WO2006040920A1 - Method of evaluating ctl induction ability and screening method using the same - Google Patents

Abstract

It is intended to provide a method whereby a specific CTL induction ability in a protein can be quickly and conveniently evaluated. This object can be achieved by providing a method of evaluating a protein-specific CTL induction ability which comprises the step of transferring a gene encoding the test protein into antigen presenting cells in such a manner as allowing the expression of the protein, the step of culturing the antigen presenting cells, into which the gene has been transferred, together with lymphocytes, and the step of examining the CTL induction efficiency in the lymphocytes, wherein use is made of, as the antigen presenting cells, cells originating in an established cell line expressing a T cell costimulator and an MHC class I molecule and being capable of presenting as the antigen the entire protein expressed in the cells or a part of the same. According to this method, an antigen protein or an antigen epitope peptide having a specific CTL induction ability can be quickly and conveniently screened and thus it becomes possible to search for a novel antigen epitope peptide.

Description

 Specification

 CTL inducibility evaluation method and method for using it, screening method

 Technical field

 The present invention relates to a method for evaluating CTL inducibility and a screening method using the same.

 Background art

 [0002] Immunity is a series of biological defense systems and other recognition systems that work against what is recognized as a foreign substance (antigen) in a living body. This system protects organisms from various diseases.

 [0003] For example, in the body of a healthy person, it is thought that most of the force that cancer cells are always generated is suppressed by the immune response and disappears without leading to illness. . However, if the power of the cancer exceeds the deterrent due to immunity as a result of something, cancer cells will continue to proliferate and eventually cancer will be established as a disease.

 [0004] In recent years, immune cell therapy that artificially enhances cellular immunity and treats various diseases (particularly cancer and viral infections) has attracted attention. The advantage of immune cell therapy is that it only requires a mild fever, even if it has very few side effects.

 [0005] Among immune cell therapies, cytotoxic T cell therapy (CTL therapy) is said to be particularly effective. Cytotoxic T Lymphocytes (hereinafter also referred to as CTL) play a central role in biological defense reactions that recognize and eliminate tumors (cancer cells) and viruses. CTL therapy is a treatment / prevention method that induces CTLs specific to these antigens for the purpose of treatment and prevention of cancer and infectious diseases, and plays a central role in the immune response to cancer and viruses. It is a treatment / prevention method aimed at inducing CTL and eliminating cancer cells and virus-infected cells.

[0006] CTL recognizes a part of a protein that is specifically expressed on or in the surface of cancer cells or virus-infected cells and is called an antigen epitope peptide. Antigen epitope peptides usually also have 9 to: L 1 amino acid residue strength. This antigen epitope peptide binds to major histocompatibility antigen complex (MHC) class I and is presented on antigen-presenting cells. Re The lymphocyte recognizes the presented antigen epitope peptide and becomes a CTL that specifically attacks cancer cells or virus-infected cells having the same antigen epitope peptide (for example, see Non-Patent Document 1). ).

 [0007] Therefore, in order to induce CTL, it is important to identify an antigen epitope peptide recognized by CTL. Based on the information of the amino acid sequence of the identified antigenic epitope peptide, for example, the epitope peptide is synthesized and directly administered as a peptide vaccine to induce CTL, It becomes possible to produce a more powerful DC vaccine by knocking into cells (hereinafter also referred to as DC) (see, for example, Non-Patent Document 2). For this reason, identification of epitope peptides has been attempted by various methods until now.

 [0008] However, the conventional cancer antigen epitope peptide search methods have the following problems, for example.

 1. A tumor-specific CTL cell line must be established.

 2. A large number of peptides with a motif structure must be synthesized and screened as candidate antigenic epitope peptides.

 3. DC must be induced every time as an antigen-presenting cell and cultured.

 4. Ability to introduce genes (DNA) into DC Very inefficient and difficult.

 Non-patent literature 1: Thierry Boon et al, Tumor antigens recognized by T cells "Immunology today 267—268 Vol. 18, 1997

 Non-Patent Document 2: Joachim L. Schultzeand et al, "From cancer genomics to cancer immunotherapy: toward second-generation tumor antigens" Trends in Immunology Vol. 22 No. 9: 516-523, 2001 Disclosure of the Invention

 Problems to be solved by the invention

[0009] The present invention has been made in view of the above circumstances, and has a specific CTL inducing ability that enables rapid and simple screening of an antigenic epitope peptide without using DC as an antigen-presenting cell. The purpose is to provide an evaluation method.

Means for solving the problem [0010] In order to solve the above-mentioned problems, a method for evaluating specific CTL inducing ability in the protein of the present invention (hereinafter also referred to as the evaluation method of the present invention) is:

 Introducing a gene encoding a test protein into an antigen-presenting cell so that the protein can be expressed;

 Co-culturing the antigen-presenting cells into which the gene has been introduced with lymphocytes, and examining the induction efficiency of CTL in the lymphocytes,

 As the antigen-presenting cell, a T cell costimulatory factor and an MHC class I molecule are expressed, and all or part of the protein expressed intracellularly can be presented as an antigen on the MHC class I molecule. This is an evaluation method using cells derived from a cell line. The invention's effect

 [0011] Among the screening methods for antigen epitope peptides, the present inventors have heretofore been used as antigen-presenting cells for screening methods using CTL specifically induced by antigen epitope peptides as an index. Instead of DC, we intensively researched the idea of using an established cell line that has excellent proliferative ability and does not require differentiation induction. As a result, if the antigen-presenting cell is derived from a cell line that expresses or has been modified to express a T cell costimulatory factor and an MHC class I molecule, it encodes a protein containing an antigen epitope peptide. It has been found that when the gene to be expressed is introduced into the antigen-presenting cell so that it can be expressed, the antigen epitope peptide is presented as an antigen on the MHC class I molecule and a specific CTL can be induced. Furthermore, the present inventors have shown that the efficiency of gene introduction into the antigen-presenting cells is superior to that of conventional DC, and therefore the specific CTL inducing ability of the introduced protein can be improved by using the antigen-presenting cells. It has been found that screening can be performed more quickly and simply than conventional methods, and by using this evaluation method, screening of antigen epitope peptides can be performed quickly and simply.

