KR20110128008A - Method for identifying novel tumor antigen by using rna-transduced antigen presenting cells - Google Patents

Abstract

PURPOSE: A method for identifying a novel tumor antigen using RNA-transduced antigen presenting cells is provided to develop a vaccine for cancer. CONSTITUTION: A method for identifying tumor antigens comprises: a step of preparing animal-derived candidate tumor antigen gene-transduced antigen presenting cell vaccine; a step of confirming immune response and anti-tumor effect of the vaccine; a step of identifying human candidate tumor antigen having sequence homology with anti-tumorous tumor antigen; and a step of detecting an apitope of the tumor antigen. The candidate tumor antigen gene contains a base sequence of sequence number 3. The antigen presenting cell vaccine is manufactured by transferring candidate tumor antigen gene RNA to a cell having an antigen presenting ability.

Description

Method for identifying novel tumor antigen by using RNA-transduced antigen presenting cells

The present invention relates to a method for the identification of new tumor antigens using RNA transfer antigen presenting cells, and more particularly, to an improved aspect using an antigen-presenting cell vaccine to which candidate tumor antigen genes which have not been identified as animal tumor antigens have been transferred. The present invention relates to a method for identifying novel tumor antigens using RNA transfer antigen presenting cells that can effectively detect human tumor antigens that can be targeted for cellular immunotherapy through an epidemiological approach.

Currently, cancer is one of the biggest diseases of mankind along with heart disease, and despite the development of medicine, the cause of cancer is still not clearly known, and the treatment method is not clear. The three major cancer treatments currently in use include surgery, radiation therapy, and chemotherapy. Each treatment method has limitations depending on the nature of the cancer. The situation is accompanied by severe side effects and pain in the patient.

Most recently, new attempts to treat cancer using immunological methods have been actively conducted mainly through animal experiments, collectively called cancer vaccines or immunological treatments. The cancer vaccine, called the fourth cancer therapy, has been suggested as a promising treatment by inducing killing T cells, which are immune response-specific cells to tumor-specific antigens, to activate immune functions in vivo and attack and remove cancer cells.

The most important focus of cancer vaccine development is to produce antigen-specific T cell responses. Cancer cells have several escape mechanisms, such as secreting immunosuppressive substances and suppressing the expression of costimulatory factors to escape the body's immune surveillance system. The purpose of the present invention is to inhibit specific activation of tumor cells by killer cells of human body including cytotoxic T cells. Thus, the success of immunotherapy for cancer depends on how certain tumor-specific antigens are effectively recognized by the body's immune system.

Clinically successful specific tumor immunotherapy requires the discovery of a number of new 'tumor antigens' that can effectively induce antitumor effects among proteins expressed in cancer cells.

The concept of classical genetics is to identify related genetic factors based on observed phenotypes. The conventional method of identifying tumor antigens is based on a cellular immune response. By identifying the T-cell clones isolated, the peptides bound to them are isolated and purified, and the sequences are analyzed to identify the proteins from which the peptides are derived. Second, a method called 'serologic analysis of recombinant cDNA expression libraries (SEREX)' is a method for identifying tumor antigens expressed in cancer cells using antigen antibodies using antibodies obtained from the serum of tumor patients. However, tumor-specific T cell clones are difficult to secure and maintain, and the complexity of identifying tumor antigens with T-cell clones requires a lot of time, resulting in low efficiency. In addition, the discovery of tumor antigens using antibodies to tumor antigens has already been studied, and the discovery of new tumor antigens using antibodies has reached the limit.

Due to the recent development of periphery, the identification of tumor antigens is made through a 'reverse immunology' approach. An immunological approach has been made possible by the development of bioinformatics and the development of sophisticated immunological analytical techniques. Advances in bioinformatics allow gene expression profiling to detect overexpressed genes compared to normal and tumor cells, and candidate tumor antigen peptides synthesized from antigenic determinants predicted based on HLA-binding motifs. I was identified.

In addition, advances in immunological techniques have enabled peptide-specific T cells to be identified as tumor antigens by quantitative and qualitative analysis at the cellular level using ELISPOT and tetramer technology.

However, the limitations of tumor antigen identification by recent immuno-immunological trials are 1) complex predicting peptides and synthesis and purification of peptides require a lot of time and cost. There is a difficulty in inducing a reaction. Therefore, there is a problem in screening candidate tumor antigen genes effectively and quickly.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for identifying tumor antigens, which can effectively discover new tumor antigens that can be targeted for cellular immunotherapy by improving the immunological method.

In order to achieve the above object, the present invention

Preparing an antigen-presenting cell vaccine to which candidate tumor antigen genes derived from animals other than humans have been transferred;

Identifying the immune response and antitumor effect of the vaccine;

Identifying a human candidate tumor antigen having sequence homology with said antitumor tumor antigen; And

It provides a method for identifying a tumor antigen comprising the step of identifying the epitope of the tumor antigen.

The present invention provides a tumor-specific T that is most important for protective tumor immune response by rapidly searching for new tumor antigen genes or tumor related gene products of known functions and characteristics through improved immuno-immunological approaches. It is expected to be a leading platform technology to establish a system for discovering new tumor antigens that target cellular immune responses.

In addition, the present invention can develop cancer vaccines or adoptive T cell immunotherapy by discovering tumor antigens as immunological targets that are widely available in most tumors.

1 shows the expression of Top2α in the tissues of various tumor cell lines and normal mice.
Figure 2 shows the T cell response specific for Top2α and survivin in mice inoculated with dendritic cells loaded with MC38 cell line lysate.
3 shows the induction of Top2α-specific immune responses by Top2αC RNA / DC inoculation.
4 depicts the activity of cytotoxic T cells on Top2α expressing cells.
5 to 7 show antitumor effects by Top2αC RNA / DC inoculation in mouse tumor models.
8 shows the effect of the absence of CD4 and CD8 T cells in the MC38 tumor model inoculated with Top2αC RNA / DC.
Figure 9 shows the production of IFN-γ by the peptide predicted in the Top2αC RNA / DC inoculated mice and the anti-tumor effect by dendritic cells loaded with p1327 peptide.
10 is a schematic representation of a target tumor antigen discovery system for cellular immune treatment via an immunoimmunological approach of the present invention.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present invention

Preparing an antigen-presenting cell vaccine to which candidate tumor antigen genes derived from animals other than humans have been transferred;

Identifying the immune response and antitumor effect of the vaccine;

Identifying a human candidate tumor antigen having sequence homology with said antitumor tumor antigen; And

It relates to a method for identifying a tumor antigen comprising the step of identifying the epitope of the tumor antigen.

