KR100669065B1 - Cytotoxic t lymphocyte - Google Patents

Abstract

(Problem) To provide a technique useful for treating and diagnosing a disease caused by the specific expression of prostate specific membrane antigen in Asians, in particular Japanese, specifically cancer, and more preferably prostate cancer.

(Solution) A T cell having the sequence of SEQ ID NO: 1 or 2 or a sequence having a mutation, and which specifically recognizes a cell presenting a complex of the peptide of this sequence and the HLA-A24 molecule on the cell surface. HLA-A24 binding antigen peptides and nucleic acids thereof which induce CTLs with receptors; CTL having this T cell receptor; CTL induction method using this HLA-A24 binding antigen peptide; CLA inducers and cancer suppressors containing HLA-A24 binding antigen peptides; CTL containing cancer inhibitors and CTL sensitive cell detectors; Antigen presenting cells presenting this complex; Antigen presenting cell-containing CTL inducers and cancer suppressors; When a change selected from the group consisting of cell lysis, cytokine release and CTL proliferation by CTL occurs by contacting the CTL with a test cell, a method for detecting CTL-sensitive cells as an indicator that the test cell is a CTL-sensitive cell. And detection agents; Detection method of HLA-A24 molecule; Target cells of CTL and a method of manufacturing the same.

T lymphocytes

Description

Cytotoxic T lymphocytes {CYTOTOXIC T LYMPHOCYTE}

Figure 1 shows the results using effector cells induced by PSMA24-3. White squares (□) indicate TISI (-) as target cells, and black circles (●) indicate TISI (+) results. The vertical axis is specific cytotoxic activity (%), and the horizontal axis is E / T ratio.

Figure 2 shows the results using effector cells induced by PSMA24-5. White squares (□) indicate TISI (-) as target cells, and black circles (●) indicate TISI (+) results. The vertical axis is specific cytotoxic activity (%), and the horizontal axis is E / T ratio.

The present invention relates to techniques useful for the treatment and diagnosis of diseases, such as cancer, specifically prostate cancer, etc., in which the prostate specific membrane antigen (hereinafter referred to as PSMA) is specifically expressed. More specifically, it has a T cell receptor capable of recognizing a complex of an antigen peptide and a major human histocompatibility antigen (HLA) molecule useful for the treatment and diagnosis of cancer, specifically PSMA, including prostate cancer. Detection of cytotoxic T lymphocytes (also known as CTLs), the antigen peptides, CTL inducers useful when inducing or inducing CTLs, methods of inducing CTLs, cancer suppressors useful in the cancer, and detection of susceptible cells of these CTLs The present invention relates to a method, a detection agent thereof, a method for detecting HLA-A24 molecules, a target cell injured by the CTL, and a method for producing the same.

Prostate cancer is the most common carcinoma in the United States, but is proliferating in all Asian countries, including Japan. Prostate cancer has been shown to be highly sensitive to hormonal therapy and radiotherapy. However, since hormone refractory cancer occurs with prolonged treatment, effective treatments for such conditions are required and immunotherapy is expected.

In CTL, the major histocompatibility gene complex (MHC), which is encoded by the antigen peptide and the major histocompatibility gene complex (MHC), is called human leukocyte antigen class I (hereinafter referred to as human leukocyte antigen class I). ) The complex, which is a combination of molecules, is recognized by a specific T cell receptor (hereinafter referred to as TCR), which may injure cells presenting the complex on the cell surface. Such a CTL is called an MHC class I molecular binding CTL in that only a target cell having the same MHC class I molecule as the CTL itself is recognized and injured.

Therefore, in order to establish this cytotoxic reaction, 1) the presence of a CTL having a specific TCR, 2) the antigen peptide presented in the HLA class I molecule and recognized by the CTL, so that the binding to the MHC class I molecule In addition, there is a need for an antigen peptide capable of forming a complex that is recognized by TCR.

