TWI392731B - Dna vaccine comprising ctgf-encoding dna construct and applications thereof - Google Patents

Description

DNA vaccine containing CTGF-encoded DNA construct and application thereof

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.

Cervical cancer is one of the leading causes of death in women. It is known that the persistent infection of high-risk human papillomavirus (HPV) such as HPV subtype 16 (HPV-16) and the development and progression of cervical cancer It is directly related. Although existing HPV vaccines can be used to prevent cervical cancer, it is not known whether they can be used to treat HPV-related 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 vaginal vulva, vulva, penis) and other HPV-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 agent that can be used to treat a variety of diseases, including as immunotherapies in certain cancers. Therapeutic DNA vaccine is introduced into the antigen presenting cells (APCs) of the recipient's immune system to represent protein antigens that are processed by major histocompatibility antigen (MHC) molecules of class I and class II. And presented to induce immune responses, such as helper T cell responses, toxic T cell responses, and humoral (antibody) reaction, which directly leads to the removal of tumor cells expressing the aforementioned antigen by the immune system.

Compared to "traditional" (protein antigen) vaccines, DNA vaccines have 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 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 DNA 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 et al ., 2001; Chen et al. , 2000); The antigen of the reverse intracellular degradation is targeted (Rodriguez et al ., 1997); it is associated with an APC receptor ligand (Boyle et al ., 1998) or with a pathogen sequence (such as a fragment of tetanus toxin). C) (King et al ., 1998) Fusion to transfer antigen to APC cells; co-injection with cytokines (Weiss et al ., 1998), and co-administering it to patients with CpG oligonucleotides (Klinman Et al ., 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 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 and thereby 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-X1, Bcl-2, XIAP, dominant negative caspase-9 or negative dominant caspase) 8) Enhance antigen-specific immunity and anti-tumor effects (Kim et al ., 2003; Kim et al ., 2004; Kim et al ., 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 inhibitors of apoptosis (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.

In view of the absence of conventional DNA vaccines, one of the objects of the present invention is to develop a DNA vaccine having improved efficacies comprising a sequence derived from a connective tissue growth factor (CTGF) gene and a derived from The sequence of the human papillomavirus E6 and/or E7 gene. The combination of CTGF and HPV sequences selected from E6 and/or E7 enhances the recipient's immune response and results in a more potent anti-tumor effect.

Another object of the present invention is to provide a pharmaceutical composition comprising the aforementioned DNA vaccine and a selective pharmaceutically acceptable carrier. A further object of the invention is to provide a method of producing the aforementioned DNA vaccine.

In order to achieve the above object, the present invention provides a DNA construct comprising: a expression vector which can be expressed in a eukaryotic cell; and a nucleotide fragment comprising a CTGF coding sequence and an HPV sequence, wherein the aforementioned HPV The sequence is selected from the group consisting of an E6 coding sequence, an E7 coding sequence, or a combination thereof.

The aforementioned E6 coding sequence and E7 coding sequence are available from all subtypes of HPV, particularly from HPV-16.

In a preferred embodiment, the aforementioned expression vector can be expressed in human cells; 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 CTGF coding sequence is SEQ ID NO: 3, the aforementioned E6 coding sequence is SEQ ID NO: 6, and the aforementioned E7 coding sequence is SEQ ID NO: 9.

The present invention also provides a DNA vaccine comprising: the above DNA construct; and a particle coated with the DNA construct.

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, the aforementioned expression vector can be expressed in a human cell; more preferably, the expression vector is selected from the group consisting of pcDNA3, pSG5 or pCMV; optimally, the aforementioned expression vector is pcDNA3.

In a preferred embodiment, the aforementioned CTGF coding sequence is SEQ ID NO: 3, the aforementioned E6 coding sequence is SEQ ID NO: 6, and the aforementioned E7 coding sequence is SEQ ID NO: 9.

The present invention further provides a pharmaceutical composition comprising the above DNA vaccine.

In a preferred embodiment, the aforementioned pharmaceutical composition further comprises a pharmaceutically acceptable carrier; more preferably, the aforementioned pharmaceutically acceptable carrier is ddH ₂ O or PBS (phosphate buffer solution).