Referring to the method of identification of tumor antigen of the present invention in detail as follows.

The first step is to prepare antigen-presenting cells loaded with candidate tumor antigen genes derived from animals other than humans.

In the present specification, "loading or pulsing" refers to an antigen presenting cell (APC), such as dendritic cells, that captures and degrades an antigen and loads it on an MHC molecule to express it on the surface. Antigen loaded cells induce the activity of potent antigen specific T lymphocytes.

The candidate tumor antigen gene may be a gene that is specifically expressed in tumor cell lines derived from animals other than humans.

Thus, the tumor antigen may include, but is not particularly limited to, a gene encoding a fragment of the Topoisomerase II alpha full length or C-terminus.

The fragment derived from the C-terminal may include the nucleotide sequence set forth in SEQ ID NO: 3 derived from a mouse. In addition, a variant sequence in which one or more bases are substituted, deleted or added to the base sequence may be used, but is not particularly limited thereto.

The candidate tumor antigen gene may be in the form of DNA or RNA. Preferably in the form of RNA.

The antigen-presenting cell vaccine can be prepared by delivering candidate tumor antigen gene RNA to a cell having antigen-presenting ability.

The antigen-presenting cells are cells expressing MHC class I or II antigens or HLA antigens capable of presenting tumor antigens on the cell surface, for example, dendritic cells, monocytes, CD4 T cells, B cells or gamma delta T cells can be used. The CD4 T cells, B cells, or γδ T cells may be used in a naive state, an activated state, or an amplified state. More preferably, dendritic cells can be used.

The dendritic cells express about 50 times higher levels of MHC molecules than macrophages, providing more peptides / MHC ligands for the employment of T receptors, and also providing fixed and costimulatory molecules that are critical for T cell activation. It is expressed at very high levels. Genes encoding T cell specific chemokines can also add strong activity of dendritic cells to induce T cell responses, resulting in effective tumor specific T cells.

Such antigen-presenting cell vaccines are, for example,

Separating lymphocytes from myeloid cells;

Mounting the gene RNA encoding candidate tumor antigens derived from animals other than humans into the cells; And

It may be prepared by the step of maturing the cells.

More specifically,

(a) adding GM-CSF and IL-4 to induce dendritic cells from bone marrow cells;

(b) loading the dendritic cells with gene RNA encoding protein fragments, peptides derived from the C-terminus of topoisomerase 2 alpha, or derivatives having functionally equivalent properties thereof; And

(C) can be prepared by the step of maturing dendritic cells by adding LPS as a mature inducer to the Top2α-C-terminal RNA loaded dendritic cells.

Top2α C-terminal RNA can be prepared by injecting Top2α C-terminal RNA into the vector and mixing it with T7 RNA polymerase to perform in vitro transcription. As a vector, pcDNA3.1 TOPO vector can be used. But not limited to this.

Top2α C-terminal RNA loading of step (b) can be carried out by electroporation, step (c) is 10-20ng / mL of GM-CSF, 10-20ng IL-4 The concentration of / mL, LPS may be carried out by adding a concentration of 1 μg / mL and incubating for 12 hours to 2 days, specifically 1 day.

The delivery of the antigen to the dendritic cells has been made in various forms, the peptide can induce an antigen-specific immune response, but because it presents only a limited portion of the tumor antigen, it is difficult to induce a strong immune response, tumor lysate Even unknown antigens can induce immune responses, but side effects due to immune responses to autologous antigens can occur, and DNA is very low in gene transfer efficiency, making antigen presentation difficult. Adenoviruses can effectively deliver antigens to dendritic cells, but require a lot of time and labor to produce adenoviruses. On the contrary, when the RNA of specific tumor antigen is delivered to dendritic cells by the electroshock method, the antigen shows high metastasis rate, less cytotoxicity, rapid expression as protein, and clinical application. In addition, tumor antigens delivered in RNA form are characterized by maximizing specific immune responses to tumor antigens by simultaneously activating cell killer T cells and secondary T cells by presenting both MHC class I and MHC class II in dendritic cells. .

In the method for identifying the tumor antigen of the present invention, the second step is to identify the tumor antigen by confirming the immune response and anti-tumor effect of the antigen-presenting cell vaccine.

The immune response of the antigen-presenting cell vaccine can be confirmed by examining cytokine secretion or tumor cell killing ability of candidate tumor antigen-specific T cells isolated from animal tumor models.

More specifically, the immune response of the antigen-presenting cell vaccine is

Vaccinating the mouse with antigen-presenting cells transfected with candidate tumor antigen RNA;

Isolating T cells from the mouse spleen and restimulating with tumor cells; And

The cytokine secretion of anti-tumor T cells can be identified through the step of investigation.

The cytokine may include IFN-γ and the like.

The cytokine secretion investigation may be carried out by ELISPOT by culturing tumor cells expressed in target cells in a plate to which a cytokine specific antibody is attached and re-stimulating T cells, but are not particularly limited thereto.

Tumor cell killing ability of the T cells may be investigated by performing a ⁵¹ Cr release assay, but is not particularly limited thereto.

In addition, the anti-tumor effect of the antigen-presenting cell vaccine can be confirmed by inoculating the vaccine into an animal tumor model or a heterogeneous tumor model by examining the volume or the number of days of survival of the tumor.

More specifically, when using an animal tumor model,

Vaccination of the antigen-presenting cells with tumor antigens in an animal model;

Injecting tumor cells into the animal model; And

The antitumor prevention effect of the antigen-presenting cell vaccine can be confirmed by examining the size or the number of days of survival of the tumor formed in the animal model.

or,

Injecting tumor cells into an animal model;

Vaccinating the animal model with antigen-presenting cells loaded with tumor antigens; And

The antitumor treatment effect of the antigen-presenting cell vaccine can be confirmed by examining the volume or the number of days of survival of the tumor formed in the animal model.

The tumor volume can be measured by a caliper meter in two perpendicular dimensions. Tumor volume can be confirmed using the following equation.

Tumor volume = (short dimension) ² × (long dimension) / 2

In addition, when using a heterogeneous tumor model,

Injecting T cell clones specific for tumor antigen into nude mice;

Injecting human-derived tumor cells into the nude mouse; And

The anti-tumor prevention effect of the antigen-presenting cell vaccine can be confirmed by examining the survival days of the nude mouse.

or,

Injecting human derived tumor cells into nude mice;

Injecting said T cell clone specific for tumor antigen into said nude mouse; And

The antitumor treatment effect of the antigen-presenting cell vaccine can be confirmed by examining the survival days of the nude mouse.