Such antigenic peptides are generated by, for example, antigens synthesized in cells of mammalian cells, processed in endoplasmic reticulum and degraded into small epitope peptides, and are associated with MHC class I molecules and presented on the cell surface. That is, in a proteosome complex consisting of many subunits, the protein is broken down into peptides consisting of 8-15 amino acids, some of which are transported from the cytoplasm to the endoplasmic reticulum by the TAP transporter. These peptides are stabilized as trimolecular complexes if they are endoplasmic reticulum and can bind heterodimers of class I / β ₂ microglobulin and are transported through the Golgi apparatus to the cell surface. Tumor cells expressing tumor-associated antigens or tumor specific antigenic proteins are capable of presenting MHC class I molecularly binding antigen peptides recognized in T lymphocytes on the surface of tumor cells.

The first tumor-specific antigen recognized in T lymphocytes was cloned and identified by P. van der Bruggen et al., A gene called melanoma antigen E (hereinafter referred to as MAGE). Van der Brgen et al., Science, 254, pp 1643-1647 (1991). P. van der Brgeneu et al. Cloning expression using a CTL clone from Melanoma patients, a series of melanoma cell lines from the same patient recognized by this CTL, and a cell line obtained from which some of these cell lines are immune-selected as if they were resistant to this CTL. MAGE was isolated by law.

That is, when a 5 kb DNA fragment prepared from a melanoma cell line recognized by CTL is transduced into a cell line that was not originally recognized by CTL, it is disclosed that the resulting transduction line is recognized by the CTL clone. The DNA fragment encodes a protein having an estimated molecular weight of about 26,000. The gene encoding this protein is called MAGE-1. In addition, it has been disclosed that 40% of cell lines derived from melanoma as well as metastatic as well as early stages express the MAGE-1 by mRNA analysis. Antigens derived from MAGE-1 protein are HLA-A1 binding, and MAGE-1 specific CTL clones are disclosed to recognize peptides consisting of 9 amino acid residues bound to an HLA-A1 molecule, which is a type of HLA class I molecule [ C. C. Traversari et al., Journal of Experimental of Medicine, 176, pp. 1453-1457 (1992).

Although there are many antigens known as tumor antigens, antigens recognized by CD8-positive CTL include MAGE-1, gp100, tyrosinase, and CEA, which are known to exist in the dozens or more, including MAGE-1. , HER2 / neu and the like are well known. Recognized by CTLs are peptides derived from tumor antigen proteins, which are presented as complexes with HLA class I molecules.

HLA class I molecules are mainly HLA-A, HLA-B, and HLA-C. The antigen peptides that are combined with these are composed of 8 to 10 amino acids, and also have a certain structural structure depending on the type of each HLA molecule. It is known that there is a feature. For example, as a peptide bound to the world's most frequent HLA-A2.1 molecule, a peptide in which Leu is placed at the 2nd position at the N-terminus and Leu or Val is placed at the C-terminus, 9 to 10 amino acids Peptides consisting of residues are best known. In addition, as a peptide bound to many HLA-A24 molecules to Asians including Japanese, any one of Tyr, Phe, Met or Trp is disposed at the 2nd position at the N-terminal, and Leu, Ile, Trp at the C-terminal position. Or a peptide consisting of 9 to 10 amino acids is best known as a peptide in which either Phe is disposed [A. Kondo et al., Journal of Immunology, Vol. 155, pp4307 ~. 4312 (1995).

Tumor antigens to which antigen peptides have been identified to date include MAGE-1 and MAGE-3 for HLA-Al; MAGE-3, MART1, tyrosinase, gp100, HER2 / neu, CEA, etc. for HLA-A2.1; MAGE-3 for HLA-Cw1, MAGE-3 for HLA-B44, and the like; MAGE-1, MAGE-2, MAGE-3, CEA, HER2 / neu, tyrosinase, β-catenin and the like for HLA-A24.