In a preferred embodiment, the aforementioned pharmaceutical composition is for treating a disease caused by HPV; more preferably, for treating a 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) or gastrointestinal cancer (such as esophageal or colorectal cancer); Good for treating 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: a expression vector which can be expressed in a eukaryotic cell; and a nucleotide fragment And comprising a CTGF coding sequence and an HPV sequence, wherein the HPV sequence is selected from the group consisting of an E6 coding sequence, an E7 coding sequence, or a combination thereof; and (2) coating the DNA construct on the surface of the particle.

In a preferred embodiment, the expression vector is pcDNA3; the aforementioned CTGF coding sequence is SEQ ID NO: 3, the aforementioned E6 coding sequence is SEQ ID NO: 6, and the aforementioned E7 coding sequence is SEQ ID NO: 9; The particles are gold particles; preferably, the gold particles have a diameter of 1.6 μm.

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 an effective amount to a patient suffering from the disease caused by the aforementioned HPV or having a risk of developing the disease Pharmaceutical composition.

In a preferred embodiment, the disease caused by the aforementioned HPV is genital cancer or precancerous lesion, head and neck cancer or gastrointestinal cancer; more preferably, the genital cancer includes cervical 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 includes oropharyngeal squamous cell tumor; and the aforementioned gastrointestinal cancer includes esophageal cancer, colorectal cancer, and anal cancer. And its precancerous lesions; the best, the disease caused by the aforementioned HPV is cervical cancer.

In summary, the present invention provides a DNA vaccine comprising a DNA construct, and the DNA construct comprises: a expression vector capable of expressing in a eukaryotic cell, and a nucleoside comprising a CTGF coding sequence and an HPV sequence. An acid fragment wherein the aforementioned HPV sequence is selected from the group consisting of an E6 coding sequence and/or an E7 coding sequence; the vaccine has excellent utility and can be used to treat diseases caused by HPV. Further, the present invention also provides a pharmaceutical composition of the aforementioned DNA vaccine and a method for producing the same.

Because the early genes of HPV are expressed throughout the life cycle of HPV, these early genes can be used as target antigens for therapeutic HPV vaccines. In particular, we want to use the HPV early genes E6 and E7 (because they have the ability to transform host cells, also known as oncogenes) for DNA vaccines to treat diseases caused by HPV, including the reproductive tract, head and neck, and gastrointestinal tract. Cancer 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 disseminated in vivo, but in useful therapeutic DNA. The handling and presentation of antigens plays an important role in the manufacture of vaccines.

Connective tissue growth factor (CTGF) is a cysteine-rich protein that was first found in the conditioned medium of human umbilical vein endothelial cells. In recent years, it has been shown that CTGF inhibits apoptosis of chicken embryonic fibroblasts and human rhabdomyosarcoma cells, and also increases endothelial cell survival. CTGF also gives cells an apoptosis-resistant phenotype, which is similar to the role of IGF-II in skeletal myoblasts.

The following examples are intended to further illustrate the importance of the invention and are not intended to limit the scope of the invention. Especially heavy It is to be noted that "E6" and "E7" as referred to in the present invention refer to the E6 and E7 genes of any subtype of human papillomavirus.

Example

DNA construction and preparation

CTGF was amplified using polymerase chain reaction (PCR) using human placental complementary DNA as a template and using the following primer pairs: 5'-CCGGTCTAGACAACCATGACCGCCGCCAGT-3' (SEQ ID NO: 1) and 5' -CCGGAAT TCGTTCAAGTTCCAGTCTAATG-3' (SEQ ID NO: 2). The amplified CTGF nucleotide sequence (SEQ ID NO: 3) was then cloned into the Xba I/ EcoR I position of the pcDNA3 vector (Invitrogen, Carlsbad, CA, USA) to produce pcDNA3-CTGF.

E6 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'-GGGGAATTCATGCACCAAAAGAGAACTGCAATGT-3' ( SEQ ID NO: 4) and 5'-CCCAAGCTTTTACAGCTGGGTTTCTCTACGTGTTCT-3' (SEQ ID NO: 5). The amplified E6 nucleotide sequence (SEQ ID NO: 6) was then cloned into the EcoR I/ Hind III positions of pcDNA3 and pcDNA3-CTGF, respectively, to produce pcDNA3-E6 and pcDNA3-CTGF/E6. In addition, E7 was amplified by PCR using the DNA of the CaSki cell line as a template, and the following primer pairs were used: 5'-CCGGAAGCTTATGCATGGAGATACACCTAC-3' (SEQ ID NO: 7) and 5'-CCCAAGCTTTTGAGAACAGA TGG-3' ( SEQ ID NO: 8). The amplified E7 nucleotide sequence (SEQ ID NO: 9) was then cloned into the Hind III positions of pcDNA3 and pcDNA3-CTGF, respectively, to produce pcDNA3-E7 and pcDNA3-CTGF/E7.