In addition, in order to characterize the anti-tumor T cells, the cell surface antigenicity can be analyzed to confirm the distribution of CD8 + cell killing T cells, CD4 ⁺ helper T cells and NK cells of the cultured cells.

The antigen-type analysis was carried out by washing the cultured anti-tumor T cells with PBS containing 1% BSA and reacting by adding antibodies to CD3, CD4, CD8, CD56, CD14, and CD19 to which the fluorescent material was bound. It is measured by using, ELISPOT can be carried out by analyzing the immunogenicity of the specific peptide to increase the immunogenic ability according to the antigen type, but is not particularly limited thereto.

In the method for identifying tumor antigens of the present invention, the third step is to identify human tumor antigens by searching for human candidate tumor antigens having sequence homology with animal tumor antigens and confirming their immune response or anti-tumor effect. .

Homology search between the animal tumor antigen and human tumor antigen is http://www.ncbi.nlm.nih.gov/blast ; This can be done via http://www.ch.embnet.org/software/LALIGN , but is not particularly limited thereto.

Identification of human candidate tumor antigens selected through the homology search

Stimulating the isolated lymphocytes with an antigen-presenting cell vaccine transfected with a human candidate tumor antigen gene to induce human tumor antigen specific cell killing T cells in vitro; And

It may include the step of examining the cytokine secretion ability or tumor cell killing ability of the T cells.

The cytokine secretion or tumor cell killing ability of the T cells may be investigated by ELISPOT, or ⁵¹ Cr release assay, but is not particularly limited thereto.

In addition, cellular immune responses against human tumor antigens, which have been demonstrated antitumor effect, can be measured using cells isolated from the blood of tumor patients.

In the method for identifying tumor antigens of the present invention, the fourth step is to induce human or animal tumor antigen specific cell killing T cells and to identify epitopes of tumor antigens from the T cells.

The tumor antigen-specific cell killer T cells can be induced and cloned to kill cell T cells that specifically respond to candidate tumor antigens in tumor patients to obtain tumor antigen-specific T cell clones.

Identifying the epitope of the tumor antigen is

Analyzing peptides having HLA antigen binding specificity in tumor antigen specific cell killing T cell clones; And

Investigating the immune response or anti-tumor effect of the peptide.

The epitope can be identified by analyzing peptides having HLA antigen binding specificity in tumor antigen specific cell killing T cell clones of humans or animals.

More specifically, tumor antigen-derived peptides according to various HLA types can be predicted and produced using bioinformatics programs (http://www.syfpeithi.de, http://www-bimas.dt.nih.gov). .

Antigen-presenting cells carrying the synthesized peptides were vaccinated in an animal model, and then induced T cells specific for human or animal tumor antigens to react with antigen-presenting cells carrying various peptides, followed by ELISPOT and ⁵¹ Cr release assay. Antigen determinants can be identified by investigating the immune response by vaccinating or vaccinating antigen-presenting cells with peptides in animal tumor models.

The epitope may include the amino acid sequence of SEQ ID NO: 5, but is not particularly limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples of the present invention, but the scope of the present invention is not limited by the following Examples.

Preliminary Experimental Example 1 Experimental Animal and Cancer Cell Culture

6-8 week old female C57BL / 6 mice (H- ^2b ) were purchased from Orient Bio (Gapyeong, Gyeonggi-do, Korea) and used.

GL26 (H-2 ^b ; glioma), MC-38 and MC-38-cea2 (H-2 ^b ; colorectal cancer), B16F10 (H-2 ^b ; malignant melanoma) and CT26 (H-2 ^d ; colon Cancer) 10% heat inactivated fetal bovine serum (fetal bovine serum, FBS, Gibco, Grand Island, NY, USA), 2 mM glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin The cells were cultured in complete DMEM (complete Dulbecco's Moified Eagle Medeum) with a streptomycin.

EL4 (H-2 ^b ; Lymphoma) and YAC-1 (H-2 ^a ; Lymphoma) were given complete RPMI under 10% heat inactivated fetal bovine serum, 2 mM glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin. Incubated in -1640 (Cambrex) medium.

GL26 is Yu (Cedars Sinai Medical Center, Los Angeles, CA, USA), MC-38-cea2 expressing human CEA Schlom (Division of Tumor Immunology and Biology, NIH, Bethesda, MD, USA), MC-38, EL4, B16F10, CT26 and YAC-1 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Preliminary Experimental Example 2 Statistical Analysis

The experimental results were expressed as mean ± standard deviation (SD) or standard error (SE). Statistical analysis was performed using Student's t- test except for survival confirmation data. Survival confirmation data were analyzed using the Kaplan-Meier test and compared with other groups using the log-rank test. The p value <0.05 was considered to be statistically significant.

Example 1 Preparation of Dendritic Cell Vaccine on Top2αC RNA

<1-1> Cloning of Top2αC

The C-terminal part of mouse Top2α used in the present invention, and is composed of amino acid residues 1263-1528 (Top2αC). Reverse-transcription mRNA for mouse Top2αC from GL26 was used as a template for polymerase chain reaction (PCR) amplification. PCR conditions were as follows: 2 minutes at 94 ° C; 30 cycles of 15 seconds at 94 ° C and one minute at 72 ° C; 10 minutes at 72 ° C.

Primers for each Top2αC were 5'-CTGAGGATGGTGCAGAAGAAAGAGCCGGT-3 '(forward primer) and 5'-TGCCTCAGAAGAGGTCGTCATCGTCATCGAA-3' (reverse primer). PCR products were injected into the pcDNA3.1 TOPO vector (Invitrogen, Grand island, NY, USA). The cloned gene was confirmed by sequencing.

<1-2> Bone marrow-derived dendritic cell culture

In order to obtain dendritic cells, bone marrow cells were collected from femur and tibia bone of 6-8 week-old female C57BL / 6 mice, followed by hypotonic buffer, 9.84 g / L NH ₄ Cl, 1 g / L KHCO ₃ , And 0.1 mM EDTA) to remove red blood cells. After washing the cells twice with serum-free RPMI-1640 medium, 20 ng / mL murine granulocyte macrophage colony-stimulating factor, GM-CSF, R & D Systems, Minneapolis, MN, USA ) And 20 ng / mL recombinant murine interleukin-4 (IL-4, R & D System) at 5 × 10 ⁶ cells / well concentration in complete RPMI-1640. After 48 hours, non-adherent cells were removed and supplemented with complete medium containing GM-CSF and IL-4. On day 6 of culture, non-adhesive and loosely adherent cells (dendritic cells) were collected and used for RNA electroporation.

