KR20100023696A - Vaccine compositions comprising tat-fusion antigen protein and adjuvants - Google Patents

Abstract

PURPOSE: A vaccine composition used as therapeutic agent is provided to enhance T cell activity and inteferon-gamma generation and to enable repetitive and safe administration. CONSTITUTION: A vaccine composition inducing antigen-specific immune reaction contains protein transduction domain(PTD) of HIV TAT(human immunodeficiency virus transactivator protein) of sequence number 1, a fusion protein containing an antigen connected to the amino terminal thereof, a nucleic acid molecule encoding the fusion protein, a recombinant vector containing polynucleotide sequence encoding the fusion protein, and an adjuvant as active ingredients. The adjuvant is single or mixed CpG ODN, and Poly I:C.

Description

Vaccine compositions comprising TAT-fusion Antigen protein and adjuvants

The present invention relates to a vaccine composition, and more particularly, to a fusion protein and an adjuvant, CpG-ODN or poly I: C, which are connected to a protein transduction domain (PTD) and antigen of HIV transactivator protein (TAT). The present invention relates to a vaccine which exhibits enhanced immune effects by combined administration.

Current cancer treatments are mainly performed by surgically removing cancer cellized tissue as much as possible, and then performing radiation therapy and chemotherapy to destroy the remaining cancer cells. However, in the case of surgical operation, there is a wide range of resection, there is a risk of recurrence due to micrometastasis, etc., there are many side effects in the case of radiation therapy and chemotherapy, and each anticancer agent is not effective for all cancers, In the case of the cancer cells exposed to the anticancer drug has a number of problems, such as gaining resistance and continue to grow, and eventually spread to other organs to fall into an untreated state.

Therefore, we cannot but realistically admit that there are limitations to conquer cancer with these therapies alone, and the anticipated treatment of new cancer treatments is immunotherapy using the immune function of our body itself.

Immunotherapy has fewer side effects than other therapies and shows excellent effects when used in combination with other therapies, and is a therapy that has become increasingly important in recent years. Among cancer treatments, surgery, chemotherapy and radiation therapy are those that directly attack cancer cells, while immunotherapy is an indirect method of treating cancer with its own immunity by acting on and activating the immunity of the patient. to be.

There are humoral and cellular immunity in the body's immune function. Humoral immunity is the production of antibodies for the decomposition and removal of antigens when bacteria or viruses enter the body. Cellular immunity, on the other hand, produces cells (lymphocytes) that respond to certain antigens (cancer cells) to act as an immune monitoring mechanism. In immunity against cancer, cellular immunity centered on lymphocytes plays a more important role than humoral immunity. As such, anti-tumor immune responses generally involve cellular immune responses, which are known to play an important role in CD8 + cytotoxic T lymphocytes (CTLs). In order to induce these anti-tumor T cells, research on tumor-associated antigen (TAA) has been continued, and the development of T cell immunotherapy for tumors with the development of recombinant DNA technology has recently been continued. It is becoming.

Dendritic cells have been studied as a powerful cellular adjuvant for tumor vaccines. The reason for using dendritic cells is to present antigens to MHC class I molecules to induce antigen-specific cytotoxic T cells that act specifically on tumor cells. Dendritic cells are powerful antigen-presenting cells. Because it can function. Among the antigen presenting cells used for antigen-specific cytotoxic T cell induction, dendritic cells originate from the bone marrow and are immature at the peripheral site, and then stimulate the T cells whose growth is inhibited by recognizing the antigen. It is known to initiate or modulate the immune response of T cells that are active against. In addition to cytotoxic T cells, dendritic cells express high levels of molecules such as MHC, CD80, CD86, lymphocyte function-associated antigen (LFA) 1 and 3, and I intracellular adhesion molecules (CAMs) 1 and 3, which are involved in immune responses. Therefore, it is preferable as an antigen presenting cell used to induce an anti-tumor immune response.

Although dendritic cells are thought to be the ideal vaccine for immunotherapy, dendritic cells are sparsely present in peripheral blood mononuclear cells (PBMCs) and do not proliferate well in vitro, so the use of dendritic cells for clinical applications There are many limitations. Thus, protein vaccines containing an effective adjuvant, which are simpler, safer and repeatable than conventional vaccines using dendritic cells, may be promising tumor antigen specific immune therapies. We use HIV-1 TAT peptides to move macromolecules into cells, and proteins act as immunogens for the cytotoxic T cell response of major histocompatibility complex (MHC) class I. A novel recombinant fusion protein was prepared so that the peptide-antigen-linked fusion protein vaccine can perform an antigen-specific immune response, and by selecting an adjuvant that maximizes its immune effect, the anti-tumor effect of the dendritic cell vaccine is comparable. The present invention has been completed by confirming that it is possible to provide an antigen-specific immunotherapeutic agent having a simple, safe and repeatable administration.

An object of the present invention is to provide a fusion protein comprising a protein transduction domain (PTD) of HIV transactivator protein (PTD) and an amino terminal thereof which induce an anti-tumor immune response.