[0012] Here, the "specific CTL inducing ability" means a protein or peptide power that is specific to the above-mentioned epitope peptide when part or all of the protein or peptide strength is presented as an epitope peptide. It means the ability to activate CTL and promote proliferation. In addition, “established cell line” means a cell line capable of producing clonal cells having proliferative ability and exhibiting the same properties and characteristics. [0013] The evaluation method of the present invention uses, as an antigen-presenting cell, the antigen-presenting cell of the present invention derived from an established cell line that proliferates and needs to be induced immediately instead of DC. Therefore, it can be performed efficiently in a short time. In addition, since the antigen-presenting cell of the present invention has excellent gene transfer efficiency, the necessary preparatory work can be efficiently performed in a shorter time. Therefore, according to the present invention, it is possible to provide a quick and simple method for evaluating CTL inducibility. Further, the gene introduction in the evaluation method of the present invention can be a rapid and simpler evaluation method of the present invention, which may be a transient introduction.

 [0014] In addition, according to the evaluation method of the present invention, even if a gene encoding the whole protein is introduced regardless of the size of the protein, it is specific to the antigen epitope peptide contained in the protein. Since the ability to induce CTL can be evaluated, a method for screening a protein containing an antigenic epitope peptide that induces a specific CTL is possible. In addition, if the gene to be introduced is a gene encoding a part of the protein and the site or length of the protein is changed, screening of antigen epitope peptides can be performed by the evaluation method of the present invention. Is possible. That is, according to the evaluation method of the present invention, for example, a protein or antigenic epitope peptide that specifically induces CTL without establishing an antigen-specific CTL cell line or synthesizing a peptide of an antigenic epitope candidate. This screening can be performed quickly and easily. Brief Description of Drawings

 [0015] FIG. 1 is an example of a flow cytometer analysis diagram of the MDA-CD80 cell line.

FIG. 2 is a diagram showing an example of RT-PCR results for the MDA-CD80 cell line.

 [FIG. 3A] FIG. 3A is an example of an example in which pTracer was introduced into an MDA-CD80 cell line for antigen presentation, co-cultured with lymphocytes, and specific CTL inducibility was measured by FACS. .

 [Figure 3B] Figure 3B shows an example of MDA-CD80 cell line introduced with pTracer-BMLF 1 for antigen presentation, co-cultured with lymphocytes, and specific CTL inducibility measured by FACS. It is.

[FIG. 3C] FIG. 3C shows an example in which pTracer—Ub—BMLF 1 was introduced into an MDA CD80 cell line for antigen presentation, co-cultured with lymphocytes, and specific CTL inducing ability was measured by FACS. It is an example figure.

 [FIG. 4A] FIG. 4A is an example of introduction of pTracer into an MDA-CD80 cell line for antigen presentation, co-culture with lymphocytes, and measurement of specific CTL inducing ability by FACS. .

 [FIG. 4B] FIG. 4B shows an example in which pTracer-Marti was introduced into an MDA-CD80 cell line for antigen presentation, co-cultured with lymphocytes, and specific CTL inducing ability was measured by FACS. is there.

 [FIG. 4C] FIG. 4C shows the antigen presentation by introducing pTracer-Ub-Mart 1 into the MDA-CD80 cell line, co-cultured with lymphocytes, and the specific CTL inducing ability was measured by FACS. FIG.

 [Fig. 5A] Fig. 5A shows the introduction of pTracer into an MDA-CD80 cell line for antigen presentation, co-culture with lymphocytes, and the ability to induce specific CTLs using INF-γ as an index. It is a figure of an example measured by.

 [Fig. 5B] Fig. 5B shows the introduction of pTracer—Ub—BMLF 1 into the MDA CD80 cell line, antigen presentation, co-culture with lymphocytes, and specific CTL induction using INF-y as one of the indicators. It is a figure of an example which measured conductivity with FACS.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0016] In the evaluation method of the present invention, the T cell costimulatory factor is preferably CD80. In the step of introducing the gene, in addition to the protein, it is preferable to introduce a gene encoding ubiquitin so that it can be expressed. More preferably, the gene encoding a fusion protein of the protein and ubiquitin is used. Is introduced in an expressible manner. The gene transfer may be a transient gene transfer. The protein may be derived from a cancer cell or a virus. The antigen-presenting cell is preferably adherent, and is preferably a cell derived from a tumor cell-derived cell line. The tumor cell-derived cell line is preferably a breast cancer-derived cell line MDA-MB-231! /.

[0017] The screening method of the present invention is a protein screening method capable of specifically inducing CTL (hereinafter also referred to as the antigen protein screening method of the present invention). Evaluate specific CTL inducibility of test protein A screening method comprising a step of identifying a protein having a typical ability to induce CTL.

 [0018] In another aspect, the screening method of the present invention is a method for screening an antigenic epitope peptide in a protein capable of specifically inducing CTL (hereinafter, the screening method for an antigenic epitope peptide of the present invention). It is also a screening method comprising at least one of the following steps (A) and (B).

 (A) Instead of the step of introducing a gene encoding a test protein into the antigen-presenting cell so that the protein can express, a polynucleotide encoding a partial peptide of the protein is introduced into the antigen-presenting cell so that it can be expressed. A step of evaluating the CTL inducing ability of the partial peptide by the evaluation method of the present invention, comprising identifying a peptide having a specific CTL inducing ability.

 (B) In place of both the steps of introducing a gene encoding a test protein into an antigen-presenting cell so that the protein can be expressed and co-culturing the antigen-presenting cell into which the gene has been introduced with lymphocytes. Then, after contacting the partial peptide of the protein with the antigen-presenting cell, or by co-culturing with lymphocytes while contacting, the CTL inducing ability of the partial peptide is evaluated by the evaluation method of the present invention, Identifying a peptide having a specific ability to induce CTL.

 [0019] In the method for screening an antigenic epitope peptide of the present invention, the test protein is preferably a protein identified by the antigen protein screening method of the present invention. In the method for screening an antigenic epitope peptide of the present invention, the partial peptide may be a peptide having 9 to L 1 residues.

[0020] The antigen-presenting cell of the present invention is an antigen-presenting cell used in the evaluation method of the present invention, and expresses a T cell costimulatory factor and an MHC class I molecule, and is expressed in the cell. Antigen-presenting cells derived from an established cell line capable of presenting all or part of the antigen on the MHC class I molecule. In addition, the antigen-presenting cell of the present invention is the antigen-presenting cell used in the screening method for the antigen protein of the present invention as another aspect, and as another aspect, the antigen-epitope peptide of the present invention is screened. The antigen-presenting cell used in the method. [0021] The kit of the present invention is an evaluation kit used in the evaluation method of the present invention, and includes the antigen-presenting cell of the present invention. In another embodiment, the kit of the present invention is a screening kit used in the method for screening an antigen protein of the present invention, and includes the antigen-presenting cell of the present invention. The kit of the present invention is a screening kit used in the screening method for an antigenic epitope peptide of the present invention as yet another embodiment.