In prostate cancer, a tumor marker called prostate specific membrane antigen (hereinafter referred to as PSMA) is expressed, and it is expected to provide antigen peptides targeting these. Regarding the antigenic peptide derived from PSMA, peptide consisting of 9 residues having the amino acid sequence of SEQ ID NO: 3 (PSM-P1: LLHETDSAV) and 9 residues having the amino acid sequence of SEQ ID NO: 4 (PSM-P2: ALFDIESKV) Two types of peptides have been identified as HLA-A2.1 binding antigen peptides (Prostate, Vol. 28, pp. 65-69 (1996)).

In addition, PSMA is known to be expressed not only in tumor cells of prostate cancer but also in neovascularization of tumor sites (Chang S.S. et al., Cancer Research, 59, pp3192 to 3198 (1999)).

Most of these tumor antigen peptides first cheat the class I binding CTLs that recognize tumor cells, identify the tumor antigens recognized by the CTLs, and then genetically analyze the CTLs in the tumor antigen proteins. By finding the minimum recognizable unit and identifying the antigenic peptide among the minimum units based on the information on the binding motif to the HLA class I molecule [Y. Kawakami et al. Proceedings of the National Academy of the Sciences of the United States of America, 91, pp. 3515-3519 (1994). First, based on the motif structure common to the HLA class I molecular binding peptide, the HLA class I molecular binding peptide in the tumor antigen protein was found, and then, the antigen-presenting cells were selected to induce CTL. Finally, antigenic peptides are determined by whether they can induce injured CTLs against tumor cells [E.Celis et al., Proceedings of the National Academy of Sciences of the USA, 91, pp. 2105-2109 (1994)], B. Gaugler et al., Journal of Experimental of Medicine, 179, pp 921-930 (1994).

However, having a HLA-A24 binding motif in a peptide is only one condition for inducing CTL, and it is currently difficult to guess whether or not it exhibits selective cytotoxic activity against cells.

HLA class I molecules, on the other hand, are classified into several subtypes, and the types of retention subtypes vary greatly from race to race. In the world, HLA-A2 is the most common, accounting for 45% of white species, and the identification of this HLA-A24 binding antigen peptide is the best.

For Japanese, the HLA-A2 accounts for 40%, but in its subtype, HLA-A ^* 0201 is 20%, which is the same as that of Caucasians, most of which are HLA-A ^* 0206. Binding peptides for these subtypes are different and the HLA-A2 being studied is HLA-A ^* 0201. On the other hand, HLA-A24 accounts for more than 60% of Japanese people, and this HLA-A2 is higher in Asians than other races.

However, there are MAGE-1, MAGE-2, MAGE-3, CEA, HER2 / neu, tyrosinase, β-catenin, etc. for the antigen peptide bound by HLA-A24, but HLA-A2.1 Compared with this, the interpretation is still delayed, and new antigenic peptide expression for various tumor antigens is required. In addition, the interpretation of CTLs that specifically affect tumor cells in Asians, especially Japanese, has been delayed, making it difficult to provide CTLs useful for tumor treatment.

An object of the present invention is useful in the treatment and diagnosis of diseases such as cancer, specifically prostate cancer, etc., caused by the specific expression of prostate specific membrane antigen (hereinafter referred to as PSMA) in Asians, especially in Japan. Technology, such as CTL. In particular, for example, the present invention provides for the treatment and diagnosis of diseases such as cancer, specifically prostate cancer, etc., in which PSMA is specifically expressed, and both HLA-A24 and PSMA selectively select positive cells, particularly tumor cells. At least one of those which can cause injury, can be used as cell medicines (cancer inhibitors, etc.) for cancer treatment, and detects the presence of positive cells, especially tumor cells, in both HLA-A24 and PSMA in ex vivo samples. It aims to provide a CTL that can achieve one. The present invention also provides an HLA- that can achieve at least one of being able to selectively induce such CTL, to be used as such CTL inducer, and to enable treatment of diseases such as cancer based on the CTL induction. It is an object to provide an A24 binding antigen peptide and a nucleic acid encoding the same. In addition, an object of the present invention is to provide an antigen-presenting cell that can achieve at least one of the use in the production of the CTL, the treatment of diseases such as cancer based on the CTL induction. It is also an object of the present invention to provide a CTL derivation method and a CTL derivating agent which can efficiently and selectively obtain the CTL of the present invention. In addition, a method of detecting CTL sensitive cells capable of efficiently detecting CTL sensitive cells, and at least one of diagnosing a disease such as HLA typing, cancer, specifically prostate cancer, and the like. And it aims at providing a detection agent. In addition, the present invention provides a target that is injured by cytotoxic T lymphocytes capable of expressing human major histocompatibility antigens and / or antigenic proteins in human major histocompatibility antigen-free and / or antigenic proteins. It is an object of the present invention to provide a cell and a method for producing this target cell which can be obtained efficiently.