The amplified E7 nucleotide sequence (SEQ ID NO: 9) was cloned into the Hind III position of pcDNA3-CTGF/E6 to produce pcDNA3-CTGF/E6/E7.

The CTGF and E6 nucleotide sequences contained in the aforementioned pcDNA3-CTGF/E6 are synthesized into a continuous open reading frame, so that the construct expresses a fusion protein comprising CTGF and E6. Similarly, pcDNA3-CTGF/E7 will display a fusion protein comprising CTGF and E7, while pcDNA3-CTGF/E6/E7 will exhibit a fusion protein comprising CTGF, E6 and E7.

Thereafter, pcDNA3-CTGF/E7 was digested with Hind III, and the GFP fragment obtained by digestion of EcoR I/ Not I by plastid pcDNA3-E7/GFP (Dr. Wu Ziwu from Johns Hopkins Medical Institutes) was used. , ligation, to produce pcDNA3-CTGF/E7/GFP. All DNA constructs were confirmed by DNA sequencing, and schematic representations of DNA constructs are shown in the first panel.

Mouse

The study described below uses six to eight week old females. C57BL/6J mice were performed in groups of five. 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 immunity

The DNA-coated gold particles were prepared, and the DNA immunoassay mediated by the particles was carried out using a helium gas gene gun, which was carried out by a low-pressure accelerated gene gun (Bio-Gal Technology Co., Ltd., Taipei, Taiwan). 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 μg of the collected gold particles were placed in an Eppendorf tube and mixed with 100 μL of 0.05 M spermidine, and the mixture was subjected to ultrasonic treatment for 10 to 20 seconds. Next, 25 μg of DNA dissolved in 25 μL of ddH ₂ O was added, and the mixture was shaken. 100 μL of 1 M CaCl _{2 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 μL of 100% ethanol. These DNA-coated gold particle suspensions serve as bullets for the gene gun. Naked DNA (naked DNA) dissolved in ddH ₂ O or PBS can also be used as a bullet. DNA-coated gold particles were delivered from the shaved abdomen to mice using a low pressure accelerated gene gun with a helium gas pressure of 50 psi.

Intracellular cytokine staining and flow cytometry

First, each group of mice was immunized with one of the following DNA vaccines: pcDNA3 (no insert), pcDNA3-E7, pcDNA3-CTGF, pcDNA3-E7+pcDNA3-CTGF, and pcDNA3-CTGF/E7 (2 μg) DNA constructs / 2 μg gold particles / mouse); all mice received boost immunity after one week. The naïve group in which the 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: 10) of 1 μg/ml or a long E7 peptide DSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRL of 10 μg/ml (aa 30-67, SEQ ID NO: 11) co-culture. 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 μL/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 1,200-1,600 rpm for 5 minutes at 4 °C. Thereafter, the cells were washed with 500 μL of FACScan buffer (0.5% BSA in PBS), and further centrifuged at 4 ° C for 5 minutes. The cells were resuspended in 1 μL (0.5 μg) of anti-CD4 or anti-CD8 antibody (PharMingen) conjugated with PE diluted in 50 μL of FACScan buffer, 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 fixation buffer and protected from light for 20 minutes on ice; next, the cells were again centrifuged and washed with 500 μL of Perm Wash Buffer (BioLegend Biotech). 1 μL (0.5 μg) of anti-IFN-γ antibody (PharMingen) or anti-IL-4 antibody (Biolegend, San) conjugated to FITC Diego, CA) was diluted into 50 μL of Perm Wash buffer, added to the aforementioned cells, and incubated on ice for 30 minutes in the dark. The cells were centrifuged and washed twice with 500 μL of Perm Wash buffer. The cells were then resuspended in 300 to 500 μL 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 results are shown in the second figure.