<1-3> RNA electric shock into dendritic cells

Top2αC / pcDNA3.1 TOPO plasmids were digested with Sma I and performed in vitro transcription (IVT) with T7 RNA polymerase (performed by manufacturer's instructions, Ambion mMESSAGE mMACHINE T7 Ultra kit, Austin, TX , USA). Production of IVT RNA was agarose gel electrophoresis and RNA concentration was measured by spectrophotometry. RNA samples were aliquoted and stored at -70 ° C. After 6 days of incubation, the dendritic cells were prepared in a Opti-MEM medium at a concentration of 2.5 × 10 ⁷ cells / mL, and 200 μl of the cell suspension was placed in a 2-mm cuvette and 20 μg of RNA was added thereto. The cell suspension was placed in an electroporator (ElectroSquarePorator, ECM 830, BTX, San Diego, Calif., USA) and given 300V current for 500 mA. The cells were immediately removed from the cuvette and the complete media containing GM-CSF, IL-4 and LPS (1 μg / mL: Sigma, Saint Louis, MO, USA) was added to allow the dendritic cells to fully mature for 24 hours. The transfer efficiency by dendritic cell electroshock method was measured by GFP RNA through FACS analysis.

<1-4> Preparation of tumor lysate

MC38, MC-38-cea2 or B16F10 cells were re-inoculated into PBS at a concentration of 1 × 10 ⁷ cells / mL. The cell suspension was frozen with liquid nitrogen and thawed in a 37 ° C. constant temperature water bath. The freeze / thaw cycle was repeated four times in rapid succession. Large particles were removed by centrifugation at 600 rpm for 10 minutes. The supernatant was passed through a 0.2 μm filter and aliquoted and stored at −70 ° C. Protein concentration of the lysate was measured using a non-cinconic acid assay (Pierce). Dendritic cells were incubated with MC-38 tumor lysate (100 μg / mL) for 16-18 hours to load MC-38 tumor lysate.

Experimental Example 1 Western Blotting

The mouse tissue is ground finely into surgical mass and made into small pieces, which are 1 mM DTT, 1 mM phenylmethanesulfonyl fluoride, 10 μg / mL aporotinin, and 5 μg / mL of leupeptin. Suspension in RIPA buffer. The suspension was homogenized using Precellys24 lyser (Bertin Technologies, Cedex, France). Cancer cells were lysed in the same buffer. Protein concentrations were measured by a bicinchoninic acid assay (Pierinch, Rockford, IL, USA). Total protein (50 μg) was isolated on 8% polyacrylamide gel, blotted onto nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) and then anti-Top2α antibody (Epitomics, Burlingame, CA, USA) Incubated with Bands were visualized by enhanced chemiluminescence (ECL) staining (Amersham Pharmacia, Freiburg, Germany).

Expression of Top2α in murine tumor cell lines was measured by Western blotting using an anti-Top2α antibody, indicating that Top2α protein is expressed in various murine tumor cell lines, including MC-38 and GL26, as shown in FIG. Could.

Top2α expression in normal murine tissues was measured in bone marrow, brain, spleen, lymph node, lung, kidney, liver, and large intestine. However, as shown in FIG. 1B, Top2α was not expressed in other tissues except the spleen.

Experimental Example 2 Isolation of CD4 ⁺ and CD8 ⁺ T Lymphocytes

For measuring CD4 ⁺ and CD8 ⁺ T lymphocyte immune responses, splenocytes were conjugated with monoclonal antibodies specific for CD4 or CD8 magnetic beads (MACS); Miltenyi Biotec, Bergisch Gladbach, Germany) at 15 ° C. for 15 minutes. After incubation, cells were washed with PBS containing 2 mM EDTA and passed through a MACS magnetic separation column. After isolation, the purity of each T lymphocyte was> 90% by FACS analysis.

Experimental Example 3 ELISPOT Assay (Enzyme-linked ImmunoSpot Assay)

The ELISPOT kit was purchased from BD Bioscience (Qume Drive, San Jose, Calif., USA) and used and tested according to the manufacturer's instructions.

Splenocytes (5 × 10 ⁴ cells / well) were seeded in 96-well plates coated with anti-mouse IFN-γ antibody. Top2αC RNA / DC, MC-38TL / DC, SuvRNA / DC and dendritic cells were put into target cells. Plates were incubated at 37 ° C. for 20 hours. The cells were removed and the plates washed three times with wash buffer. Biotin labeled anti-mouse IFN- [gamma] antibodies were then added to the cells and incubated for 2 hours at room temperature. After three washes, streptavidin-Holradish peroxidase was added to each well and incubated for 1 hour at room temperature. After washing a chromogenic substrate (3-amino-9-ethyl carbazole [AEC]) was added to each well. After spots were formed the reaction was quenched with distilled water and the plate was dried overnight in the dark. The number of spots corresponding to IFN-γ secreting cells was measured using an automatic AID-ELISPOT-reader (Strassberg, Germany).

To determine whether mice vaccinated with DC (dendritic cells) (MC-38TL / DC) loaded with MC-38 tumor lysate produced an immune response corresponding to Top2α, the number of IFN-γ secreting cells was counted. Measurements were made using an EILSPOT assay. Top2α used in this experiment is the C-terminal site and amino acid residues 1263-1528. MC-38 was vaccinated against C57BL / 6 mice because MC-38 was shown to express several tumor antigens including survivin and Top2α. One week after the second vaccination, the splenocytes were extracted and activated into various target cells.

When stimulated with Top2αC RNA / DC, the number of IFN-γ secreting cells was found to be significantly increased than when stimulated with dendritic cells without antigen. When MC-38TL / DC and survivin were stimulated with RNA transfected DC (SurRNA / DC), also the higher levels of IFN-γ secreting cells in splenocytes were detected (see FIG. 2). DCs loaded with CEA, unrelated tumor antigens, and unloaded DCs were used as Top2α negative cells.

The results indicate that the immune response to several tumor antigens in mice is induced by vaccination of MC-38TL / DC, and Top2αC RNA / DC can present Top2α epitopes.

To measure the immune response induced by Top2αC RNA / DC in vivo, Top2α specific IFN-γ secreting T lymphocytes after Top2αC RNA tertiary vaccination were quantified by ELISPOT. When activated by Top2αC RNA / DC, the frequency of IFN-γ secreting cells in splenocytes, CD4 ⁺ and CD8 ⁺ T lymphocytes was significantly higher in mice vaccinated with Top2αC RNA / DC compared to vaccinated DC. appear. The frequency of Top2αC-specific CD4 ⁺ T lymphocytes was higher than that of CD8 ⁺ T lymphocytes.