Still another object of the present invention is to provide a vaccine composition comprising the fusion protein and an adjuvant.

In order to achieve the object of the present invention, the present invention provides a fusion protein comprising a protein transduction domain (PTD) of the HIV transactivator protein (PTD) and its amino terminus to induce an enhanced immune response do.

According to one embodiment of the present invention, in order to prepare the fusion protein, the amplification of the antigen using a primer in the pET15b plasmid, and cloned using a cleavage enzyme of Xho I and BamH I, cloning the PTD of HIV TAT fusion protein Prepare a recombinant expression vector for expression. The recombinant expression vector is introduced into BL21 competent cell, transformed cell line is cultured in LB ampicillin liquid medium and IPTG (isopropyl-β-thiogal-actoside) is introduced during expression to induce mass production of protein. The fusion protein was purified using the binding of His-Taq and Ni-NTA resin of pET15b, and the expression and size of the protein were confirmed by SDS-PAGE and Western blot. (Fig. 1 (b) and Fig. 6 (b)). )

remind PTD used the 47-57th residue 'YGRKKRRQRRR' (SEQ ID NO: 1). Since these amino acid residues can deliver proteins into cells and also have cation properties, they bind to the adjuvant (CpG, polyIC, etc.) used in the present invention and are concentrated at the site where the adjuvant and antigen are injected without being diluted in vivo. It acts as a potentiating immune response. The antigens linked thereto are all or part of tumor antigens, infectious agents, and the like, and in one embodiment of the present invention, the D1 domain (SEQ ID NO: 2, SEQ ID NO: 9) and service of a carcinoembryonic antigen (CEA) Bin (SURVIVIN) (SEQ ID NO: 3, SEQ ID NO: 10) was used as the antigen, which was amplified using an appropriate primer and cloned into the pET15b plasmid.

Tumor-associated antigens can be used to induce antigen-specific cytotoxic T cells that specifically act on tumor cells, and the carcinoembryonic antigen (CEA) used in one embodiment of the present invention is 180 kDa. It is a cancerous glycoprotein and a soluble tumor marker. Cancer fetal antigens are expressed at least 95% in colorectal cancer, gastric cancer and pancreatic cancer, and are widely expressed in about 50% of breast cancers and 70% of non-small cell lung cancers.

The sequence encoding human cancer fetal antigens has been fully identified ( Thompson JA , Grunert F, Zimmermann W. Carcinoembryonic antigen gene family : molecular biology and clinical perspectives . J Clin Lab Anal 5: 344-66, 1991.), In the present invention, the D1 domain of the fetal antigen of SEQ ID NO: 2 was cloned to prepare a TAT-CEA D1 domain (hereinafter referred to as "TAT-CEA"). The D1 domain of the fetal antigen of SEQ ID NO: 2 used in the present invention is a site that is easy to produce in bacteria while having the antigenicity of the fetal antigen.

Survivin (SURVIVIN) is a component of the IAPs (inhibitor of apoptosis) protein family that inhibits apoptosis and can be fused with HIV TAT in the present invention to induce anti-tumor immune responses in vivo.

In addition, the present inventors observed the antitumor immune response following administration of the TAT-antigen fusion protein alone and in combination with the adjuvant.

 "Adjuvant" refers to a substance that enhances an immune response. The adjuvant of the present invention can be selected from one or more of CpG-ODN, Poly I: C interferon, interleukin or cyclosporin A, etc. In the specific embodiment of the present invention, CpG-ODN and Poly I: C were used.

Cytosine-phosphorothioate-guanine-oligodeoxynucleotides (CpG-ODNs) are non-methylated immunomodulators and adjuvants that contain oligodeoxynucleotides (ODNs) and are like toll like receptors in antigen presenting cells (APCs). It is a substance that induces Th1-type immune response by binding to 9). In addition, CpG-ODN is a substance that secretes various cytokines such as IL-6, IL-12, TNF-α, IFN-γ by activating B cells, NK cells, dentritic cells and the like.

The effect of increasing the antitumor immune effect of TAT-antigen fusion protein according to the combined use of CpG-ODN was examined.

C57BL / 6 mice were injected subcutaneously twice a week at intervals of one week, and then, after one week, the mice were sacrificed to remove spleen and purified T cells were isolated. After re-stimulation of these T cells with MC38 / CEA2 target cells immobilized in advance, the activity of cytotoxic T cells specific to tumors and the degree of IFN-γ production were confirmed. PBS was used as a control, and each experimental group was administered with antibody alone, TAT-antigen fusion protein-administered group, a combination co-administration group of antibody and CpG-ODN, and a combination co-administration group of TAT-antigen fusion protein and CpG-ODN were used. In the case of carcinoembryonic antigen (CEA), carcinoembryonic antigen (CEA) sensitized dendritic cell vaccine was used as a positive control to compare the antitumor immune responses.