Figure 2a shows the flow cytometric analysis of E7-specific IFN-γ secreting CD8 ⁺ T lymphocytes in each immunized group, while the bar graphs in the second and second graphs c depict each 3.5 ×10 The number of E7-specific IFN-γ secreting CD8 ⁺ T lymphocytes and E7-specific IFN-γ secreting CD4 ⁺ T lymphocytes in ⁵ spleen cells. In addition, the second bar graph in FIG d depict CD4 T lymphocyte number per 3.5 × 10 ⁵ E7-specific spleen cell was in the secretion of IL-4 ^+. These data show that mice immunized with CTGF/E7 produced more E7-specific IFN-γ secreting CD8 ⁺ T lymphocytes and E7-specific IL-4 secreting CD4 than the other groups. ⁺ T lymphocytes. In other words, CTGF/E7 activates the poisonous T lymphocytes (Fig. 2b) and the Th2 pathway (Fig. d), but does not activate the Th1 pathway (Fig. 2c).

Similarly, the other five groups of mice were also immunized and sacrificed using the above procedure, but the following DNA vaccines were used: pcDNA3-CTGF/E7, pcDNA3-CTGF/E6, pcDNA3-CTGF/E6/E7, and pcDNA3-CTGF/E6+ pcDNA3-CTGF/E7. The spleen cells of these mice were then co-administered with a short E7 peptide of 1 μg/ml (ie SEQ ID NO: 10) or a short E6 peptide LCIVYRDG (aa 50-57, SEQ ID NO: 12) of 1 μg/ml. The cells were cultured for 16 to 20 hours, and subjected to the above treatment to determine the number of E6 and/or E7-specific IFN-γ secreting CD8 ⁺ T lymphocytes per 3.5 × 10 ⁵ spleen cells. The third panel a and the third panel b show flow cytometric analysis of E7-specific IFN-γ secreting CD8 ⁺ T lymphocytes and E6 specific IFN-γ secreting CD8 ⁺ T lymphocytes in the above immunized group, respectively. . In the third panel c, the E7-specific IFN-γ secreting CD8 ⁺ T lymphocytes increased in the pcDNA3-CTGF/E7, pcDNA3-CTGF/E6/E7 and pcDNA3-CTGF/E6+pcDNA3-CTGF/E7 groups. However, the E6-specific IFN-γ secreting CD8 ⁺ T lymphocytes of the pcDNA3-CTGF/E6, pcDNA3-CTGF/E6/E7, and pcDNA3-CTGF/E6+pcDNA3-CTGF/E7 groups were increased. These data show that physical linkage between CTGF and E6 or E7 is necessary to enhance E6 or E7 specific IFN-[gamma] secreting CD8 ⁺ T lymphocyte activity. In other words, CTGF/E6/E7 can also activate toxic T lymphocytes. Similarly, it activates the Th2 path but does not activate the Th1 path (data not shown).

Anti-E7 antibody enzyme-labeled immunosorbent assay (ELISA)

At 14 days after the last immunization, sera from all mice were taken and used to detect HPV-16 E7-specific antibodies by the following direct enzyme-labeled immunosorbent assay.

100 μL of the HPV-16 E7 protein (0.5 μg/ml) obtained from bacteria was coated on a 96-microplate and incubated overnight at 4 °C. The wells were then blocked in PBS containing 20% fetal bovine serum at 37 °C for 2 hours, after which 100 μL of serum diluted 1:100, 1:500 or 1:1,000 in PBS was added and the wells were added. The plate was incubated at 37 ° C for 2 hours. These wells were then washed with PBS containing 0.05% Tween 20 and incubated for 1 hour at room temperature with a 1:2,000 dilution of rabbit anti-mouse IgG antibody (Zymed, San Francisco, CA) conjugated with peroxidase. . Washed well plate to 1-Step Turbo TMB-ELISA ( Pierce, Rockford, IL) to color, then 1M H ₂ SO ₄ to stop the reaction, and read in a standard ELISA ELISA plate analyzer at 450 nm.

Figure 2e shows E7-specific antibodies in mice immunized with various DNA vaccines. Mice immunized with the CTGF/E7 DNA vaccine will have higher titers than the other groups.

In vivo tumor protection experiment

To determine if we were able to convert the enhanced E7-specific T cell immunity to a significant 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-CTGF, pcDNA3-E7+pcDNA3-CTGF, or pcDNA3-CTGF/E7 (2 μg/mouse), and all mice were Reinforce a week later. One week after the reinforcement, mice were subcutaneously injected with 5 × 10 ⁴ TC-1 cells/mouse for challenge. These mice will be monitored until 60 days after TC-1 challenge. The original group in which the mice were not immunized was used as a negative control group.

