen cell in 3.5xl05 The number of E7-specific IFN-γ secreting CD4+ T lymphocytes and E7-specific IL-4 secreting CD4+ T lymphocytes. These data show that mice immunized with IL-6/E7 produced more E7-specific IFN-γ secreting CD4+ T lymphocytes and E7-specific IL-4 secretion than other groups. CD4+ T lymphocytes. It is easy to say that IL-6/E7 activates the Th1 path (Fig. 2b) and the Th2 path (Fig. 2c). Figure 2d shows the flow cytometric analysis of E7-specific IFN-γ secreting CD8+ T lymphocytes' and the bar graph of the second panel e depicts E7-specific IFN-γ secreting CD8+ T lymphocytes Number: IL-6/E7 activates the poisonous T lymphocytes (Fig. e). Regardless of which E7 peptide we used in these experiments, CD4+ IFN-γ secretion and CD8+ IFN- in mice immunized with IL-6/E7 compared to mice immunized with E7 alone. The number of gamma secreting E7-specific T lymphocytes is significantly higher. Anti-marker free 疬叨p assay (ELISA) All mice were sera taken 14 days after the last immunization and used for debt testing by the following direct enzyme-labeled immunosorbent assay 1344989 : E7-specific antibody to HPV-16 on April 29, 1995. 100 pL of HPV-16 E7 protein (〇.5 pg/ml) obtained from bacteria was coated on a 96-microwell plate and cultured overnight at 4 °C. These wells were then incubated at 37 with PBS containing 20% fetal bovine serum. (: Block for 2 hours, then add 100 pL of serum diluted in PBS at a ratio of 1: 1 1, 1: 5 〇〇 or 1 ··1,000, and then incubate the plate at 37 ° C for 2 hours. The holes were then washed with PBS containing 0.05% Tween 20 and then anti-mouse antibodies from rabbits that were conjugated to peroxidase (

San Francisco, CA) 1 : 2,000 dilutions were incubated for 1 hour at room temperature. • Wash wells and develop with Up Turbo TMB-ELISA (Pierce, Rockford, IL), then stop the reaction with 1 M H2S〇4 and read the ELISA plate at 450 nm using a standard ELISA analyzer. Figure f shows the mouse heterologous antibody immunized with various DNA vaccines. Mice immunized with the 1L_6/E7 vaccine will have higher titers than the other groups. Swollen body

It was observed that the enhancement of Ε7-specific tau cell immunity was marked by a significant Ε7-specific protective anti-tumor effect (helical insert sequence) in vivo tumor protection experiments. Each group of mice was immunized with PcDNA3 + Ϊ, Ρ _ Α 3 · Ε 7, (10), 3 剔, 帅 E E7 (2- u 'PCDNA3_MCM/E7 or PCDNA3_IL- (4) tonic one 怂 怂 ΐ ΐ ΐ ΐ ΐ 怂 怂 怂Accept the reinforcement. In (chat 5X10 ^ to li. The mouse is not immunized the original state _ ' as a negative 15 1344989 The date of application: April 29, the Republic of China 曰 TC-1 cell line in the supplement has 1% (ν 〇ι/ν〇ι) fetal bovine serum, 50 units/mL penicillin/streptomycin, 2 mM glutamic acid, 1 mM sodium citrate, 2 ml V [Gibco company and 0.4 MGMI-1640 of mg/mL G418 was grown at 370C and 5% C02. On the day of tumor challenge, TC-1 cells were collected by trypsinization, washed twice with lxPBS, and finally suspended in TC-1. LxHanks buffered saline solution for poisoning. After challenge with TC-1 tumor cells, 1% of mice receiving IL-6/E7 DNA vaccine remained tumor-free at 6 days. Only 40 % of mice receiving the Mcl-1/E7 DNA vaccine remained tumor-free, while all mice in the other groups (including the E7 group) developed swelling within 14 days after TC-1 challenge. The results are shown in Figure 3. The in vivo antibody depletion experiment determines whether the lymphoid subpopulations (CD4+ T lymphocytes, CD8+ T lymphocytes, and NK lymphocytes) play an important role in antitumor effects. We performed an in vivo antibody depletion experiment. The mice were immunized with IL-6/E7 DNA vaccine, reinforced one week later, and then subjected to depletion of lymphocyte subpopulations, including monoclonal antibody GK1.5. For CD4 depletion (Berkeley Antibody Company) 'Single antibody 2.43 is for CD8 depletion (Berkeley Antibody Company), and monoclonal antibody PK136 is for Natural Killer 1.1+ depletion (Berkeley Antibody Company) 〇 These individual antibodies were delivered to mice by intraperitoneal injection. After one week of exhaustion, all mice in the group were challenged with 5xl〇4 TC-1 cells/mouse. Flow cytometry It was shown that 99% of these specific lymphocyte subpopulations were depleted, but the other subpopulations remained at normal numbers (data not shown). All mice terminated the experiment on the 40th day after tumor challenge. April 29, 100 / / There is a small gas of f main sorrow and all the exhaustion of CD8 + T cells are η go / grow tumor within 14 days. In addition, '20% CD4+ Τ fine I τγ 1, 4〇% natural killer cells 1.1+ cells depleted mice, ancient TS surnames ^ zhi poor ^ 60 days to develop tumors. The results are shown in the third panel, ί ‘/ϋ γ»μΓ two CD8+ T cells, CD4+ T cells and NK cells are important for the anti-tumor immunity produced by the seedlings. In vivo tumor treatment