And the antigen-presenting cell of the present invention.

 [0022] The production method of the present invention is a method for producing an antigenic epitope peptide having a specific CTL inducing ability, and the method for screening an antigenic epitope peptide of the present invention allows the antigen epitope of the protein to be produced. A production method comprising the step of identifying a peptide. The production method of the present invention preferably further includes at least one of a step of separating the antigen epitope peptide identified with the protein strength and a step of synthesizing the identified antigen epitope peptide.

 [0023] As described above, the evaluation method of the present invention uses the antigen-presenting cell of the present invention which is a cell line that expresses a T cell costimulatory factor and an MHC class I molecule, and the test protein is applied to the antigen-presenting cell. One of the technical features is to introduce the encoded gene and present the antigen.

 [0024] In the antigen-presenting cell, transcription and translation of the introduced gene is performed, and a test protein is synthesized. The synthesized test protein is decomposed into several fragments by intracellular proteolytic enzymes (hereinafter also referred to as proteasomes). When the test protein contains an epitope site, it binds to the MHC class I molecule and is displayed on the cell surface

[0025] The T cell receptor (hereinafter also referred to as TCR) present on the surface of T lymphocytes recognizes a complex of MHC class I molecules and epitope peptides presented on the surface of antigen-presenting cells. To do. T lymphocytes with TCR binding to epitopes are activated to CTL.

[0026] That is, according to the evaluation method of the present invention, an antigen that can bind to an MHC class I molecule in a test protein and is recognized by TCR is confirmed by confirming a specific CTL inducing ability. It is possible to easily confirm the presence of the pitope peptide. [0027] An example of the evaluation method of the present invention will be specifically described below.

 [0028] (1. Gene introduction process)

 First, a vector incorporating a gene encoding a test protein for evaluating specific CTL inducing ability is prepared and introduced into the antigen-presenting cell of the present invention.

 [0029] The test protein is not particularly limited and refers to a protein in which an amino acid has a peptide bond and has a linear force S, and may be a simple protein that has only an amino acid force. It may be a complex protein (eg, glycoprotein, nucleoprotein, lipoprotein, heme protein, metal protein, etc.). Further, the number of amino acid residues is not particularly limited, and may be, for example, an oligopeptide or a polypeptide.

 [0030] The method for preparing the vector into which the gene is incorporated is not particularly limited, and a conventionally known method can be applied. For example, after obtaining cDNA (complementary DNA; complementary deoxyribonucleic acid) of a gene to be expressed. For example, a method of amplifying cDNA by PCR method (Polymerase Chain Reaction method; polymerase chain reaction method) and incorporating it into an appropriate expression vector can be mentioned. The expression vector is not particularly limited, and examples thereof include a plasmid vector and a virus vector.

 [0031] The size of the gene is not particularly limited. For example, even when a gene encoding the full length of the test protein is used, the procedure is very simple because it undergoes proteolysis in the cell and presents the epitope site as an antigen. Further, the gene may be a polynucleotide encoding a part of the test protein.

[0032] As the test protein, for example, a protein derived from cancer cells can be used. Examples of target cancer cells include liver cancer, stomach cancer, colon cancer, lung cancer, breast cancer, uterine cancer, brain tumor and the like. According to the evaluation method of the present invention, as will be described later, for example, an antigen epitope peptide is identified and the peptide is synthesized, and the synthesized peptide is administered to a cancer patient as a peptide vaccine. By inducing CTL or pulsing the synthetic peptide into rod cells (DC), it becomes possible to produce a DC vaccine and administer the DC vaccine to a patient. It is also possible to administer a gene encoding a protein or peptide sequence containing an antigenic epitope peptide as a vaccine in the form of a polynucleotide such as DNA (for example, a DNA vaccine). [0033] As the test protein, for example, a virus-derived protein can be used. Examples of the virus include HIV (Human Immunodeficiency Virus), HBV (Hepatitis B Virus), HCV (Hepatitis C Virus), SARS (Sever Acute Respiratory Syndro Syndrome). me; Severe acute respiratory syndrome) According to the evaluation method of the present invention, as described later, for example, it is possible to identify an antigen epitope peptide, synthesize the peptide, and use the synthesized peptide as a vaccine against a viral infection. Therefore, the evaluation method of the present invention can be used as a vaccine development system for viral infections.

 [0034] The prepared vector is introduced into the antigen-presenting cell of the present invention having antigen-presenting ability.

[0035] The antigen-presenting cell of the present invention expresses a T cell costimulatory factor and an MHC class I molecule, and all or part of the protein expressed in the cell is placed on the MHC class I molecule. It is a cell derived from an established cell line capable of presenting an antigen.

 [0036] Antigen presenting cells have conventionally used DC or the like. Peripheral blood force DC obtained cannot be proliferated in small numbers, so it is difficult to secure the number to be used as antigen presenting cells. There are many cases. In addition, when isolating peripheral blood isostatic DC progenitor cells, it is necessary to induce differentiation using site force in. In contrast, the antigen-presenting cell of the present invention is advantageous in that, for example, it is easy to proliferate and it is not necessary to induce differentiation, so that preparation of the antigen-presenting cell is easy.

 [0037] In addition, in the case of a test of CTL inducibility using conventional DC, cells must be collected from the donor each time the test is performed. Furthermore, certain results are difficult to obtain because the cell properties differ from donor to donor. In contrast, in the case of the antigen-presenting cell of the present invention, for example, uniform cells can be obtained by subculture, so that a certain result can be obtained by appropriately setting the conditions, whereby the evaluation method of the present invention. There are advantages such as improved reproducibility.

[0038] The antigen-presenting cell of the present invention is preferably an adherent cell. When DC is used as an antigen-presenting cell, there is a problem that the introduction efficiency of the test gene is very low. Antigen When the display cell is an adherent cell, for example, there is an advantage that the vector introduction efficiency by the ribofusion method or the like is improved. In addition, when co-cultured with lymphocytes, it is advantageous that the antigen-presenting cells are attached to the bottom surface of a shear or the like, so that the pulse efficiency to lymphocytes is increased.