As a result, mice vaccinated using a combination of TAT-antigen fusion protein and CpG-ODN were antigen-specific in cytotoxic T lymphocyte activity and interferon- γ- secreting T cells compared to antigen- and CpG-ODN-combined vaccine or TAT-antigen vaccine. The immune response was increased, and it can be seen that the immune activity was increased by 60 to 90 times or more than the immune response using only the TAT-antigen fusion protein. In addition, the combination of the TAT-antigen fusion protein and the CpG-ODN mixed vaccine induced a Th1 type immune response, resulting in a high ratio of antigen-specific IgG2a / IgG1 antibodies. In the tumor model using MC38 / CEA2, the survival rate of TAT-antigen fusion protein and CpG-ODN concomitant vaccine was increased. Responded. These results show that the use of CpG-ODN adjuvant in combination with the TAT-antigen fusion protein can be a potent agent for increasing antitumor effects.

The antitumor vaccine composition according to the present invention exhibits antitumor immune effects by increasing cytotoxic T cell activity, increasing interferon-γ (IFN-γ) production, and inducing Th1 type immune responses, and conventional dendritic cells. It can be used as a promising vaccine treatment because it is inexpensive, simple, safe and can be administered repeatedly compared to the vaccine using.

Hereinafter, preferred examples and experimental examples are presented to help understand the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the Examples.

<Example 1>

1-1. Cell Lines and Laboratory Animals

Preparation of all cell lines and experimental animals used in the examples below is as follows.

Six to eight weeks female C57BL / 6 (H-2Kb) mice were purchased from Orient (Gapyeong, Gyeonggi, Korea). The MC38 / CEA2 adenocarcinoma cell line was obtained from Dr J. Schlom (Oncology and Biology, NIH, USA). Mouse colon adenocarcinoma cells expressing human CEA (MC38 / CEA2) were prepared by transforming MC38 cells with RNA tumor virus transfection with CEA cDNA. MC38 / CEA2 cells, MC38 cells and GL26 cells were completed DMEM (Dulbecco ') containing 10% heat inactivated FBS (fetal bovine serum), 100 U / ml penicillin, and 100 μg / ml streptomycin and 2 mM L-glutamine. s Modified Eagle Medium; BioWhittaker, USA).

To enhance the expression of major histocompatibility complex (MHC) I, MC38 / CEA2 cells were treated with 50 IU / mL of murine interferon (IFN) -γ; Endogen prior to cytotoxicity measurements of restimulation and cytotoxic T cells. Inc., USA) for 48 hours at 37 ° C.

1-2. Adjuvant CpG - ODN

Sequence 1826 (5′-TCC ATG ACG TTC CTG ACG TT-3 ′ ( Hso- Chi Chaung . CpG) oligodeoxynucleotides as DNA adjuvants in vertebrates and their applications in immunotherapy. international immunopharmacology 6: 1586-1596,2006) have adjuvant activity with protein antigens. It was purchased from CpG-ODN (Bioneer, Korea) and synthesized as a nuclease-resistant (phosphothioate backbone). CpG-ODN is water soluble and has undetectable intracellular toxicity.

1-3. Through in vitro transcription CEA RNA  produce

CEA-pcDNA3 vector was linearized cleaved with Sma I, mMessage in mMachine ™ Ultra T7 kit (Ambion Corporation, USA) in vitro transcription as directed by the manufacturer (in in vitro transcription). mRNA concentration and quality were measured using spectrophotometer and agarose gel electrophoresis. RNA samples were periodically checked for size and integrity using formaldehyde / agarose gel electrophoresis and stored at 70 ° C.

1-4. CEA RNA Introduced Dendritic  Ready

Dendritic cells were expressed by Kim CH (Kim CH, et. al ., Cancer Immunol Immunother 2006; 55 : 130919) and the like. The mouse bone marrow was prepared using 20 ng / mL of recombinant recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) and 20 ng / mL of IL-4 (manufactured by R & D Systems, USA). Before 7 days of electroporation, immature dendritic cells were washed twice with serum-free medium and resuspended to a final concentration of 1 × 10 ⁷ cells / mL. 200 μL of cell suspension was preincubated in a 0.2-cm hole electroporation cuvette for 5 minutes on ice. Next, 20 μg of RNA was added, and the cells were subjected to electromagnetic waves with an ECM 830 electroporator (BTX, USA) at a voltage of 300 V and a time interval of 50 μs. After electroporation, the cells were immediately transferred to maturation medium and incubated at 37 ° C for 24 hours. After electroporation, the cells showed more than 80% cell activity. Transfection efficiency according to electroporation in dendritic cells was determined by enhanced green fluorescent protein (EGFP) RNA using fluorescenceactivated cell sorting (FACS) analysis.