^ Since the model of lung hematogenous spread is similar to cancer metastasis, this model is used to evaluate the therapeutic efficacy of IL-6/E7, a conjugate DNA vaccine (chimedc IL 6/E7 DNA confidence) (see Chenge) /a/.,2〇〇5a). In this experiment, 5x1〇4 TC-1 cells were injected from the tail vein into mice. Two days later, each group of mice was pcDNA3 (no insert), pcDNA3_E7, pcDNA3-IL-6 'pcDNA3-E7 + pcDNA3- IL-6 >

Immunization (16 pg/mouse) was performed with pcI^NA3_Mcl, l/E7 or pcDNA3-IL-6/E7, followed by a booster immunization every 7 days. T (>1 after 28 days of 'sacrificial mice' and their lungs were removed. The experimental group of the sample group was not known to estimate and calculate the lung tumor nodules of each group of mice. The mice were not immunized The original group was used as a negative control group. Representative lung tumor nodules of the immunized mice of each group are shown in the fourth panel a' and the lung weight average and the number of lung tumor nodules in the immunized mice of each group. They are shown in Figure 4b and Figure 4c, respectively. Compared with other groups of mice, mice immunized with IL-6/E7 had much lower lung weight, and their lung tumor nodules. It is also significantly less common than all other groups of mice. For direct comparison of chimeric DNA vaccines, mice were grouped and immunized using three different vaccine combinations: IL-6/E7 only 17 1344989 Date of application: DNA on April 29, 100, using only ETA(dII)/E7 DNA (Hung W α/., 2001) or a combination of IL-6/E7 and ETA(dII)/E7 DNA, after Reinforce every 7 days = third to kill these mice after 28 days of TC-1 challenge and estimate pulmonary swelling as described above Nodules. The results are shown in Figure 5 and Figure 5b. In the three groups of mice, mice immunized with IL-6/E7 and ETA(dII)/E7 had the lowest lung weight and lung tumors. The nodules were the least. The results are shown in the fifth figure. Inguinal lymph node preparation of fish A immunized mice CDllc+榭突纟@PcDNA3-GFP, Lu pcDNA3-E7/GFP ' pcDNA3-Mcl-l in the abdomen of mice with a gene gun Intradermal injection of /E7/GFP or pcDNA3-IL-6/E7/GFP. Inguinal lymph nodes of all mice were collected 1 or 5 days after immunization. CDllc (N418) microparticles were used (Miltenyi Biotec, Auburn) , CA ) enriched dendritic cells with CD1 lc+ expression on the surface of the cells from the lymph nodes. Annexin V-PE apoptosis detection kit (BD Bioscience, San) Diego, CA) measured the apoptotic cells in GFP+CD11c+ cells, and calculated the percentage of apoptotic cells by flow cytometry. The untreated group of mice was used as a negative control group. 2x1004 of the above enriched CDllc+ dendritic cells and 2×106 E7-specific CD8+ T Strain co-culture (see Kim e / α /., 2003). After the surface of these cells do CD8 two staining and intracellular IFN-γ, the above-described another example analyzed to flow cytometry. The results of the flow cytometry analysis are shown in Figure 6a, and the results of the apoptosis detection kit are shown in Figure 6 b-d. As shown in the sixth panel b, there was no significant difference in the number of GFP+ CD 1 lc+ cells in the inguinal lymph nodes of each group on the first day after immunization. However, on day 5, we found that compared with the group immunized with E7/GFP and only GFP, 18 1344989, the date of application: April 29, 100, in MCM/E7/GFP and The percentage of GFP+CDllc+ cells in the lymph nodes received by mice immunized with IL_6/E7/GEP compared the stomach. We further determined the apoptosis of GFP CD 11 c cells obtained from the inguinal lymph nodes of each group of mice. Percentage of apoptotic cells in mice immunized with DNA with Mcl-1/E7/GFP compared to mice immunized with ορρ戎E7/GFP