 [0039] The antigen-presenting cell of the present invention is preferably derived from a tumor cell-derived cell line. Although tumor-derived cells are not easily established, they can always grow semipermanently and their properties are stable. In addition, a cell line obtained by modifying a tumor cell-derived cell line so as to express an MHC class I molecule or a T cell costimulatory factor may be used as the antigen-presenting cell of the present invention. Such antigen-presenting cells of the present invention can be prepared by appropriately using conventionally known methods. For example, reference can be made to the literature [Anal Biochem. 1993 Feb 1; 208 (2): 352-6. Maximal expression of Recombinant cDNAs IN COS cell for use IN expression cloning. Kluxen FW, Lubbert H.].

 [0040] Specifically, for example, the antigen-presenting cell of the present invention can be prepared using the following procedure.

 1) MHC class I molecules and T cell costimulatory factors are expressed. Amplify the gene of the molecule to obtain cDNA, and then amplify it by PCR and incorporate it into an appropriate expression vector.

 2) The expression vector is introduced into the cell line to be used, cultured in a medium, and then selected in a medium containing antibiotics.

 3) Furthermore, using antibody-bound beads, express recombinant protein (the gene product of the gene of the molecule you want to express) and concentrate the cells.

 4) Select the cells concentrated by the beads, and clone the cells by limiting dilution to select a stable cell line.

[0041] Specific examples of the antigen-presenting cell of the present invention include the MDA-CD80 cell line. Here, the MDA-CD80 cell line refers to a tumor cell line derived from a breast cancer expressing MHC class I molecule, MDA-MB-231, into which a gene encoding CD80 (Cluster Differentiation 80) is introduced. A cell line modified so that the molecule is highly expressed on the cell surface. The MDA-MB-231 cell line can be obtained, for example, from ATCC. In addition, as a tumor cell-derived cell line that can be used for the antigen-presenting cell of the present invention, For example, TUHR10TKB cell line derived from renal cancer, JR-st cell line derived from gastric cancer, and the like.

 [0042] Examples of the T cell costimulatory factor include CD80 (B7.1) molecule and CD86 (B7.2) molecule. These molecules are one of the molecules that transmit signals that activate the immune function of lymphocytes. When these molecules bind to the CD28 molecule on the surface of lymphocytes, the lymphocytes are activated and CTLs are activated. Be guided. In the antigen-presenting cell of the present invention, it is more preferable that either CD80 molecule or CD86 molecule is expressed. CD 80 and CD86 molecules can be expressed simultaneously.

 [0043] When the gene encoding the test protein is introduced into the antigen-presenting cell by a vector, it is preferable to introduce a gene encoding ubiquitin in addition to the gene encoding the test protein so that it can be expressed. More preferably, a gene encoding a fusion protein of the test protein and ubiquitin is preferably introduced so as to allow expression. By doing so, the transition to the antigen presentation pathway is promoted, the antigen presentation ability is improved, and the CTL induction ability is improved accordingly. As a result, it is possible to evaluate the inducibility of CTL with high sensitivity, that is, to detect the presence of a highly sensitive epitope peptide.

 [0044] Here, ubiquitin is a protein having a strength of 76 amino acid residues, and binds to a target protein to serve as a marker for degradation. Further, ubiquitin is added to the protein to which ubiquitin is bound, and the target protein is added with a polyubiquitin chain. This is a signal for degradation, and the target protein is rapidly degraded by intracellular proteasomes.

 [0045] The method for introducing the vector into the antigen-presenting cell is not particularly limited, and a general method can be used. For example, the calcium phosphate method, the lipofussion method, the DEAE (dimethylaminoethyl) dextran method, the electo-poration method, the microinjection method and the like are preferable from the viewpoint of high power introduction efficiency. The lipofusion method is a method in which a complex of ribosome, which is a lipid bilayer vesicle, and DNA to be introduced is formed, and the target gene is introduced into the cell by phagocytosis or membrane fusion.

[0046] (2. Co-culture process) Next, lymphocytes are co-cultured with antigen-presenting cells into which a gene encoding a test protein has been introduced.

 [0047] After the introduction of the vector, antigen-presenting cells are cultured to allow antigen presentation. The culture period is preferably 24 hours to 48 hours, for example. In the case of longer culture, the ability to induce CTL may decrease. Hereafter, antigen-presenting cells that have been presented with antigens are also called stimulator cells. /

 [0048] In the evaluation method of the present invention, since CTL induction can be detected by expression by transient gene introduction without obtaining a cell line that stably expresses the introduced gene, preparation of stimulator cells is possible. The period can be significantly shortened. In transient expression strains, there may be a problem that the expression level of the antigen protein is small. For example, as described above, the above problem can be solved by using a fusion protein with ubiquitin.

 [0049] As the lymphocytes, it is preferable to use human lymphocytes capable of using various lymphocytes. The lymphocyte is preferably a lymphocyte that shares at least one MHC class I molecule with the antigen-presenting cell of the present invention, and more preferably a lymphocyte that matches the MHC class I molecule. The method for collecting lymphocytes is not particularly limited, and examples thereof include a method for recovering peripheral blood mononuclear cells (Peripheral Blood Mononuclear Ce 11, hereinafter also referred to as PBMC) from blood by density centrifugation gradient method. Hereinafter, lymphocytes used for co-culture with the stimulator cells are also referred to as responder cells (reactive cells).

 [0050] After the co-culture, the stimulator cells and the responder cells are mixed and cultured. The mixing ratio is not particularly limited, but for example, stimulator cells: resp onder cells = 1: 4 is preferred! / ヽ.

 [0051] As the culture conditions for culturing the mixed cells, the temperature condition is, for example, 34 ° C to 38 ° C, preferably 37 ° C, and the CO condition is, for example, 2 to 10%. In the presence of CO

 twenty two

 Preferably, it is in the presence of 5% CO.

 2

 [0052] The culture period of the co-culture is preferably about 5 to 7 days. If cultured for more than 5 days, CTL can be induced, and if cultured for a long time without applying a new stimulus, the cell may die.

[0053] As the medium for co-culture, AIM-V medium (manufactured by Invitrogen), RPMI-1640 Medium (manufactured by Invitrogen), Dulbecco's modified Eagle medium (manufactured by Invitrogen), TIL (manufactured by Immunobiological Laboratories, Inc.), epidermal keratinocyte medium (manufactured by Kojin Bio Inc.), Iskov medium (manufactured by Invitrogen), etc. Commercially available media used for cell culture can be used. If necessary, 5-20% urine serum, fetal calf serum (hereinafter also referred to as FCS), human plasma, and the like may be added. Also, if necessary, you can add various site strengths.