Example 2 Preparation of TAT-CEA Fusion Protein

2-1. TAT - CEA Fusion protein  Preparation of Expression Vectors

CEA cDNA is a CEA domain 1 (A1-B1) sense primer containing Xho I site 5′-ACG CCA TAT GCT GCT GAT CCA GAA CA-3 ′ (SEQ ID NO: 4) and CEA domain 1 antisense comprising BamH I site Amplified from LoVo cell line by RT-PCR (reverse transcriptase polymerase chain reaction) using primer 5′-ATA TGG ATC CCG TCG TGA CTG TGG TCC T-3 ′ (SEQ ID NO: 5). The obtained RT-PCR product was digested with Xho I and BamH I, and the cleaved DNA fragments were gel separated.

CEA DNA fragments were cloned into the Xho I and BamH I sites of the pET-15b vector (manufactured by Novagen, USA). This plasmid is called Escherichia coli Was introduced into DH5α (Real Biotech Corp. Taiwan) and screened for ampicillin. The intracellular adhesion domain of HIV-1 TAT was constructed with plasmid pNL4-3 as a template, and a small TAT peptide, 'YGRKKRRQRRR', was produced by PCR at Nde I and Xho I restriction sites at 5 'and 3' ends. The fragment was digested with Nde I and Xho I and subcloned into the corresponding restriction enzyme site of pET15b-CEA domain 1. As a result, a plasmid pET15b-TAT-CEA domain 1 vector of the cleavage map shown in FIG. 1A was prepared, and the vector was purified, introduced into BL21 cells, and the final 100 μg / mL concentration of LB containing ampicillin. (Luria-Bertani) medium was incubated.

2-2. TAT - CEA Fusion protein  production

The BL21 strain of E. coli can express genes present in an expression vector comprising a bacteriophage T7 promoter. BL21 cells transformed with the pET-15b plasmid of Example 2-1 induced protein expression with 1.5 mmol of IPTG (isopropyl β-D thiogalactoside) and incubated for 3 hours. Cells were collected and sonicated. Sonicated cell supernatant RT (room temperature) Buffer Z and 10 mmol pre-balanced with imidazole ^{(imidazole) (Ni 2 + (} Ni-NTA superflow in; applied Qiagen Corporation, USA) was isolated by gravity Next, a refolding buffer was added, gradually decreasing the concentration of urea from 6.0 to 1 mol, and increasing the concentration of imidazole.The final product was dialyzed overnight to remove the remaining base. (overnight dialysis) was performed with 0.1 mol urea and 1 mol of imidazole.

For western blot analysis, purified proteins were separated using 12.5% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then transferred to nitrocellulose membrane. Membranes were incubated with 5% nonfat milk in phosphate buffered saline (PBS) and then incubated with anti-His monoclonal antibody (mAb) overnight at 4 ° C. After washing, the membrane was incubated with alkaline phosphatase-conjugated Gout anti-mouse IgG antibody (Amersham Biosciences, UK) for 1 hour at room temperature. Immune response bands were detected using the ECL Western Blotting Assay System (Amersham Biosciences, UK) (FIG. 1B).

Example 3 Measurement of Cytotoxic T Cell Activity

In vivo (in For the induction of the initial cytotoxic T cells in vivo), of 50 μg to (syngeneic) rats Group of Winter CEA protein [CEA], of the 50 μg CEA and 2 μmol of CpG-ODN [CEA + CpG] , 50 μg TAT-CEA protein [TAT-CEA] except CpG-ODN was administered and PBS [PBS] was administered as a control. Vaccination of dendritic cells [CEA peptide / DC] containing CEA peptide was performed according to the method used by Oh ST et al. (Oh ST et. al . Vaccine 2006; 24 : 28608). On day 0 of the first dose and on day 7 the mice were vaccinated in the same manner as above. Seven days after the second vaccine administration on day 7, the mice were killed to obtain splenocytes, homogenized, and red blood cells (RBCs) were lysed with low osmotic buffer.

Non-adhesive splenocytes were separated from dendritic cells, macrophages and monocytes through adhesion to plastic for 90 minutes and used as effector cells.

Intermediate killing measurements of target cells were performed in vitro using standard 4 hour chromium assays with various effector to target cell ratios.

Splenocytes (2 × 10 ⁷ ) were re-stimulated for 5 days with 4% paraformaldehyde and immobilized MC38 / CEA2 cells in the presence of 10 IU / mL of interleukin (IL) -2. MC38 / CEA2, MC38, and GL26 tumor cells were labeled for 1 hour in 3.7 mBq of [51Cr] sodium chromate / 10 ⁶ cells, washed five times, and multiplied by four times the bottom microtiter plates, varying the percentage of effector cells. Was added to the wells. The ratio of effector cells (E) and target cells (T) (E: T) was tested at 10: 1, 20: 1, and 40: 1, respectively. After 4 hours of incubation at 37 ° C., 100 μL of supernatant was collected in each well and radioactivity was measured using a gamma counter. Ratio of the specific dissolution Kim CH, et al. (Kim CH , et al ., Cancer Immunol Immunother 2006; 55: 130919) and the like.