Significantly lower' as shown in Figure 6c. It is easy to say that the cell apoptosis of dendritic cells is inhibited by IL-6/E7 or Mcl-1/E7 DNA immunization. We also estimated the ability of these enriched CDUc+ 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. Activation of CD 11 c dendritic cells isolated from mice immunized with IL-6/E7/GFP or Mcl-1 /E7/GFP compared to mice immunized with GFP and E7/GFP E7-specific CD8+ T cell lines are more effective in secreting IFN-γ. Peripheral blood mononuclear cells were obtained from HLA-A2-positive human volunteers using CaSki cells as target cells, and exposed to granule-macrophage colony-stimulating factor (GM-CSF) (800 U/ml) and IL-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/1. The lysate of 293 DbKb cells was pulsed, and then each group of shocked DC cells were co-cultured with fresh peripheral blood mononuclear cells from the same human volunteer to produce four E7-specific CD8+ T cells. . Then, the CTL assay was carried out, in which the application date was 19 1344989: on April 29, 100, 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 e/a/., 2005b). The effector cells and target cells (lxl〇4 target cells per well) were mixed at various ratios (1:1, 5: Bu 15:1 and 45:1) with a final volume of 200 μί. These cell mixtures are at 37. (: Incubate for 5 hours, then collect 50 μί of the medium to estimate the amount of LDH in the medium, and calculate the percentage of cell lysis according to the instructions of the CytoTox assay kit (Promega, Madison, WI). For the three repeated experiments. As shown in the seventh figure, the CaSki cell specificity caused by the IL-6/E7 protein-impacted T cells compared with the E7-specific toxic T cell. The percentage of lysis is significantly higher. Statistical analysis All data represented by mean SEM represent at least two different experiments. Intracellular cytokine staining data and tumor treatment experimental data obtained by flow cytometry are based on the number of variances. Analyze (ANOVA) to estimate. [Simplified schematic] The first figure is a schematic diagram of the DNA construct pcDNA3-IL-6/E7. The second panel shows the E7-specific immune status of immunized mice. (a) A representative of flow cytometry analysis of E7-specific IFN-γ secreting CD4+ T lymphocytes in each group. (b) The histogram depicts E7 specificity per 3.5 x 105 spleen cells. Sexual IFN-γ 20 1344989 Shen Fu 曰: The number of CD4+ τ lymphocytes in the secretion of the CD4+ τ lymphocytes (average soil SEM, /><〇.〇% ^曰, subvariant analysis ( One_way ANOVAW. (4) The E7-specific IL-4 secretory CD in 3.5x10 spleen cells was inserted into the map.