[0054] (3. CTL induction efficiency measurement process)

 Finally, collect the cells and confirm the presence or absence of specific CTL inducibility. The confirmation method is not particularly limited, and is a force that can be applied to a conventionally known method.For example, in the case where the antigen epitope peptide sequence is divided, it is a tetramer of MHC class 1 / antigen peptide complex. CTL quantification method using tetramer can be applied. Further, when the antigen epitope peptide sequence is not known, ELISPOT method (Enzyme-Linked Immunospot Assay), a method for measuring intracellular site force in (interferon γ), etc. can be applied.

 [0055] By performing the evaluation method of the present invention as described above, it is possible to confirm whether or not an antigenic epitope peptide having a specific CTL-inducing ability is present in a test protein. Inducibility can be evaluated.

 [0056] The antigen protein screening method of the present invention comprises a protein containing an antigenic epitope peptide that specifically induces CTL (hereinafter referred to as an antigen protein) by using the evaluation method of the present invention. It is also a screening method including a step of identifying. With such a method, for example, a protein containing an antigenic epitope peptide can be easily searched from various cancer cell-derived proteins and virus proteins. Further, the identified protein can be targeted for identification for identification of a new antigen epitope peptide, for example, as described later.

 [0057] The method for screening an antigenic epitope peptide of the present invention comprises the step (i) and the step

(Ii) A screening method including at least one of the steps. In the method for screening an antigenic epitope peptide of the present invention, the test protein is identified by the screening method for an antigenic protein of the present invention, which may be a known antigenic protein. It may be a novel antigen protein. The test protein is preferably the latter antigen protein.

 [0058] The step (A) is a part of a known antigen protein whose antigen protein or antigen epitope peptide is unknown, for example, identified by the antigen protein screening method of the present invention. This is a step of performing the evaluation method of the present invention using various partial peptides as test proteins (hereinafter also referred to as test partial peptides), and identifying partial peptides containing the antigenic epitope peptides from the test partial peptides. By performing such a method by changing the length or cleavage site of the test partial peptide introduced into the antigen-presenting cell, it becomes possible to narrow down the antigen epitope peptide, and finally, the antigen epitope The peptide can be identified. The number of amino acid residues of an antigenic epitope peptide is generally 9 to: L1 residues.

 [0059] In the above step), for example, various test partial peptides are synthesized based on the antigen protein identified by the antigen protein screening method of the present invention, and the synthesized test partial peptide is converted to the antigen of the present invention. In the step of co-culturing with lymphocytes after contacting with the presenting cells or in contact with them, examining the induction efficiency of specific CTLs in the lymphocytes, and identifying the antigen epitope peptide from the test partial peptide is there. By changing the length and cutting position of the test partial peptide, it becomes possible to narrow down the antigen epitope peptide, and finally the antigen epitope peptide can be identified. The number of amino acid residues of an antigen epitope peptide is generally 9 to 11 residues.

[0060] The kit of the present invention is an evaluation kit used in the method for evaluating CTL inducibility of the present invention, and comprises the antigen-presenting cell of the present invention. In another aspect, the kit of the present invention is a screening kit for use in the screening method for the antigen protein and Z or antigen epitope peptide of the present invention, comprising the antigen-presenting cell of the present invention. Let's say. Conventionally, in order to evaluate the ability to induce CTL, it is necessary to prepare antigen-presenting cells each time, and such a kit is difficult. However, by using the antigen-presenting cells of the present invention, a kit can be prepared, whereby a simple and highly accurate method for evaluating the ability to induce CTLs and a method for screening antigen proteins and Z or antigen epitope peptides. It becomes possible. In addition to the antigen-presenting cell of the present invention, the kit of the present invention can be used for culture. Necessary items such as a culture medium and a reagent can be included.

 [0061] The method for producing an antigenic epitope peptide having the ability to induce CTLs of the present invention includes a step of identifying an antigenic epitope peptide by the method for screening an antigenic epitope peptide of the present invention, and other steps. Is not particularly limited. It is preferable that the production method of the present invention further includes a step of separating the antigenic epitope peptide identified as the protein force and a step of synthesizing the identified antigenic epitope peptide.

[0062] [Example]

 Hereinafter, the present invention will be described in more detail with reference to examples. However, it goes without saying that the present invention is not limited to these examples.

 Example 1

 [0063] <Establishment of MDA-CD80 cell line>

 The MDA-CD80 cell line, which is a cell line modified from the MDA-MB 231 cell line so as to express human CD80 molecule constitutively, was established by the following method.

 [0064] Cell Power Expressing CD80 Molecule Using the extracted RNA, RT-PCR method was used to prepare a cDNA fragment in the form of a cage, and primers C 080 and 1187-1 consisting of the following two types of synthetic oligo DNAs: : 1 ^ was used to amplify the CD80 gene fragment by PCR. The underlined part in the primer base sequence below indicates the sequence of the restriction enzyme site (Hindlll; CD80: F, Xbal; hB7-1: R) for insertion into the vector.

 [0065] [Chemical 1] Primer CD80: F

 5 '-TTCAAGCTTACCATGGGCCACACACGGAGGCAGGGAACATCACC-3' (SEQ ID NO: 1) Primer hB 7-1: R

 5 '-TAATCTAGATGCGGACACTGTTATACAGG-3' (SEQ ID NO: 2)

PCR is performed using LATaq DNA polymerase (Takara) for 3 minutes at 94 ° C, then for 30 seconds at 94 ° C, 30 seconds at 55 ° C, and 30 seconds at 72 ° C. The reaction was performed for 30 cycles as a cycle, and finally at 72 ° C for 5 minutes. The DNA fragment specifically amplified by the PCR method was excised by 1.5% agarose gel electrophoresis, It was treated with restriction enzymes Hindlll and Xbal, and inserted between the Hindlll site and Xbal site of plasmid pRcZCMV (Invitrogen) to obtain pRcZCMV-CD80. When the nucleotide sequence of the inserted human CD80 cDNA was confirmed by a DNA sequencer, it was found to be complete with the cDNA sequence of human CD80 reported in the literature [Free man GJ et al. (J. Immunol.) 143 2714-2722 (1989)]. Matched.