When activated with DC loaded with tumor lysate of MC38, a tumor cell line expressing Top2α, it was observed that IFN-γ secreting cells show high frequency in splenocytes (FIG. 3A).

Cytotoxicity of Top2αC-specific CTLs (cytotoxic T lymphocytes) was measured in various target cells. For T lymphocytes from C57BL / 6 mice vaccinated with Top2αC RNA / DC, MC-38 (H-2 ^b ), GL26 (H-2 ^b ) and Top2αC as Top2α-expressing cells at an E / T ratio of 40 It showed killing activity in response to RNA / DC (H- ^2b ), but showed only very low lysis for DCs that were not loaded as Top2α-unexpressing cells (FIGS. 4A-4D).

YAC-1 (H-2 ^a ) showed low cytolysis, indicating that killing activity corresponding to target cells was not affected by NK cells (FIG. 4E). CT26, a Top2α- cells expressing H-2 ^d singular body will not melt because it is limited to Top2α- specific T lymphocytes are MHC class I molecule H-2 ^b from the C57BL / 6 mouse.

The results show that Top2α-specific immune responses were successfully induced in vivo through Top2αC RNA / DC vaccination.

Experimental Example 4 Cytotoxicity Analysis

Standard ⁵¹ Cr-emission analysis was performed. Splenocytes were extracted from each mouse and reactivated with 4% paraformaldehyde-fixed MC-38 cells in vitro for 5 days before being used as effector cells. Targets MC-38, GL26, Top2αC RNA / DC, DC, YAC-1, and CT26 labeled with 100% Ci [ ⁵¹ Cr] -sodium chromate per 1 × 10 ⁶ cells at 5% CO ₂ , 37 ° C. for 1 hour. Used as a cell. 100 μl of the supernatant of each well was collected and radioactivity was measured with a gamma counter.

Specific lysis rate = 100 × [(experimental release-voluntary release) / (maximum release-voluntary release)]

The spontaneous release and maximal release were measured for radioactivity in the presence of medium and 2% Triton X100, respectively.

Experimental Example 5 Tumor Model and Dendritic Vaccination

To prepare the MC-38 tumor model, C57BL / 6 mice (6-8 weeks old) were injected subcutaneously with MC-38 cells (2 × 10 ⁵ cells). Two days after MC-38 cell inoculation, mice were injected subcutaneously with 1 × 10 ⁶ cells Top2αC RNA / DC, MC38TL / DC, p1327 / DC, or DC once a week for three weeks.

To prepare the MC-38-cea2 tumor model, C57BL / 6 mice (6-8 weeks old) were injected subcutaneously with MC-38-cea2 cells (1 × 10 ⁶ cells). At 10, 17 and 24 days after MC-38-cea2 inoculation, mice were injected subcutaneously with 1 × 10 ⁶ cells Top2αC RNA / DC / DC, MC-38-cea2TL / DC, or DC.

To prepare a B16F10 tumor model, C57BL / 6 mice (6-8 weeks old) were injected subcutaneously with MC-38 cells (2 × 10 ⁵ cells). One day after B16F10 inoculation, mice were injected subcutaneously with 1 × 10 ⁶ cells Top2αC RNA / DC, B16F10TL / DC, or DC once a week for three weeks.

To prepare the GL26 tumor model, GL57 cells (1 × 10 ⁶ cells) were subcutaneously injected into C57BL / 6 mice (6-8 weeks old). On day 14 and day 7 before GL26 cell inoculation, mice were injected subcutaneously with 1 × 10 ⁶ cells Top2αC RNA / DC, SuvRNA / DC, GFPRNA / DC, or DC.

For intracranial transplantation of GL26 glioma cells, C57BL / 6 mice (6-8 weeks old) were anesthetized with ketamine / xylazine. The head was shaved and the skull exposed. The experimental animals were placed in a stereotactic frame with small animal earbars. A dremel drill made burr holes approximately 2 mm behind and 1 mm behind the intersection of the coronal and sagittal sutures (bregma).

GL26 cells (1 × 10 ⁴ cells) were injected at a depth of 3 mm in a volume of 4 μl using a Hamilton syringe. Mice were vaccinated subcutaneously at 4, 11 and 18 days after GL26 implantation with 1 × 10 ⁶ cells Top2αC RNA / DC or DC. DCs that had undergone metastasis without antigen were used as negative controls. Tumor growth or survival reduction was compared to the negative control.

Because Top2α is expressed in various tumor cell lines, including MC-38, MC-38-cea2, B16F10, and GL26, MC-38, MC-38-cea2, B16F10 subcutaneous tumor models and established GL26 intracranial and subcutaneous tumor models The anticancer effect of Top2αC RNA / DC at was measured.

In the MC-38, MC-38-cea2, and B16F10 tumor models, mice vaccinated with Top2αC RNA / DC showed inhibition of tumor growth compared to DC vaccinated mice ( p <0.05, Figures 5a, b, 6c). ). Four days after GL26 inoculation, Top2αC RNA / DC inoculated mice appeared to survive significantly longer than DC inoculated mice ( p <0.05). 20% of Top2αC RNA / DC inoculated mice survived more than 85 days after tumor transplantation (FIG. 6D). In the GL26 subcutaneous tumor model, tumor development was inhibited in 5 of 7 mice in Top2αC RNA / DC-inoculated mice, but all 7 tumors were produced in DC-inoculated mice (FIG. 7).

Experimental Example 6 Absence of T Lymphocyte Subgroups in Vivo

To determine what type of T lymphocytes are involved in anticancer effects in the MC-38 tumor model, anti-CD4 monoclonal antibodies (GK1.5; eBioscience) and anti-CD8 monoclonal antibodies (2.43: a gift) from Prof. Byoung S. Kwon in Ulsan University, Ulsan, Korea) to vaccinate the MC-38 tumor model on day 0, 7, 14 to remove CD4 ⁺ and CD8 ⁺ T lymphocytes (antibodies specific for CD4 and CD8) FACS analysis was used to demonstrate that CD4 and CD8 disappeared (> 95%) during treatment, dendritic cells without tumor antigen were vaccinated for normal tumor growth as a control.

Tumor growth inhibition by Top2αC RNA / DC vaccination disappeared almost completely when CD8 ⁺ T lymphocytes disappeared (FIG. 8). However, the removal of CD4 ⁺ T lymphocytes did not affect the anticancer efficacy by Top2αC RNA / DC vaccination. Based on these results, it can be seen that CD8 ⁺ T lymphocytes are involved in the inhibition of tumor growth in vivo by Top2αC RNA / DC vaccination.