Combination of TAT-CEA with CpG and cytotoxicity experiments on syngeneic MC38 / CEA2 target cells in the spleen of mice immunized with CEA peptide / DC, as shown in FIG. 2B, immunization of different control groups Significantly higher than the cytotoxicity of the rats. This cytotoxic T cell (CTL) activity is CEA specific in that it does not show cytotoxic activity against CEA-free MC38 or irrelevant GL26, as shown in FIGS. 2C and 2D. In other words, it was confirmed that the combination of TAT-CEA and CpG-ODN in combination with the present vaccine increased tumor specific cytotoxic T cell (CTL) response in vivo. Cell killing ability was not observed in effect cells vaccinated into the control group. In addition, effector cells did not lyse YAC-1 target cells, meaning that the killer activity did not pass through NK-cells. (data not shown).

<Example 4> Measurement of IFN-γ production by ELISPOT (Enzyme-linked immunospot) assay.

ELISPOT kit (manufactured by AID, Germany) was used according to the manufacturer's method. Briefly, pre-fixed MC38 / CEA2 cells and re-stimulated splenocytes for 5 days were coated with anti-mouse IFN-γ antibody at a concentration of 1 × 10 ⁵ cells / well in cell medium. Seeds in 96-well microplates. Stimulated with CEA RNA-electroporated dendritic cells (5 × 10 4 cells / well) for 18 hours. Plates were incubated at 37 ° for 24 hours. After the spot appeared the reaction was stopped with distilled water and the plate was dried overnight in the dark. The number of antigen-specific (tumor specific) cytotoxic T cells producing IFN-γ was determined using an automatic AID-ELISPOT-Reader.

Figure 3 shows the average number of IFN-γ per 1 × 10 ⁵ splenocytes, splenocytes of mice immunized with a combination vaccine of TAT-CEA and CpG-ODN and CEA peptide / dendritic cells IFN-γ The number of secretory T cells can be seen to increase significantly. In contrast, other experimental and control groups do not produce IFN-γ significantly. These results show that immunized mice with a combination of TAT-CEA and CpG-ODN increase the CEA specific immune response.

Example 5 Immunoglobulins in Rats Isotyping

Mouse Immunoglobulin Screening / Isotyping Kit (Zymed Laboratories, Invitrogen Immunodetection) was used to test for specific immunoglobulin subclass antibodies. Briefly, 96-well flat bottom microplates were coated with 500 ng / well of CEA protein, diluted with PBS and incubated overnight at 4 ° C. Plates were blocked with 200 μL PBS containing 1% bovine serum albumin (BSA) at 37 ° C. for 1 hour.

Serum diluted 1:10 in 1% PBS was added to each well 50 μL and incubated at 37 ° C. for 30 minutes. Plates were placed in a blank (reference) with 50 μL of PBS, a negative control of the biotinylated antibody included in the kit and IgG1, IgG2a, IgG2b, IgG3, Ig A, And a drop of antibodies of IgM were incubated at room temperature for 15 minutes and further incubated with horseradish peroxidase (HRP) treptavidin for 15 minutes at room temperature.

Finally, 100 μL of working substrate solution containing ABTS (2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) and hydrogen was added to the citrate buffer provided in the kit. Measurement was performed at 405 nm using an ELISA reader (manufactured by Molecular Devices, USA).

The present inventors measured the amount of IgG1 and IgG2a, and as shown in FIG. 4A, it was confirmed that the amount of IgG2a was significantly higher in the serum administered with the combination of TAT-CEA and CpG-ODN than the other groups. Mice vaccinated with a combination of TAT-CEA and CpG-ODN showed higher IgG2a / IgG1 ratios than the other groups (FIG. 4B). Since IgG1 is a Th2-associated antibody and IgG2a is a Th1-associated antibody, the experimental results shown in FIGS. 4A and 4B show a combination of TAT-CEA and CpG-ODN in vivo through the ratio of Th1 and Th2 cells. Means through the antigen-specific antibody response control effect. In conclusion, CpG-ODN enhances the Th1 type of immune response induced by TAT-CEA.

Example 6 Antitumor Immune Effect Measurement

Since the combined co-administration of TAT-CEA and CpG-ODN induces tumor specific cytotoxic T cells, the present inventors examined the antitumor immune effects following vaccination. Mice were injected with 1 × 10 ⁶ MC38 / CEA2 tumor cells. Seven rats were used for each group. On the 10th day of tumor cell inoculation, the rats were treated with TAT-CEA and CpG-ODN combination vaccines, CEA peptide / DC, CEA, TAT-CEA, and PBS at weekly intervals for 5 weeks. (FIG. 5A), the tumor volume and survival rate are shown in FIGS. 5B and 5C, respectively. When the tumor volume and survival rate were observed, the tumors in the TAT-CEA and CpG-ODN combination vaccine administration group As the volume is reduced the most, the combination of TAT-CEA and CpG-ODN combination vaccines inhibits tumor growth. As the survival time increases, the survival rate is excellent due to the antitumor effect.