 [0067] MDA—MB—231 cells were mixed with a 10% FCS (CELLect

 The cells were cultured in RPMI 1640 medium containing Gold Fetal Bovine Serum (manufactured by ICsN Biomedicals, Inc.) for 4 to 6 hours. 6 μg of the pRcZCMV-CD80 was introduced into the cells by the ribofusion method. 48 hours after introduction, it was confirmed that the CD80 molecule was transiently expressed using a flow cytometer (EPICS XL / MCL, manufactured by Beckman Coulter, Inc.). It was also confirmed. At the same time, the medium was changed to a medium in which the antibiotic G418 was added to the medium, and cells having the plasmid integrated into the chromosome were selected. Furthermore, the selected cells were also clotted by single cell force by the limiting dilution method to obtain a cell line MDA-CD80 that stably expresses human CD80 molecules.

 [0068] It was confirmed that the MDA-CD80 expressed a CD80 molecule by FACS using an anti-CD80 antibody and RT-PCR using mRNA extracted from the MDA-CD80. An example of the results is shown in FIGS. In FIG. 1, the vertical axis represents the number of cells, and the horizontal axis represents the intensity of fluorescence (expression frequency of CD80 molecule) in one cell. The white mountain represents the control (MDA-MB-231) cells, and the black mountain represents the MDA-CD80 cells. As shown in the figure, FACS confirmed that the CD80 molecule was expressed on the MDA-CD80. In FIG. 2, lane a is a marker, lane b is MDA-MB-231, and lane c is MDA-CD80. As shown in the figure, it was confirmed that the MDA-CD80 (lane c) showed higher expression of CD80 mRNA than its parent cell MDA-MB-231 (lane b)! .

 [0069] <Induction of CTL by BMLF1 gene transfer>

RT-PCR was performed using RNA extracted from cells expressing BMLF1! / And a cDNA library was prepared. The BMLF1 is derived from EBV (Epstein-Barr Virus) It is a protein. The BMLFl gene fragment was selected as much as possible from the prepared cDNA library, and the obtained BMLF1 fragment was amplified by PCR. The amplified gene fragment was found at 1,317 bp.

 [0070] pTracer ™ —SV40 (trade name, manufactured by Invitrogen, hereinafter also referred to as pTracer) and the ubiquitin gene (hereinafter also referred to as Ub) were cleaved with restriction enzymes Aflll and Kpnl to restrict ρΤ racer. Ub was inserted between the enzyme cleavage sites to create pTracer-Ub.

 [0071] The pTracer-Ub and the BMLF1 fragment were cleaved with restriction enzymes Kpnl and Notl, and BMLF1 was inserted between the restriction enzyme cleavage sites of pTracer-Ub to prepare pTracer-Ub-BMLF1.

 [0072] Without introducing Ub, in some cases, the pTracer and the BMLF1 fragment are cleaved with restriction enzymes Kpnl and Notl, and BMLF1 is inserted between the restriction enzyme cleavage sites of pTracer, p

Tracer-BMLF 1 was prepared.

[0073] 1 x 10 ⁶ cells of MDA—CD80 cell line was cultured on 6-well plate (Sumitomo Bakelite) for 4-12 hours, and then pTracer—Ub—BMLFl (4 μg) was added by lipofusion method. To the MDA-CD80 cells.

[0074] After 24 hours, the transfection efficiency was confirmed by measuring the expression level of GFP (Green Fluorescent Protein) by FACS.

 [0075] pTracer— Ub— BMLFl-introduced MDA— CD80 cells per 10 I X 10 ° cells

Treated with 0 μg of mitomycin (2 mg for mitomycin injection, manufactured by Kyowa Hakko Kogyo Co., Ltd.) for 30 minutes to prepare stimulator cells.

[0076] On the other hand, a blood cell centrifuge (Lymphoprep, manufactured by AXIS—SHIELD PoC AS)

PBMCs were prepared by a centrifugation method using) and used as responder cells.

The stimulator cells (5 × 10 ⁵ cells) and the responder cells (2 × 10 ⁶ cells) were mixed and cultured at 37 ° C. under 5% CO for 7 days. At this time, IL-2 (PROLEUKIN,

 2

 CHIRON) was added to 100 U / ml. As the medium, AIM-V medium supplemented with 10% FCS was used.

[0078] After 7 days of culture, cells were collected, so that 100 1 per 5 X 10 ⁵ cells PBS (phos Suspended in phate buffered saline, phosphate buffer, Dulbecco PBS (—), manufactured by Nissui Pharmaceutical Co., Ltd.

 [0079] In the cell suspension, 5 µ 1 of anti-CD80-FITC (CD8 a, IMMUNOTECH) and 51 BMLF1-specific T-select MHC classl tetramer-PE (T-sele ct HLA— A * 0201 EBV Tetramer—GLCTLVAML—PE, manufactured by Medical and Biological Laboratories Co., Ltd.) was added and reacted at room temperature for 15 minutes.

 [0080] After the cells were washed with PBS and suspended again in PBS, the cell population expressing CD8 (CD8-positive) and recognizing BMLF1 (tetramer-positive) was measured by FACS. Ability to express CD8 and bind tetramer. Specific CTL against BMLF1.

 [0081] Examples of the FACS measurement results are shown in FIGS. Fig. 3A is an example of the result of Mock treatment in which only pT racer is introduced into MDA-CD80. Fig. 3B is an example of the result of introduction of p Tracer-BMLF1 into MDA-CD80. Fig. 3C is an example of MDA- This is an example of the result of introducing pTracer-Ub-BMLFl into CD80. 3A to C, the horizontal axis shows 1S FITC-CD8 signal, and the vertical axis shows PE-Tetramer signal. As shown in FIG. 3A and FIG. 3B, it was confirmed that by introducing a gene encoding a full-length protein derived from virus into MDA-CD80, antigens were presented and specific CTLs could be induced. In addition, as shown in Fig. 3C, when the ubiquitin-antigen peptide fusion gene was introduced, the specific CTL induction 'detection efficiency was 3 It was confirmed that it improved more than twice.

 Example 2

 [0082] <Induction of CTL by introducing Marti gene>

MDA-CD80 cell line of 1 × 10 ⁶ cells (prepared in Example 1) was cultured on 6-well plates for 4-12 hours.

[0083] RT-PCR was performed using RNA extracted from cells expressing Marti! /, And cDN

A library was prepared. Marti is an antigenic protein of melanoma (malignant melanoma).