The TC-1 cell line is supplemented with 10% (vol/vol) fetal bovine serum, 50 units/mL penicillin/streptomycin, 2 mM L -glutamic acid, 1 mM sodium pyruvate, 2 mM non-essential amine Gibco company and 0.4 mg/mL G418 in RPMI-1640 were grown at 37 ° C and 5% CO ₂ . On the day of tumor challenge, TC-1 cells were collected by trypsinization, washed twice with 1×PBS, and finally resuspended in 1×Hanks buffered saline solution for TC-1 challenge.

After challenge with TC-1 tumor cells, 100% of mice receiving the CTGF/E7 DNA vaccine remained tumor-free on day 60, while all mice in the other groups (including the E7 group) were Tumors developed within 14 days after TC-1 challenge. The result is shown in the fourth figure a.

Similarly, as shown in Figure 4b, the other five groups of mice were also immunized and challenged using the above procedure, but the following DNA vaccines were used: pcDNA3-E7, pcDNA3-CTGF/E6, pcDNA3-CTGF/E7, pcDNA3- CTGF/E6/E7, and pcDNA3-CTGF/E6+pcDNA3-CTGF/E7. 100% of mice receiving CTGF/E6, CTGF/E7, CTGF/E6/E7 or CTGF/E6+CTGF/E7 vaccines at 60th It stays in a state of no tumor at all times. These results show that fusion of CTGF with antigen E6 and/or E7 is essential for the anti-tumor activity of TC-1 tumor cells expressing E6 and/or E7.

In vivo antibody depletion experiment

To determine whether lymphocytic subpopulations (CD4 ⁺ T lymphocytes, CD8 ⁺ T lymphocytes, and NK lymphocytes) play an important role in antitumor effects, we performed an in vivo antibody depletion assay. The mice were immunized with CTGF/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), monoclonal antibody 2.43 For the CD8 depletion (Berkeley Antibody Company), the monoclonal antibody PK136 was used for Natural Killer 1.1 ⁺ depletion (Berkeley Antibody Company).

These monoclonal antibodies were administered to mice by intraperitoneal injection. After one week of exhaustion, all groups of mice were challenged with 5 x 10 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 terminated the experiment on day 40 after tumor challenge.

All mice in the normal group and all CD8 ⁺ T cell depleted mice developed tumors within 14 days after TC-1 challenge. Conversely, as shown in Figure 5, all mice that were not depleted, and all CD4 ⁺ T cells depleted or natural killer cells 1.1 ⁺ cells depleted in immunized mice after TC-1 tumor challenge It remained in a tumor-free state at 60 days. These results indicate that this result indicates that CD8 ⁺ T cells are important for the anti-tumor immunity produced by the CTGF/E7 DNA vaccine.

In vivo tumor treatment experiment

Since the model of lung hematogenous spread is similar to cancer metastasis, the efficacy of the CTGF/E7 chimeric DNA vaccine (chimeric CTGF/E7 DNA vaccine) is evaluated by this model (see Cheng et al ., 2005a). In this experiment, 5 x 10 ⁴ TC-1 cells were injected from the tail vein into mice. Two days later, each group of mice was immunized with pcDNA3 (no insert), pcDNA3-E7, pcDNA3-CTGF or pcDNA3-CTGF/E7 (16 μg/mouse), followed by booster immunization every 7 days. After 28 days of TC-1 challenge, the mice were sacrificed and their lungs were removed. The lung tumor nodules of each group of mice were estimated and calculated from the experimenters who did not know the sample group. The original group in which the mice were not immunized was used as a negative control group.

Representative lung tumor nodules of each group of immunized mice are shown in panel a, and the number of lung tumor nodules in each group of immunized mice is shown in panel 6b. Compared with the other groups of mice, mice immunized with CTGF/E7 had significantly fewer lung tumor nodules.

Preparation of CD11c from inguinal lymph nodes of immunized mice ⁺ Dendritic cell

Intradermal injection of pcDNA3-GFP, pcDNA3-E7/GFP or pcDNA3-CTGF/E7/GFP was performed in the abdomen of mice with a gene gun. Inguinal lymph nodes of all mice were collected 1 or 5 days after immunization. Dendritic cells with CD11c ⁺ expression on the cell surface were enriched from the lymph nodes using CD11c (N418) microparticles (Miltenyi Biotec, Auburn, CA). The annexin V-PE apoptosis detection kit (BD Bioscience, San Diego, CA) was used to detect apoptotic cells in GFP ⁺ CD11c ⁺ cells, and the flow was Cell method to calculate the percentage of apoptotic cells. The original group in which the mice were not immunized was used as a negative control group. In addition, 2 x 10 ⁴ of the above enriched CD11c ⁺ dendritic cells were co-cultured with 2 x 10 ⁶ E7-specific CD8 ⁺ T cell lines (see Kim et al ., 2003). These cells were then stained with both surface CD8 and intracellular IFN-γ, and analyzed by flow cytometry as described above.