Example 7 Preparation of TAT-Servivin (SURVIVIN) Fusion Protein

7-1. TAT - Survivin ( SURVIVIN ) Fusion protein  Preparation of Expression Vectors

The TAT-Servibin (SURVIVIN) fusion protein expression vector was constructed in the same manner as described in Example 2-1. Service blank (SURVIVIN) cDNA are services including a Xho I site blank (SURVIVIN) sense primer 5'- CGAACTCGAGATGGGTGCCCCGACGTTG-3 '(SEQ ID NO: 6) and service blank (SURVIVIN) an antisense primer containing a BamH I site 5'- GCAAGGATCCTCAATCCATGGCAGCCAG RT-PCR (reverse transcriptase polymerase chain reaction) using 3 ′ (SEQ ID NO: 7) Amplification from LoVo cell line. The obtained RT-PCR product was digested with Xho I and BamH I, and the cleaved DNA fragments were gel separated.

SURVIVIN fragments were cloned into the Xho I and BamH I sites of the pET-15b vector (manufactured by Novagen, USA). This plasmid is called Escherichia coli DH5α (Real Biotech Corp. Taiwan) was introduced and selected with ampicillin. The intracellular adhesion domain of HIV-1 TAT was constructed with plasmid pNL4-3 as a template, and a small TAT peptide, 'YGRKKRRQRRR', was produced by PCR at Nde I and Xho I restriction sites at 5 'and 3' ends. The fragment was digested with Nde I and Xho I and subcloned into the corresponding restriction enzyme site of pET15b-Servibin (SURVIVIN) to prepare a plasmid pET15b-TAT-Servibin (SURVIVIN) vector, which was purified. It was again introduced into BL21 cells and cultured in LB (Luria-Bertani) medium containing ampicillin at a final 100 μg / mL concentration.

7-2. TAT - Survivin ( SURVIVIN ) Fusion protein  production

The BL21 strain of E. coli can express genes present in an expression vector comprising a bacteriophage T7 promoter. BL21 cells transformed with the pET-15b plasmid of Example 7-1 induced protein expression with 1.5 mmol of IPTG (isopropyl β-D thiogalactoside) and incubated for 3 hours. Cells were collected and sonicated. The sonicated cell supernatants were separated by gravity by applying Ni2 + (Ni-NTA superflow; manufactured by Qiagen, USA) pre-balanced with Buffer Z and 10 mmol imidazole at room temperature. Gradually, the concentration of urea was reduced from 6.0 to 1 mol and a refolding buffer was added with increasing concentration of imidazole.The final product was overnight to remove residual base. dialysis) was performed and obtained with 0.1 mol urea and 1 mol of imidazole.

For western blot analysis, purified proteins were separated using 12.5% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then transferred to nitrocellulose membrane. Membranes were incubated with 5% nonfat milk in phosphate buffered saline (PBS) and then incubated with anti-His monoclonal antibody (mAb) overnight at 4 ° C. After washing, the membrane was incubated with alkaline phosphatase-conjugated Gout anti-mouse IgG antibody (Amersham Biosciences, UK) for 1 hour at room temperature. Immune response bands were detected using an ECL Western blotting assay system (Amersham Biosciences, UK).

Example 8 Induction and Identification of Cytotoxic T Cells by Combination Administration of TAT-Survivin (SURVIVIN) Fusion Protein and CpG-ODN

In vivo (in vivo) cytotoxic T cells to the induction of the first, (syngeneic) CpG-ODN in mice 50 μg of empty TAT- services (SURVIVIN) protein and 2 μmol of copper were vaccinated in. The rat group consisted of 50 μg of SURVIVIN protein [CEA], 50 μg of Survivin (SURVIVIN) and 2 μmol of CpG ODN [CEA + CpG] and 50 μg of CpG-ODN (SURVIVIN). ), Or PBS was vaccinated. Vaccination with dendritic cells containing SURVIVIN peptide (SURVIVIN peptide / DC) is as previously described. At day 0 of the first dose and on day 7 the mice were dosed with increased vaccination in the same manner as above. After 7 days of increased vaccine administration on day 7, rats were killed and spleens were removed, and purified T cells were isolated using nylon mesh. These T cells were re-stimulated in the incubator with MC38 / CEA2 target cells fixed in advance. The response of cytotoxic T cells was confirmed by 51Cr-release assay and IFN-γ ELISPOT assay. As shown in FIG. 7, the IFN-γ-expressing lymphocyte frequency was induced by the combined combination of TAT-Servinbin (SURVIVIN) and CpG rather than PBS or TAT-Servinbin (SURVIVIN) -induced effect cells. A large number of spots were observed to develop red color in effect cells.

In addition, the combination of TAT-CEA protein and Poly I: C administration showed antitumor response similar to that of CpG ODN in combination, and it was shown as a result of measuring the cell killing ability of CEA-specific T cells.

The effector cells of the experimental mice induced by co-administration of TAT-CEA and poly I: C were similar to the positive control Tat-CEA + CpG, with respect to MC38CEA2 target cells. Strong killing activity. In addition, NK-sensitive YAC-1 cells did not show killing activity by these lymphocytes. CTL was not induced in lymphocytes of control group induced by other controls.