[0084] Marti gene fragments selected from the cDNA library prepared above were selected and Marti obtained. The gene fragment was amplified by PCR. The amplified gene fragment was 357 bp.

[0085] The pTracer-Ub and the Marti gene fragment were cleaved with restriction enzymes Kpnl and Spel, and Marti was inserted between the restriction enzyme cleavage sites of pTracer-Ub to prepare pTracer-Ub-Martl. .

 [0086] In the case where Ub was not introduced, the pTracer and the Marti gene fragment were cleaved with restriction enzymes Kpnl and Spel, and Marti was inserted between the restriction enzyme cleavage sites of pTracer. Was made.

[0087] The pTracer—Ub—Marti was converted to MDA by the same lipofussion method as in Example 1.

— Transfection efficiency was confirmed by measuring the expression level of GFP by FACS 24 hours after introduction into CD80 cells.

[0088] MDA-CD80 cells into which pTracer-Ub-Martl was introduced were treated with 100 μg of mitomycin per 1 × 10 ⁶ cells for 30 minutes to form stimulator cells.

[0089] On the other hand, a blood cell centrifuge (Lymphoprep, manufactured by AXIS—SHIELD PoC AS)

PBMCs were prepared by a centrifugation method using) and used as responder cells.

The stimulator cells (5 × 10 ⁵ cells) and the responder cells (2 × 10 ⁶ cells) were mixed and cultured at 37 ° C. under 5% CO for 7 days. At this time, IL-2 becomes lOOUZml

 2

 I ordered it as follows. As the medium, AIM-V medium supplemented with 10% FCS was used.

[0091] After culturing for 7 days, the cells were collected and suspended in PBS so that 5 × 10 ⁵ cells per 100 1 were obtained.

 [0092] In the cell suspension, 5 μ 1 of anti-CD80-FITC and 5 μ 1 of Marti-specific T—select MHC classl tetramer-PE (T-select HLA—A water 0201 Marti Tet ramer— ELAGIGILTV—PE, manufactured by Medical and Biological Laboratories Co., Ltd.) was added and allowed to react at room temperature for 15 minutes.

 [0093] After the cells were washed with PBS and suspended again in PBS, the cell population expressing CD8 (CD8-positive) and recognizing Marti (tetramer-positive) was measured by FACS. The ability to express CD8 and bind to tetramer. Specific CTL against Marti.

[0094] Examples of the FACS measurement results are shown in FIGS. Figure 4A shows MDA—CD80 with pT Fig. 4B shows an example of the result of introducing p Tracer-Marti into MDA-CD80, and Fig. 4C shows the result of introducing pTracer Ub-Marti into MDA-CD80. It is an example of a result. 4A to C, the horizontal axis represents the FITC-CD8 signal, and the vertical axis represents the PE-Tetramer signal. As shown in FIG. 4A and FIG. 4B, it was confirmed that by introducing a gene encoding a full-length protein derived from tumor cells into MDA-CD80, antigens were presented and specific CTLs could be induced. In addition, as shown in Fig. 4C, when a fusion gene of ubiquitin and an antigenic peptide is introduced, the specific CTL induction 'detection efficiency power 2. 7 times improvement was confirmed.

 Example 3

 [0095] Evaluation of CTL inducibility by measuring intracellular site force in (interferon γ)>

 By introducing the BMLF1 gene into MDA—CD80 cells to induce CTL and measuring interferon γ (hereinafter also referred to as IFN—y) produced by the induced CTL, BMLF

1 CTL inducibility was evaluated.

[0096] In the same manner as in Example 1, pTracer-Ub-BMLF1 and pTracer were introduced into MDA-CD80 to prepare stimulator cells, and responder cells were prepared in the same manner as in Example 1.

The stimulator cells (5 × 10 ⁵ cells) and the responder cells (2 × 10 ⁶ cells) were mixed and cultured at 37 ° C. and 5% CO for 7 days. At this time, IL-2 becomes lOOUZml

 2

 I ordered it as follows. As the medium, AIM-V medium supplemented with 10% FCS was used. Here, the stimulator cells are MDA-CD80 introduced with pTracer-Ub-BMLF 1 and MDA-CD80 introduced with pTracer.

 [0098] After 7 days of culture, responder cells were collected and washed with PBS (-). As described above, since the stimulator cells have been treated with mitomycin in advance, only about responder cells remain in the collected cells after 7 days of culture.

[0099] The CTL induced by the first stimulator cell stimulation reduces the amount of IFN-y produced during the 7-day culture of IFN-γ. To facilitate the detection of IFN-y production, a second stimulation was performed. The first stimulation already induced CTL Because it is being performed, the second stimulation may be brief. For the second stimulation described below, use MDA-CD80 pulsed with epitope peptide! /, But if the epitope peptide is unknown, stimulator cells, for example, ubiquitin antigen MDA-CD80 in which a protein fusion protein is introduced using pTracer or the like can be used

[0100] The collected responder cells (2 × 10 ⁵ cells) were suspended in 100 μl of AIM-V medium supplemented with 10% FCS. As a second stimulator cell, MDA-CD80 (1 × 10 ⁵ cells) pulsed with epitope peptide was suspended in AIM-V medium supplemented with 10% FCS at 100 / z 1 and prepared.

 [0101] The responder cells and the stimulator cells were mixed in a 96-well round bottom plate (manufactured by Sumitomo Beichikrite), and 50 UnitZml IL-2 and 40 μgZml BefeldinA (manufactured by Sigma Chemicals) were mixed. 4% at 37 ° C and 5% CO.

 Incubated for 2-6 hours

. The BefeldinA is a reagent that inhibits the release of cytodynamic force in the cell. Therefore, IFN-γ produced in the cell is accumulated in the cell.

[0102] Cells were collected, washed with PBS (-), suspended in 100 1 PBS (-), added with 5 1 Anti-C D8FITC and 5 μ 1 Anti-CD3-PC5, respectively, at room temperature. Allowed to react for 15 minutes

[0103] 100 1 IntraPrep Reagent 1 (cell membrane permeabilization reagent, manufactured by Beckman Coulter, Inc.) was added. The IntraPrep Reagent is a drug for fixing cells. The sample was vortexed to mix well and left at room temperature for 15 minutes. The cells were collected, washed with PBS (-), and suspended in 100 1 PBS (-). Then, 1001 IntraPrep Reagent 2 (cell membrane permeabilization reagent, manufactured by Beckman Coulter, Inc.) was added and allowed to stand at room temperature * location for 5 minutes. The IntraPrep Reagent 2 is a reagent for making a hole in the fixed cell surface so that the antibody can react with intracellular IFN-y.