The results of the apoptosis detection kit are shown in Figure 7 ac. As shown in the seventh panel a, there was no significant difference in the number of GFP ⁺ CD11c ⁺ cells in the inguinal lymph nodes of each group on the first day after immunization. However, on day 5, we found that GFP ⁺ CD11c in the lymph nodes collected from mice immunized with CTGF/E7/GFP compared to the group immunized with E7/GFP and only GFP. ^{+ The} percentage of cells will be higher.

We further determined the apoptosis of GFP ⁺ CD11c ⁺ cells obtained from the inguinal lymph nodes of each group of mice. The percentage of apoptotic cells in mice immunized with DNA with CTGF/E7/GFP was significantly lower compared to mice immunized with GFP or E7/GFP, as shown in Figure 7b. In other words, apoptosis of dendritic cells is inhibited by CTGF/E7 immunity.

We also estimated the ability of these enriched CD11c ⁺ dendritic cells to stimulate IFN-γ secretion. As shown in Figure 7c, we compared enriched CD11c ⁺ dendritic cells at 1 and 5 days after immunization with the gene gun. CD11c ⁺ dendritic cells isolated from mice immunized with CTGF/E7/GFP secreted IFN in activated E7-specific CD8 ⁺ T cell lines compared to mice immunized with GFP and E7/GFP The -γ aspect is more effective.

Since the E6 and E7 proteins are shown to be ideal targets for the development of HPV therapeutic vaccines, DNA vaccines targeting both E6 and E7 proteins will be better than vaccines directed to E6 or E7 alone. Epidemic response and anti-tumor effects. Our previous studies also showed that the two anti-tumor effects of E6 and E7 DNA vaccines against HPV E6 and E7 tumors were significantly better than those with E6 DNA or E7 DNA alone. . These results show that a better therapeutic effect is achieved when targeting E6/E7 tumors with E6 and E7 as targets.

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 analysis of variance (ANOVA).

The first picture is a schematic representation of a DNA construct.

The second panel shows the E7-specific immunological profile of the immunized mice. (a) A representation of flow cytometric analysis derived from E7-specific IFN-γ secreting CD4 ⁺ T lymphocytes in each group. (b) The histogram depicts the number of E7-specific IFN-γ secreting CD8 ⁺ T lymphocytes per 3.5 × 10 ⁵ spleen cells (mean ± SEM, P < 0.01, single factor analysis) One-way ANOVA)). (c) The histogram depicts the number of E7-specific IFN-γ secreting CD4 ⁺ T lymphocytes per 3.5 × 10 ⁵ spleen cells (mean ± SEM, P > 0.05, single factor analysis) . (d) The histogram depicts the number of E7-specific IL-4 secreting CD4 ⁺ T lymphocytes per 3.5 × 10 ⁵ spleen cells (mean ± SEM, P < 0.01, single factor variance analysis) . (e) The histogram shows the anti-E7 antibody titers in mice immunized with various DNA vaccines (mean ± SEM, P < 0.01, single factor variance analysis).

The third panel shows the E7-specific immune status of mice immunized with the following various DNA vaccines: CTGF/E7, CTGF/E6, CTGF/E6/E7, CTGF/E6+CTGF/E7. (a) A representation of flow cytometric analysis derived from E6-specific IFN-γ secreting CD8 ⁺ T lymphocytes in each group. (b) Flow cytometric analysis representative of E7-specific IFN-γ secreting CD8 ⁺ T lymphocytes from each group. (c) The histogram depicts the number of E6-specific and/or E7-specific IFN-γ secreting CD8 ⁺ T lymphocytes per 3.5 × 10 ⁵ spleen cells (mean ± SEM, P < 0.01, Single factor variance analysis).