Statistical analysis

The results are expressed as mean ± standard deviation. Statistical analysis was performed using ANOVA test. Survival data were analyzed using the KaplanMeier test and compared using the log-rank test. The data are statistically significant at P <0.05.

The vaccine composition according to the present invention exhibits a vaccine immune effect by increasing cytotoxic T cell activity, increasing interferon-γ (IFN-γ) production, inducing a Th1 type immune response, and using conventional dendritic cells. Compared to the vaccine, it can be used as a powerful antigen-specific immunotherapy because it is simpler, safer and can be repeatedly administered.

1 is a schematic diagram of cloning of TAT and CEA Domain 1 and expression of TAT-CEA fusion protein using western blot according to an embodiment of the present invention.

Figure 2 shows the effect of cytotoxic T cells specific for tumors, Figure 2 (a) shows a vaccine scheme for cytotoxicity test, (b) to (d) is a target cell expressing CEA, respectively The activity of cytotoxic T cells in MC38 / CEA2 (b) and target cells MC38 (c), GL26 (d) that do not express CEA is shown.

Figure 3 is a measure of the interferon-γ production of immunized splenocytes, re-stimulated immunized splenocytes when cultured with dendritic cells injected with CEA RNA by electroporation method of 1 × 10 ⁵ using the ELISPOT kit As a result of measuring the average number of IFN-γ per splenocytes, splenocytes immunized with a combination vaccine of TAT-CEA and CpG-ODN and CEA peptide / dendritic cells were found to have a higher number of IFN-γ-secreting T cells. Markedly increased.

Figure 4 shows the effect of vaccination on immunoglobulin, Figure 4 (a) shows the result of measuring the antibody of IgG1 and IgG2a in the plasma of the mouse using ELISA a week after two vaccinations, Figure 4 (b) shows the ratio of IgG2a / IgG1 of each group.

Figure 5 shows the antitumor effect following vaccination, (a) shows the schedule of vaccine administration, (b), (c) shows the tumor volume and survival rate following vaccination respectively.

Figure 6 shows the cloning overview of TAT and survivin (SURVIVIN) according to an embodiment of the present invention and the expression of TAT-survivin (SURVIVIN) fusion protein using western blot. In Figure 6 (b) lane 1 is an extract of E. coli strain BL21 containing pET, lane 2 is pET-nSVV (SURVIVIN), lane 3 is IPTG-induced pET-tatSVV, lane 4 Recombinant nSVV is shown, and lane 5 represents tatSVV separated with Ni-NTA resin.

Figure 7 shows IFN-γ production of cytotoxic T cells using IFN-γ ELISPOT assay according to the combination of TAT-Survivin (SURVIVIN) fusion protein and TAT-Survivin (SURVIVIN) fusion protein and CpG-ODN combination vaccine. do.