Number of lymphocytes (mean SEM, P &lt; 0.01, single factor variant T analysis). (4) A flow cytometric representative map derived from E7-specific IFN-γ secreting CD8+ τ-fractions in each group. (e) The histogram depicts the number of E7-specific IFN-γ secreting CD8+r potential bucks in 3.5 x 10 spleen cells (mean ± 8 £)^, corpse <〇.〇1, single Factor Variance Octupan (1) This histogram shows the small immunization with various DNA vaccines.

E7-specific antibodies (mean ± 8 £ 14, corpse &lt; 〇 〇 单 on the analysis). Oral Mannosis The third panel shows the results of in vivo tumor protection experiments and in vivo depletion experiments in mice. (a) In vivo tumor protection experiments for immunization with various DNA vaccines. In vivo antibody depletion experiments in mice immunized with IL_6/E7 DNA vaccine. Immune immunity

The fourth panel shows the results of treatment with high therapeutic doses of live tumors in mice. (a) Representative lung segments of immunized mice of each group. 1 : naive, 2 : no insertion sequence, 3 : E7, 4 : 1] ^ 6 ° 5 : E7 + IL-6 ' 6 : McM/E7, 7 : IL_6/E7. (b) Lung weight of mice in each group (mean soil SEM, p&lt;〇.〇01, single factor variable analysis). (c) Number of lung tumor nodules in each group of immunized mice Mean soil SEM ’P&lt;〇.〇5, single factor variance analysis). Figure 5 shows the results of in vivo tumor treatment experiments comparing anti-tumor effects in mice treated with different chimeric DNA vaccines. (a) Lung weight of each group of immunized mice (mean SEM, 'single factor 34 heterologous analysis). (8) Number of lung σ|5 tumor nodules in each group, immunized mice (mean ± 8 £ 1 \4). Figure 6 shows the flow cytometric analysis of dendritic cells transfected with DNA transfected in the inguinal lymph nodes of immunized mice by 21 1344989, and from 29 years of the Republic of China, and from immunized mice. The hallucinogenic CD8+ T cell-activated condition isolated from the inguinal lymph nodes. (a) Representative flow cytometry data for the percentage of GFP-transfected CD i i c+ cells in the g monp population. (b) The β-small histogram depicts the percentage of cd 11 c+ GFP + mononuclear spheres in the early nuclear population (mean soil SEM Ρ &lt; 〇.〇ι, single factor variance analysis). (c) The histogram depicts the percentage of apoptotic cells in CD1 lc+ GFP+ cells (mean SEM, P &lt; 0.01, single factor variance analysis). (d) The histogram depicts the percentage of E7-specific iFN-γ secreting CD8+ T cells per 1×1 〇5 cells (mean SEM, P&lt;0.001, single factor variation analysis). · Figure 7 shows the results of CTL assays for E7, IL-6 and IL-6/E7, in which E7-specific CD8+ T cell lines impinged by E7, IL-6 or IL-6/E7 proteins are used as effector cells. CaSki cells are used as target cells. E : T ratio is the ratio of effector cells to target cells' IL-6 is interleukin-6 〇 [Key component symbol description] Benefits ",, 22 1344989 Shen Fu period: Republic of China, April 29, 曰 References

Boyle et al. (1998) Enhanced responses to a DNA vaccine encoding a fusion antigen that is directed to sites of attenuation induction. Nature 392: 408-411 Chen et al. (2000) Enhancement of DNA vaccine potency by linkage of antigen gene to An HSP70 gene. Cancer Res 60; 1035-1042