Experimental Example 7 Peptide Epitopes

Using the peptide motif scoring system (BIMAS; Bioinformatics and Molecular Analysis Section of NIH; http://www-bismas.cit.nih.gov. And SYFPEITHI; http://www.syfpeithi.de ) We examined potential Top2αC derived peptides that could potentially bind H-2K ^b . Two programs were used to select and analyze five peptides with high expected scores (Table 1). Synthetic peptides were purified to 90% minimum purity by high-performance liquid chromatography (HPLC). CEA526 (526-533: EAQNTTYL) peptide was used as a negative control.

Five Top2αC-derived peptides that bind high scores to MHC class I molecules (H-2Kb) were selected with the BIMAS and SYFPEITHI programs and spleens from mice vaccinated with Top2αC RNA / DC to determine T2 lymphocyte epitopes of Top2αC Cells were stimulated with Top2αC derived peptides and IFN-γ production was measured by ELISA. Compared to other peptides, splenocytes activated with P1327 (DSDEDFSGL) produced high levels of IFN-γ (FIG. 9A). Vaccination with p1327 pulsed dendritic cells (p1327 / DC) showed significantly suppressed tumor growth of MC-38 as compared to DC without peptide (FIG. 9B). To measure the immune response induced by p1327 / DC in vivo, Top2α specific IFN-γ-secreting T lymphocytes were quantitated using ELISPOT assays after tertiary vaccination with p1327 / DC. The frequency of IFN-γ-secreting T lymphocytes was significantly higher when stimulated with Top2αC RNA / DC and p1327 / DC compared to when stimulated with unloaded DC and CEA526 peptide-loaded DC (CEA526 / DC). Increased. Splenocytes from mice vaccinated with p1327 / DC showed cytotoxicity against MC-38 tumor cells (FIG. 9D). Based on these results, it can be seen that p1327 may be one of T lymphocyte epitopes located at the Top2αC site.

Experimental Example 8 ELISA (Enzyme-linked immunosorbent assay)

Spleen lymphocytes (2 × 10 ⁵ cells / 200 μl) of mice vaccinated with Top2αC RNA / DC were incubated in round bottom 96-well plates with 1 μg / mL of peptide. After 3 days, the plates were centrifuged and 100 μl of supernatant from each well was removed. IFN- [gamma] production levels were measured by ELISA (eBioscience, San Diego, USA).