8 shows the results of measuring cell killing ability of CEA-specific T cells.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> VACCINE COMPOSITIONS COMPRISING TAT-FUSION ANTIGEN PROTEIN AND          ADJUVANTS <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 11 <212> PRT <213> HUMAN IMMUNO DEFICIENCY VIRUS 1 TAT PTD <400> 1 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg   1 5 10 <210> 2 <211> 209 <212> PRT <213> CEA D1 DOMAIN <400> 2 Leu Leu Ile Gln Asn Ile Ile Gln Asn Asp Thr Gly Phe Tyr Thr Leu   1 5 10 15 His Val Ile Lys Ser Asp Leu Val Asn Glu Glu Ala Thr Gly Gln Phe              20 25 30 Arg Val Tyr Pro Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser          35 40 45 Lys Pro Val Glu Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu      50 55 60 Thr Gln Asp Ala Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser Leu Pro  65 70 75 80 Val Ser Pro Arg Leu Gln Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu                  85 90 95 Phe Asn Val Thr Arg Asn Asp Thr Ala Ser Tyr Lys Cys Glu Thr Gln             100 105 110 Asn Pro Val Ser Ala Arg Arg Ser Asp Ser Val Ile Leu Asn Val Leu         115 120 125 Tyr Gly Pro Asp Ala Pro Thr Ile Ser Pro Leu Asn Thr Ser Tyr Arg     130 135 140 Ser Gly Glu Asn Leu Asn Leu Ser Cys His Ala Ala Ser Asn Pro Pro 145 150 155 160 Ala Gln Tyr Ser Trp Phe Val Asn Gly Thr Phe Gln Gln Ser Thr Gln                 165 170 175 Glu Leu Phe Ile Pro Asn Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr             180 185 190 Cys Gln Ala His Asn Ser Asp Thr Gly Leu Asn Arg Thr Thr Val Thr         195 200 205 Thr     <210> 3 <211> 142 <212> PRT <213> HUMAN SURVIVIN <400> 3 Met Gly Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp   1 5 10 15 His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Cys Ala              20 25 30 Cys Thr Pro Glu Arg Met Ala Glu Ala Gly Phe Ile His Cys Pro Thr          35 40 45 Glu Asn Glu Pro Asp Leu Ala Gln Cys Phe Phe Cys Phe Lys Glu Leu      50 55 60 Glu Gly Trp Glu Pro Asp Asp Asp Pro Ile Glu Glu His Lys Lys His  65 70 75 80 Ser Ser Gly Cys Ala Phe Leu Ser Val Lys Lys Gln Phe Glu Glu Leu                  85 90 95 Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys Asn Lys             100 105 110 Ile Ala Lys Glu Thr Asn Asn Lys Lys Lys Glu Phe Glu Glu Thr Ala         115 120 125 Lys Lys Val Arg Arg Ala Ile Glu Gln Leu Ala Ala Met Asp     130 135 140 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> SENSE PRIMER FOR CEA D1 <400> 4 acgccatatg ctgctgatcc agaaca 26 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> ANTISENSE PRIMER FOR CEA D1 <400> 5 atatggatcc cgtcgtgact gtggtcct 28 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> SENSE PRIMER FOR SURVIVIN <400> 6 cgaactcgag atgggtgccc cgacgttg 28 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> ANTISENSE PRIMER FOR SURVIVIN <400> 7 gcaaggatcc tcaatccatg gcagccag 28 <210> 8 <211> 20 <212> DNA <213> CPG-ODN SEQUENCE 1826 <400> 8 tccatgacgt tcctgacgtt 20 <210> 9 <211> 627 <212> DNA <213> HUMAN CEA DOMAIN 1 <400> 9 ctgctgatcc agaacatcat ccagaatgac acaggattct acaccctaca cgtcataaag 60 tcagatcttg tgaatgaaga agcaactggc cagttccggg tatacccgga gctgcccaag 120 ccctccatct ccagcaacaa ctccaaaccc gtggaggaca aggatgctgt ggccttcacc 180 tgtgaacctg agactcagga cgcaacctac ctgtggtggg taaacaatca gagcctcccg 240 gtcagtccca ggctgcagct gtccaatggc aacaggaccc tcactctatt caatgtcaca 300 agaaatgaca cagcaagcta caaatgtgaa acccagaacc cagtgagtgc caggcgcagt 360 gattcagtca tcctgaatgt cctctatggc ccggatgccc ccaccatttc ccctctaaac 420 acatcttaca gatcagggga aaatctgaac ctctcctgcc acgcagcctc taacccacct 480 gcacagtact cttggtttgt caatgggact ttccagcaat ccacccaaga gctctttatc 540 cccaacatca ctgtgaataa tagtggatcc tatacgtgcc aagcccataa ctcagacact 600 ggcctcaata ggaccacagt cacgacg 627 <210> 10 <211> 429 <212> DNA <213> HUMAN SURVIVIN <400> 10 atgggtgccc cgacgttgcc ccctgcctgg cagccctttc tcaaggacca ccgcatctct 60 acattcaaga actggccctt cttggagggc tgcgcctgca ccccggagcg gatggccgag 120 gctggcttca tccactgccc cactgagaac gagccagact tggcccagtg tttcttctgc 180 ttcaaggagc tggaaggctg ggagccagat gacgacccca tagaggaaca taaaaagcat 240 tcgtccggtt gcgctttcct ttctgtcaag aagcagtttg aagaattaac ccttggtgaa 300 tttttgaaac tggacagaga aagagccaag aacaaaattg caaaggaaac caacaataag 360 aagaaagaat ttgaggaaac tgcgaagaaa gtgcgccgtg ccatcgagca gctggctgcc 420 atggattga 429  

Claims (9)

As a vaccine composition capable of inducing an antigen specific immune response, (a) a fusion protein comprising a protein transduction domain (PTD) of the HIV transactivator protein (TAT) of SEQ ID NO: 1 and an antigen linked to an amino terminus thereof, (b) a nucleic acid molecule encoding the fusion protein, or (c) a recombinant vector comprising a polynucleotide sequence encoding said fusion protein; And A vaccine composition comprising the adjuvant as an active ingredient. The vaccine composition of claim 1, wherein the antigen is all or part of a cancer fetal antigen (CEA). The vaccine composition of claim 1, wherein the antigen is a cancer fetal antigen (CEA) D1 domain as set forth in SEQ ID NO: 2. The vaccine composition of claim 1, wherein the antigen is SURVIVIN as set forth in SEQ ID NO: 3. The vaccine composition of claim 1, wherein the recombinant vector has a cleavage map of FIG. 1. The vaccine composition of claim 1, wherein the recombinant vector has a cleavage map of FIG. 6. The vaccine composition of claim 1, wherein the adjuvant is CpG ODN, Poly I: C alone or in combination. 8. The vaccine composition of claim 1, wherein said vaccine composition increases cytotoxic T cell activity and increases interferon-γ (IFN-γ) production. 9. 8. The vaccine composition of claim 1, wherein the vaccine composition induces an Th1 type immune response. 9.

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