Cheng et al. (2001) Tumor-specific immunity and antiangiogenesis generated by a DNA vaccine encoding calreticulin linked to a tumor antigen. JC/m Invest 108: 669-678 Cheng et al. (2005a) Characterization of DNA vaccines encoding the domains of Calcine 25: 3864-3874 Cheng et al. (2005b) Induction of human papillomavirus type 16-specific immunologic responses in a normal and an human papillomavirus-infected populations. Immunology 115: 136-149 Hung et al. (2001) Cancer immunotherapy using a DNA vaccine encoding the translocation domain of a bacterial toxin linked to a tumor antigen. Cancer Res 61: 3698-3703 Kim et al (2003) Enhancing DNA vaccine potency by coadministration of DNA encoding antiapoptotic proteins. J Clin Invest 112: 109-117 Kim et al. (2004) Enhancement of DNA vaccine potency by coadministration of a tumor antigen gene and DNA encoding serine protease inhibitor-6. Cancer Res 64: 400-405 Kim et al. (2005) Modification of professional antigen-presenting cells with small interfering RNA in vivo to enhance cancer vaccine potency. Cancer Res 65: 309-316 King et al. (1998) DNA vaccines with single-chain Fv fused to fragment C of tetanus toxin inhibitor protective immunity against lymphoma and myeloma. Nat Med 4: 1281-1286 Klinman et al (1997) Contribution of CpG motifs to the immunogenicity of DNA vaccines. J Immunol 158: 3635-3639

Rodriguez et al. (1997) DNA immunization: ubiquitination of a viral protein enhances cytotoxic T-lymphocytes induction and antiviral protection but abrogates antibody induction. J Virol 71; 8497-8503

Weiss et al. (1998) A plasmid encoding murine granulocyte-macrophage colony-stimulating factor increase protection conferred by a malaria DNA vaccine. J Immunol 161: 2325-2332 23 1344989 Date of application: April 29, 1989, sequence list &lt 110] National Taiwan University &lt;120&gt; DNA vaccine containing IL-6-encoded DNA construct and its application &lt;130&gt; 07P0597 &lt;160&gt; 8 &lt;170&gt; Patentln version 3.4 &lt;210> 1 &lt;211&gt; 29 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;223&gt; Introduction &lt;400&gt; 1 29 ccgctcgaga ggagcccagc tatgaactc &lt;210&gt; 2 &lt;211&gt; 29 &lt;212&gt; DNA &lt;213&gt; Artificial sequence 1344989 Application date: April 29, 100, &lt;220&gt;&lt;223&gt; Introduction &lt;400&gt; 2 ccggaattcg accagaagaa ggaatgccc 29 &lt;210> 3 &lt;211&gt; 700 &lt;212&gt; DNA Lu &lt;213>Human&lt;400&gt; 3

aggagcccag ctatgaactc cttctccaca agcgccttcg gtccagttgc cttctccctg 60 gggctgctcc tggtgttgcc tgctgccttc cctgccccag tacccccagg agaagattcc 120 aaagatgtag ccgccccaca cagacagcca ctcacctctt cagaacgaat tgacaaacaa 180 attcggtaca tcctcgacgg catctcagcc ctgagaaagg agacatgtaa caagagtaac 240 atgtgtgaaa gcagcaaaga ggcactggca gaaaacaacc tgaaccttcc aaagatggct 300 gaaaaagatg gatgcttcca atctggattc aatgaggaga cttgcctggt gaaaatcatc 360 actggtcttt tggagtttga ggtataccta gagtacctcc agaacagatt tgagagtagt 420 gaggaacaag ccagagctgt gcagatgagt acaaaagtcc tgatccagtt cctgcagaaa 480 2 1344989 DEU multiplexing date: Republic 100, April 29, said aaggcaaaga atctagatgc aataaccacc cctgacccaa ccacaaatgc cagcctgctg 540 acgaagctgc aggcacagaa ccagtggctg caggacatga caactcatct cattctgcgc 600 agctttaagg agttcctgca gtccagcctg agggctcttc ggcaaatgta gcatgggcac 660 ctcagattgt tgttgttaat gggcattcct tcttctggtc 700 &lt; 210 &gt; 4 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Introduction &lt;400&gt; 4 30 ccggaagctt atgcatg Gag atacacctac &lt;210> 5 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220 &lt;223&gt; 引引 1344989 Application date: April 29, 100 &lt;400&gt; 5 cccaagcttt tgagaacaga Tgg &lt;210&gt; 6 &lt;211&gt; 288 &lt;212&gt; DNA &lt;213&gt; Human papillomavirus 16 &lt;400&gt; 6 23 atgcatggag atacacctac attgcatgaa tatatgttag atttgcaacc agagacaact 60 gatctctact gttatgagca attaaatgac agctcagagg aggaagatga aatagatggt 120 ccagctggac aagcagaacc ggacagagcc cattacaata ttgtaacctt Ttgttgcaag 180 tgtgactcta cgcttcggtt gtgcgtacaa agcacacacg tagacattcg tactttggaa 240 gacctgttaa tgggcacact aggaattgtg tgccccatct gttctcaa 288 &lt;210&gt; 7 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Human papillomavirus 16 &lt;400&gt;