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Method for identifying novel tumor antigen by using          RNA-transduced antigen presenting cells <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 1083 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (1080) <400> 1 atg aca ttt att gaa gaa ttg gag gct gtt gaa gcc aag gaa aaa caa 48 Met Thr Phe Ile Glu Glu Leu Glu Ala Val Glu Ala Lys Glu Lys Gln   1 5 10 15 gat gaa caa gtc gga ctt cct ggg aaa ggg ggg aag gcc aag ggg aaa 96 Asp Glu Gln Val Gly Leu Pro Gly Lys Gly Gly Lys Ala Lys Gly Lys              20 25 30 aaa aca caa atg gct gaa gtt ttg cct tct ccg cgt ggt caa aga gtc 144 Lys Thr Gln Met Ala Glu Val Leu Pro Ser Pro Arg Gly Gln Arg Val          35 40 45 att cca cga ata acc ata gaa atg aaa gca gag gca gaa aag aaa aat 192 Ile Pro Arg Ile Thr Ile Glu Met Lys Ala Glu Ala Glu Lys Lys Asn      50 55 60 aaa aag aaa att aag aat gaa aat act gaa gga agc cct caa gaa gat 240 Lys Lys Lys Ile Lys Asn Glu Asn Thr Glu Gly Ser Pro Gln Glu Asp  65 70 75 80 ggt gtg gaa cta gaa ggc cta aaa caa aga tta gaa aag aaa cag aaa 288 Gly Val Glu Leu Glu Gly Leu Lys Gln Arg Leu Glu Lys Lys Gln Lys                  85 90 95 aga gaa cca ggt aca aag aca aag aaa caa act aca ttg gca ttt aag 336 Arg Glu Pro Gly Thr Lys Thr Lys Lys Gln Thr Thr Leu Ala Phe Lys             100 105 110 cca atc aaa aaa gga aag aag aga aat ccc tgg tct gat tca gaa tca 384 Pro Ile Lys Lys Gly Lys Lys Arg Asn Pro Trp Ser Asp Ser Glu Ser         115 120 125 gat agg agc agt gac gaa agt aat ttt gat gtc cct cca cga gaa aca 432 Asp Arg Ser Ser Asp Glu Ser Asn Phe Asp Val Pro Pro Arg Glu Thr     130 135 140 gag cca cgg aga gca gca aca aaa aca aaa ttc aca atg gat ttg gat 480 Glu Pro Arg Arg Ala Ala Thr Lys Thr Lys Phe Thr Met Asp Leu Asp 145 150 155 160 tca gat gaa gat ttc tca gat ttt gat gaa aaa act gat gat gaa gat 528 Ser Asp Glu Asp Phe Ser Asp Phe Asp Glu Lys Thr Asp Asp Glu Asp                 165 170 175 ttt gtc cca tca gat gct agt cca cct aag acc aaa act tcc cca aaa 576 Phe Val Pro Ser Asp Ala Ser Pro Pro Lys Thr Lys Thr Ser Pro Lys             180 185 190 ctt agt aac aaa gaa ctg aaa cca cag aaa agt gtc gtg tca gac ctt 624 Leu Ser Asn Lys Glu Leu Lys Pro Gln Lys Ser Val Val Ser Asp Leu         195 200 205 gaa gct gat gat gtt aag ggc agt gta cca ctg tct tca agc cct cct 672 Glu Ala Asp Asp Val Lys Gly Ser Val Pro Leu Ser Ser Ser Pro Pro     210 215 220 gct aca cat ttc cca gat gaa act gaa att aca aac cca gtt cct aaa 720 Ala Thr His Phe Pro Asp Glu Thr Glu Ile Thr Asn Pro Val Pro Lys 225 230 235 240 aag aat gtg aca gtg aag aag aca gca gca aaa agt cag tct tcc acc 768 Lys Asn Val Thr Val Lys Lys Thr Ala Ala Lys Ser Gln Ser Ser Thr                 245 250 255 tcc act acc ggt gcc aaa aaa agg gct gcc cca aaa gga act aaa agg 816 Ser Thr Thr Gly Ala Lys Lys Arg Ala Ala Pro Lys Gly Thr Lys Arg             260 265 270 gat cca gct ttg aat tct ggt gtc tct caa aag cct gat cct gcc aaa 864 Asp Pro Ala Leu Asn Ser Gly Val Ser Gln Lys Pro Asp Pro Ala Lys         275 280 285 acc aag aat cgc cgc aaa agg aag cca tcc act tct gat gat tct gac 912 Thr Lys Asn Arg Arg Lys Arg Lys Pro Ser Thr Ser Asp Asp Ser Asp     290 295 300 tct aat ttt gag aaa att gtt tcg aaa gca gtc aca agc aag aaa tcc 960 Ser Asn Phe Glu Lys Ile Val Ser Lys Ala Val Thr Ser Lys Lys Ser 305 310 315 320 aag ggg gag agt gat gac ttc cat atg gac ttt gac tca gct gtg gct 1008 Lys Gly Glu Ser Asp Asp Phe His Met Asp Phe Asp Ser Ala Val Ala                 325 330 335 cct cgg gca aaa tct gta cgg gca aag aaa cct ata aag tac ctg gaa 1056 Pro Arg Ala Lys Ser Val Arg Ala Lys Lys Pro Ile Lys Tyr Leu Glu             340 345 350 gag tca gat gaa gat gat ctg ttt taa 1083 Glu Ser Asp Glu Asp Asp Leu Phe         355 360 <210> 2 <211> 360 <212> PRT <213> Homo sapiens <400> 2 Met Thr Phe Ile Glu Glu Leu Glu Ala Val Glu Ala Lys Glu Lys Gln   1 5 10 15 Asp Glu Gln Val Gly Leu Pro Gly Lys Gly Gly Lys Ala Lys Gly Lys              20 25 30 Lys Thr Gln Met Ala Glu Val Leu Pro Ser Pro Arg Gly Gln Arg Val          35 40 45 Ile Pro Arg Ile Thr Ile Glu Met Lys Ala Glu Ala Glu Lys Lys Asn      50 55 60 Lys Lys Lys Ile Lys Asn Glu Asn Thr Glu Gly Ser Pro Gln Glu Asp  65 70 75 80 Gly Val Glu Leu Glu Gly Leu Lys Gln Arg Leu Glu Lys Lys Gln Lys                  85 90 95 Arg Glu Pro Gly Thr Lys Thr Lys Lys Gln Thr Thr Leu Ala Phe Lys             100 105 110 Pro Ile Lys Lys Gly Lys Lys Arg Asn Pro Trp Ser Asp Ser Glu Ser         115 120 125 Asp Arg Ser Ser Asp Glu Ser Asn Phe Asp Val Pro Pro Arg Glu Thr     130 135 140 Glu Pro Arg Arg Ala Ala Thr Lys Thr Lys Phe Thr Met Asp Leu Asp 145 150 155 160 Ser Asp Glu Asp Phe Ser Asp Phe Asp Glu Lys Thr Asp Asp Glu Asp                 165 170 175 Phe Val Pro Ser Asp Ala Ser Pro Pro Lys Thr Lys Thr Ser Pro Lys             180 185 190 Leu Ser Asn Lys Glu Leu Lys Pro Gln Lys Ser Val Val Ser Asp Leu         195 200 205 Glu Ala Asp Asp Val Lys Gly Ser Val Pro Leu Ser Ser Ser Pro Pro     210 215 220 Ala Thr His Phe Pro Asp Glu Thr Glu Ile Thr Asn Pro Val Pro Lys 225 230 235 240 Lys Asn Val Thr Val Lys Lys Thr Ala Ala Lys Ser Gln Ser Ser Thr                 245 250 255 Ser Thr Thr Gly Ala Lys Lys Arg Ala Ala Pro Lys Gly Thr Lys Arg             260 265 270 Asp Pro Ala Leu Asn Ser Gly Val Ser Gln Lys Pro Asp Pro Ala Lys         275 280 285 Thr Lys Asn Arg Arg Lys Arg Lys Pro Ser Thr Ser Asp Asp Ser Asp     290 295 300 Ser Asn Phe Glu Lys Ile Val Ser Lys Ala Val Thr Ser Lys Lys Ser 305 310 315 320 Lys Gly Glu Ser Asp Asp Phe His Met Asp Phe Asp Ser Ala Val Ala                 325 330 335 Pro Arg Ala Lys Ser Val Arg Ala Lys Lys Pro Ile Lys Tyr Leu Glu             340 345 350 Glu Ser Asp Glu Asp Asp Leu Phe         355 360 <210> 3 <211> 801 <212> DNA <213> Mouse <220> <221> CDS (222) (1) .. (798) <400> 3 atg gtg cag aag aaa gag cca ggt gca aag aag cag act aca ttg ccg 48 Met Val Gln Lys Lys Glu Pro Gly Ala Lys Lys Gln Thr Thr Leu Pro   1 5 10 15 ttt aag cct gtc aaa aaa ggg agg aag aaa aac ccc tgg tct gac tct 96 Phe Lys Pro Val Lys Lys Gly Arg Lys Lys Asn Pro Trp Ser Asp Ser              20 25 30 gag tca gac gtg agc agt aat gaa agt aac gtt gat gtc cct cct cga 144 Glu Ser Asp Val Ser Ser Asn Glu Ser Asn Val Asp Val Pro Pro Arg          35 40 45 caa aaa gag caa aga agt gct gca gca aaa gcc aaa ttc aca gtg gat 192 Gln Lys Glu Gln Arg Ser Ala Ala Ala Lys Ala Lys Phe Thr Val Asp      50 55 60 tta gac tcg gat gaa gat ttc tca ggt ttg gat gag aag gat gag gat 240 Leu Asp Ser Asp Glu Asp Phe Ser Gly Leu Asp Glu Lys Asp Glu Asp  65 70 75 80 gaa gat ttc ctc cca tta gat gct act cca cct aag gcc aaa atc ccc 288 Glu Asp Phe Leu Pro Leu Asp Ala Thr Pro Pro Lys Ala Lys Ile Pro                  85 90 95 cca aaa aat act aaa aaa gca ctg aag acc cag gga agc tcc atg tcg 336 Pro Lys Asn Thr Lys Lys Ala Leu Lys Thr Gln Gly Ser Ser Met Ser             100 105 110 gtt gat ctt gaa agt gat gtt aag gac agt gtg cca gct tct cca ggc 384 Val Asp Leu Glu Ser Asp Val Lys Asp Ser Val Pro Ala Ser Pro Gly         115 120 125 gtt cct gct gct gac ttc cca gcg gaa act gaa cag tca aag cca tcc 432 Val Pro Ala Ala Asp Phe Pro Ala Glu Thr Glu Gln Ser Lys Pro Ser     130 135 140 aaa aag acc gtg gga gtg aag aag aca gca acc aaa agc cag tct tca 480 Lys Lys Thr Val Gly Val Lys Lys Thr Ala Thr Lys Ser Gln Ser Ser 145 150 155 160 gtc tcc act gct ggt acc aaa aag aga gct gcg cca aag gga acc aaa 528 Val Ser Thr Ala Gly Thr Lys Lys Arg Ala Ala Pro Lys Gly Thr Lys                 165 170 175 tca gat tca gcc ttg agt gct cgt gtc tcg gaa aaa cct gct cct gcc 576 Ser Asp Ser Ala Leu Ser Ala Arg Val Ser Glu Lys Pro Ala Pro Ala             180 185 190 aaa gcc aag aac agt cgc aaa agg aag cca tct tct tct gat agc tct 624 Lys Ala Lys Asn Ser Arg Lys Arg Lys Pro Ser Ser Ser Asp Ser Ser         195 200 205 gac tct gat ttt gag aga gca att tct aaa ggt gcc acg agc aag aaa 672 Asp Ser Asp Phe Glu Arg Ala Ile Ser Lys Gly Ala Thr Ser Lys Lys     210 215 220 gca aag gga gag gaa cag gac ttc cct gtg gac ttg gaa gac acg ata 720 Ala Lys Gly Glu Glu Gln Asp Phe Pro Val Asp Leu Glu Asp Thr Ile 225 230 235 240 gct cct cga gcc aaa tct gac cgg gcg agg aag cca att aag tac ctg 768 Ala Pro Arg Ala Lys Ser Asp Arg Ala Arg Lys Pro Ile Lys Tyr Leu                 245 250 255 gag gag tcc gat gac gat gac gac ctc ttc tg a 801 Glu Glu Ser Asp Asp Asp Asp Asp Leu Phe             260 265 <210> 4 <211> 266 <212> PRT <213> Mouse <400> 4 Met Val Gln Lys Lys Glu Pro Gly Ala Lys Lys Gln Thr Thr Leu Pro   1 5 10 15 Phe Lys Pro Val Lys Lys Gly Arg Lys Lys Asn Pro Trp Ser Asp Ser              20 25 30 Glu Ser Asp Val Ser Ser Asn Glu Ser Asn Val Asp Val Pro Pro Arg          35 40 45 Gln Lys Glu Gln Arg Ser Ala Ala Ala Lys Ala Lys Phe Thr Val Asp      50 55 60 Leu Asp Ser Asp Glu Asp Phe Ser Gly Leu Asp Glu Lys Asp Glu Asp  65 70 75 80 Glu Asp Phe Leu Pro Leu Asp Ala Thr Pro Pro Lys Ala Lys Ile Pro                  85 90 95 Pro Lys Asn Thr Lys Lys Ala Leu Lys Thr Gln Gly Ser Ser Met Ser             100 105 110 Val Asp Leu Glu Ser Asp Val Lys Asp Ser Val Pro Ala Ser Pro Gly         115 120 125 Val Pro Ala Ala Asp Phe Pro Ala Glu Thr Glu Gln Ser Lys Pro Ser     130 135 140 Lys Lys Thr Val Gly Val Lys Lys Thr Ala Thr Lys Ser Gln Ser Ser 145 150 155 160 Val Ser Thr Ala Gly Thr Lys Lys Arg Ala Ala Pro Lys Gly Thr Lys                 165 170 175 Ser Asp Ser Ala Leu Ser Ala Arg Val Ser Glu Lys Pro Ala Pro Ala             180 185 190 Lys Ala Lys Asn Ser Arg Lys Arg Lys Pro Ser Ser Ser Asp Ser Ser         195 200 205 Asp Ser Asp Phe Glu Arg Ala Ile Ser Lys Gly Ala Thr Ser Lys Lys     210 215 220 Ala Lys Gly Glu Glu Gln Asp Phe Pro Val Asp Leu Glu Asp Thr Ile 225 230 235 240 Ala Pro Arg Ala Lys Ser Asp Arg Ala Arg Lys Pro Ile Lys Tyr Leu                 245 250 255 Glu Glu Ser Asp Asp Asp Asp Asp Leu Phe             260 265 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Peptide name: p1327 <400> 5 Asp Ser Asp Glu Asp Phe Ser Gly Leu   1 5