 [0104] 5 µ 1 of Anti-IFN-γ-FITC (manufactured by IMMUNOTECH) was added and allowed to react at room temperature for 15 minutes. Cells were collected, washed with PBS (—), and CD3-positive, CD8-positive and IFN-γ-positive cells were measured by FACS.

[0105] Examples of the results are shown in Figs. Figure 5 shows that only pTracer is installed on MDA—CD80. Fig. 5B shows an example of the result of introducing pTracer-Ub-BMLF1 into MDA-CD80. 5A and 5B, the horizontal axis represents the FITC-CD8 signal, and the vertical axis represents the IFN-γ signal. As shown in Fig. 5 and Fig. 5, when MDA-CD80 with pTracer—Ub—BMLF 1 is used for the first stimulation, compared to the case where only IFN—γ-quantity pTracer produced is introduced. It was confirmed that the price was getting higher. That is, it was confirmed that by introducing the BMLF1 gene, antigen presentation was performed and specific CTLs were induced.

 Industrial applicability

[0106] As described above, according to the present invention, specific CTL inducibility can be evaluated quickly and easily, so that screening of antigen proteins and antigen epitope peptides can be performed quickly and easily. Therefore, the present invention is useful, for example, in the field of searching for novel antigen epitope peptides, and in the medical field including immune cell therapy for the treatment Z prevention of cancer, infectious diseases and the like.

 Sequence listing free text

[0107] SEQ ID NO: 1 primer CD80: F

 SEQ ID NO: 2primer hB7—1: R

Claims

The scope of the claims  [I] A method for evaluating a specific ability to induce CTL in a protein,  Introducing a gene encoding a test protein into an antigen-presenting cell so that the protein can be expressed;  Co-culturing the antigen-presenting cells into which the gene has been introduced with lymphocytes, and examining the induction efficiency of CTL in the lymphocytes,  As the antigen-presenting cells, T cell costimulatory factors and MHC class I molecules are expressed, and all or part of the proteins expressed intracellularly can be presented on the MHC class I molecules. Evaluation method using cells derived from the same cell line. 2. The evaluation method according to claim 1, wherein the T cell costimulatory factor is CD80. [3] The evaluation method according to [1], wherein in the step of introducing the gene, the gene encoding ubiquitin is introduced into the protein in an expressible manner. 4. The evaluation method according to claim 1, wherein, in the step of introducing the gene, a gene encoding a fusion protein of the protein and ubiquitin is introduced so as to be expressed instead of the gene encoding the protein. 5. The evaluation method according to claim 1, wherein the gene introduction is a transient gene introduction. 6. The evaluation method according to claim 1, wherein the protein is derived from a cancer cell or a virus. 7. The evaluation method according to claim 1, wherein the antigen-presenting cell is an adherent cell. 8. The evaluation method according to claim 1, wherein the antigen-presenting cell is a cell derived from a tumor cell-derived cell line.  9. The evaluation method according to claim 8, wherein the tumor cell-derived cell line is a breast cancer-derived cell line MDA-MB-231.  [10] A method for screening a protein capable of specifically inducing CTL, wherein the test protein has a specific CTL inducing ability by evaluating the specific CTL inducing ability of the test protein according to the evaluation method according to claim 1. A screening method comprising a step of identifying a protein. [II] A screening method for an antigen epitope peptide in a protein capable of specifically inducing CTL, the screening method comprising at least one of the following steps (A) and (B): (A) Instead of the step of introducing a gene encoding a test protein into the antigen-presenting cell so that the protein can be expressed, a polynucleotide encoding a partial peptide of the protein is introduced into the antigen-presenting cell so that the protein can be expressed. The step of evaluating the CTL inducing ability of the partial peptide by the evaluation method according to claim 1, wherein the antigenic epitope peptide having a specific CTL inducing ability is identified.  (B) In place of both the steps of introducing a gene encoding a test protein into the antigen-presenting cell so that the protein can be expressed and co-culturing the antigen-presenting cell into which the gene has been introduced with lymphocytes The method according to claim 1, further comprising the step of co-culturing the partial peptide of the protein with lymphocytes after or after contacting with the antigen-presenting cell. A step of evaluating and identifying an antigenic epitope peptide having a specific ability to induce CTL.  [12] The screening method according to claim 11, wherein the protein identified by the method according to claim 10 is used as the test protein. [13] The screening method according to [11], wherein the partial peptide is a peptide consisting of 9 to 11 amino acid residues.  [14] An antigen-presenting cell used in the method for evaluating a specific CTL inducing ability of the protein according to claim 1, which expresses a T cell costimulatory factor and an MHC class I molecule, and is expressed in the cell. An antigen-presenting cell derived from an established cell line capable of presenting all or part of the protein on the MHC class I molecule as an antigen.  [15] An antigen-presenting cell for use in the method for screening a protein capable of specifically inducing CTL according to claim 10, wherein the cell presents a T cell costimulatory factor and an MHC class I molecule, and is expressed intracellularly. An antigen-presenting cell derived from an established cell line capable of presenting all or part of the expressed protein on the MHC class I molecule. [16] An antigen-presenting cell for use in the method for screening an antigenic epitope peptide in a protein capable of specifically inducing CTL according to claim 11, which expresses a T cell costimulatory factor and an MHC class I molecule. And an antigen derived from an established cell line capable of presenting all or part of the protein expressed in cells on the MHC class I molecule as an antigen. Original presentation cell.  [17] An evaluation kit for use in the method for evaluating a specific CTL inducing ability of the protein according to claim 1, comprising the antigen-presenting cell according to claim 14. 18. A screening kit for use in the method for screening a protein capable of specifically inducing CTL according to claim 10, comprising the antigen-presenting cell according to claim 15. [19] A screening kit for use in the method for screening an antigenic epitope peptide in a protein capable of specifically inducing CTL according to claim 11, comprising the antigen-presenting cell according to claim 16.  [20] A method for producing an antigenic epitope peptide having a specific ability to induce CTL, the method for screening an antigenic epitope peptide in a protein capable of specifically inducing CTL according to claim 11 The manufacturing method including the process of identifying the antigenic epitope peptide of the said protein by.  21. The production method according to claim 20, further comprising at least one of a step of separating the identified antigenic epitope peptide identified as a protein force and a step of synthesizing the identified antigenic epitope peptide.