Arg Ala His Tyr Asn lie Val Thr Phe 4 51344989 Application date: April 29, 100

&lt;210&gt; 8 &lt;211&gt; 38 &lt;212&gt; PRT &lt;213> Human papillomavirus 16 &lt;400&gt;

Asp Ser Ser Glu Glu Glu Asp Glu lie Asp Gly Pro Ala Gly Gin Ala 1 5 10 15

Glu Pro Asp Arg Ala His Tyr Asn lie Val Thr Phe Cys Cys Lys Cys 20 25 30

Asp Ser Thr Leu Arg Leu 35 5

Claims (1)

1344989 (4) Year f month z repair (different run 丨 announcement! Ten, the scope of application for patent: It is expressed in eukaryotic cells, which can express one of the epitope '6 (IL-6) coding sequences, and a human milk. 1. A DNA construct, its vector, a vector, a human interleukin-6 The viral E7 (HPVE7) coding sequence is composed of the above; wherein the human IL-6 coding sequence is SEQidn〇:3, and the aforementioned coding sequence is (4)1 耻^ The above expression vector can express human IL-6 protein and HPV in eukaryotic expression. E7 protein. 2. The DNA construct of claim 1, wherein the expression vector is expressed in a human cell. 3. The DNA construct of claim 2, wherein the expression vector is selected from the group consisting of pCDNA3, pSG5 or pCMV. A DNA vaccine comprising: the DNA construct according to claim 1; and a gold particle coated with the DNA construct. 5. The DNA vaccine according to claim 4, wherein the aforementioned gold particles have a diameter of 1.6 μm. 6. The 2DNA vaccine as described in claim 4, wherein the aforementioned expression vector is expressed in human cells. 7. The DNA vaccine of claim 6, wherein the aforementioned expression vector is selected from the group consisting of pcDNA3, PSG5 or PCMV. 8. A pharmaceutical composition comprising the DNA vaccine of claim 4 of the patent application. 9. The pharmaceutical composition of claim 8 further comprising a pharmaceutically acceptable carrier. # 10. The pharmaceutical composition as described in claim 8 is used to treat diseases caused by HPV. 11. The pharmaceutical composition as described in Section 1 of the patent application is for use in a right-hand one. Date of application: April 29, 2010, cancer, vaginal cancer, vulvar cancer or penile cancer. 12. Treat cervical cancer as described in claim U. A composition of the composition of the present invention is used in the medical composition described in the above paragraph 10, which is used for the precancerous lesion of the vulva or vagina of the neck. m method, the DNA vaccine of the fourth aspect of the patent scope of the patent application, wherein the aforementioned DNA construction system is represented by its m-丄/fine cell--expression carrier, human whitening _6 total μ mail/horse sequence and human papillomavirus E7 (HPVE7) encode f, 歹L consists of 'the aforementioned human IL_6 coding sequence is SEQ ID N0: 顼·ι^ΗΡνΕ7 sequenced horse sequence is SEQ ID NO: 6 The foregoing table volume accounts for the human IL_6 protein and HPVE7 protein in eukaryotic expression; and 15 (;: the body is coated on the surface of the aforementioned gold particle. The method described in item 14 (4), wherein the aforementioned performance is ranked 14th. The method described in the above, the aforementioned gold particles