Claims (12)

Preparing an antigen-presenting cell vaccine to which candidate tumor antigen genes derived from animals other than humans have been transferred;
Identifying the immune response and antitumor effect of the vaccine;
Identifying a human candidate tumor antigen having sequence homology with said antitumor tumor antigen; And
Method for identifying a tumor antigen comprising the step of identifying the epitope of the tumor antigen.
The method of claim 1,
The candidate tumor antigen gene is a method for identifying a tumor antigen comprising a gene encoding a fragment of the Topoisomerase II alpha full length or C-terminus.
The method of claim 1,
Candidate tumor antigen gene is a method for identifying a tumor antigen comprising the nucleotide sequence of SEQ ID NO: 3.
The method of claim 1,
Candidate tumor antigen genes are DNA or RNA forms.
The method of claim 1,
An antigen-presenting cell vaccine is a method for identification of tumor antigens prepared by delivering candidate tumor antigen gene RNA to cells with antigen-presenting ability.
The method of claim 5,
A cell having an antigen-presenting ability is at least one selected from the group consisting of dendritic cells, monocytes, CD4 T cells, B cells and γδ T (gamma delta T) cells.
The method of claim 6,
CD4 T cells, B cells, or γδ T cells are naïve, activated, or amplified state.
The method of claim 5,
Gene transfer is a method for identifying tumor antigens that are carried out by electroshock method.
The method of claim 1,
Immune response of antigen-presenting cell vaccines is a method of identification of tumor antigens confirmed by examining the cytokine secretion or tumor cell killing ability of candidate tumor antigen-specific T cells isolated from animal tumor models.
The method of claim 1,
Antitumor effect of the antigen-presenting cell vaccine is confirmed by inoculating the vaccine into an animal tumor model or a heterogeneous tumor model and examining the tumor volume or the number of days of survival.
The method of claim 1, wherein the step of identifying human candidate tumor antigens is
Stimulating the isolated lymphocytes with an antigen-presenting cell vaccine transfected with a human candidate tumor antigen gene to induce human tumor antigen specific cell killing T cells in vitro; And
Method of identifying a tumor antigen comprising the step of investigating the cytokine secretion capacity or tumor cell killing ability of the T cells.
The method of claim 1, wherein the determining the epitope of the tumor antigen is
Analyzing peptides having HLA antigen binding specificity in tumor antigen specific cell killing T cell clones; And
Method for identifying a tumor antigen comprising the step of examining the immune response or anti-tumor effect of the peptide.

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