The fourth panel shows the results of in vivo tumor protection experiments in mice. (a) In vivo tumor protection experiments in mice immunized with the following various DNA vaccines: naïve, no insert, E7, CTGF, E7+CTGF, CTGF/E7. (b) In vivo tumor protection experiments in mice immunized with the following various DNA vaccines: native, E7, CTGF/E6, CTGF/E7, CTGF/E6/E7, CTGF/E6+CTGF/E7.

Figure 5 shows the results of an antibody depletion experiment in vivo in mice.

Figure 6 shows the results of in vivo treatment with high therapeutic doses in mice. (a) Representative lung tumor nodules of each group of immunized mice. 1: native (naïve), 2: no insertion sequence, 3: E7, 4: CTGF, 5: CTGF/E7. (b) Number of lung nodules in the immunized mice of each group (mean ± SEM, P < 0.001, single factor variance analysis).

Figure 7 shows flow cytometric analysis of DNA-transfected dendritic cells in inguinal lymph nodes of immunized mice, and E7-specific CD8 ⁺ T cell activation isolated from inguinal lymph nodes of immunized mice. situation. (a) The histogram depicts the percentage of CD11c ⁺ GFP ⁺ mononuclear spheres in the mononuclear sphere population (mean ± SEM, P < 0.001, one-way variance analysis). (b) The histogram depicts the percentage of apoptotic cells in CD11c ⁺ GFP ⁺ cells (mean ± SEM, P < 0.001, one-way variance analysis). (c) the bar graph depicting the percentage per 1 × 10 ⁵ cells in the E7-specific IFN-γ secreting CD8 ⁺ T cells (mean ± SEM, P <0.001, one-way ANOVA analysis).

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<400> 7

<210> 8

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400> 8

<210> 9

<211> 288

<212> DNA

<213> Human papillomavirus 16

<400> 9

<210> 10

<211> 9

<212> PRT

<213> Human papillomavirus 16

<400> 10

<210> 11

<211> 38

<212> PRT

<213> Human papillomavirus 16

<400> 11

<210> 12

<211> 8

<212> PRT

<213> Human papillomavirus 16

<400> 12

Claims (19)

A DNA construct consisting of a expression vector, a connective tissue growth factor (CTGF) coding sequence, and a human papillomavirus (HPV) sequence, which can be expressed in eukaryotic cells; wherein the aforementioned CTGF coding sequence is SEQ ID NO: 3, the aforementioned HPV sequence is one selected from the group consisting of an E6 coding sequence and SEQ ID NO: 9, and the E6 coding sequence is SEQ ID NO: 6. The DNA construct of claim 1, wherein the expression vector is expressed in a human cell. 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 of claim 1; and a particle coated with the DNA construct. The DNA vaccine of claim 4, wherein the aforementioned particles are gold particles. The DNA vaccine according to claim 5, wherein the gold particles have a diameter of 1.6 μm. The DNA vaccine of claim 4, wherein the aforementioned expression vector is expressed in a human cell. The DNA vaccine of claim 5, wherein the expression vector is selected from the group consisting of pcDNA3, pSG5 or pCMV. The DNA vaccine according to claim 8, wherein the expression vector is pcDNA3. A pharmaceutical composition comprising the DNA vaccine of claim 4 of the patent application. The pharmaceutical composition of claim 10, further comprising a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 10, which is for treating a disease caused by HPV. The pharmaceutical composition according to claim 12, which is for treating cervical cancer, lung cancer, oropharyngeal cancer, vaginal cancer, vulvar cancer or penile cancer. The pharmaceutical composition according to claim 13, which is for treating cervical cancer. The pharmaceutical composition according to claim 12, which is for treating a precancerous lesion of the cervix, vulva or vagina. A method for producing a DNA vaccine according to claim 4, which comprises: (1) providing a DNA construct, wherein the DNA constructing system comprises a performance vector which can be expressed in eukaryotic cells. The vector, a connective tissue growth factor (CTGF) coding sequence, and a human papillomavirus (HPV) sequence, wherein the aforementioned CTGF coding sequence is SEQ ID NO: 3, and the aforementioned HPV sequence is an E6 coding sequence and an E7 coding sequence. In one of the above, the E7 coding sequence is SEQ ID NO: 9, the E6 coding sequence is SEQ ID NO: 6; and (2) the DNA construct is coated on the surface of the aforementioned particles. The method of claim 16, wherein the foregoing table The current vector is pcDNA3. The method of claim 16, wherein the particles are gold particles. The method of claim 18, wherein the gold particles have a diameter of 1.6 μm.