Means to solve the problem

[1] the following (a) or (b):

(a) the amino acid sequence as set forth in SEQ ID NO: 1 or 2 or

(b) the following ① to ③ in SEQ ID NO: 1 or 2:

① deletion of at least one residue of amino acid,

② substitution of at least one residue of an amino acid with another amino acid or an amino acid analog, and

③ has an amino acid sequence of an amino acid sequence having one or more mutations selected from the group consisting of addition of at least one residue of other amino acids or amino acid analogs, and

Having a property of inducing CTL having a T cell receptor capable of specifically recognizing a cell presenting a complex of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2 and the HLA-A24 molecule on the cell surface Human major histocompatibility antigen (HLA) -A24 binding antigen peptide,

[2] A cytotoxic T lymphocyte having a T cell receptor capable of specifically recognizing a cell presenting a complex of the HLA-A24 binding antigen peptide and the HLA-A24 molecule described above in the cell surface,

[3] A method of inducing cytotoxic T lymphocytes, wherein the cytotoxic T lymphocytes are induced by using the HLA-A24 binding antigen peptide described in [1] above.

[4] (1) The antigen-presenting cell is obtained by contacting a cell having one type of antigen-producing ability selected from the group consisting of the HLA-A24-binding antigen peptide, macrophage, B cell and dendritic cell described in [1] above. To obtain effector cells by giving antigen stimulation to cells having antigen-presenting ability using the obtained antigen-presenting cells.

(2) contacting effector cells obtained in step (1) and target cells injured in cytotoxic T lymphocytes described in [2], and detecting the presence of cytotoxic activity;

(3) the induction method according to the above [3], comprising the step of selecting effector cells having cytotoxic activity as cytotoxic T lymphocytes in the step (2);

[5] The induction method according to the above [4], wherein the antigen-presenting cells are contacted with one lymphocyte at a ratio of 0.01 to 1 in the presence of IL-7.

[6] In the presence of IL-7 and keyhole limpet hemocyanin, 1 ng to 100 μg of the HLA-A24 binding antigen peptide described in [1] is added to 1 ml of the solution containing the peripheral blood monocytes to peripheral blood monocytes. The induction method described in [4],

[7] a CTL inducer for obtaining cytotoxic T lymphocytes (CTL) according to the above [2], containing the HLA-A24 binding antigen peptide according to the above [1] as an active ingredient;

[8] an anticancer agent comprising the HLA-A24 binding antigen peptide according to the above [1] as an active ingredient,

[9] an cancer inhibitor comprising the cytotoxic T lymphocytes according to the above [2] as an active ingredient;

[10] an antigen-presenting cell which presents a complex of the HLA-A24-binding antigen peptide and HLA-A24 molecule according to the above [1], on the cell surface;

[11] CTL inducer for obtaining cytotoxic T lymphocytes (CTL) according to [2], comprising the antigen-presenting cell according to [10] above as an active ingredient;

[12] an cancer suppressant containing the antigen-presenting cell according to the above [10] as an active ingredient;

[13] When a change selected from the group consisting of cell lysis, cytokine release and CTL proliferation by CTL occurs by contacting the cytotoxic T lymphocytes described above with the test cell, the test cell is characterized in that [ 2) A method for detecting susceptible cells of cytotoxic T lymphocytes, characterized in that the cells are susceptible to cytotoxic T lymphocytes (sensitive cells).

[14] An agent for detecting susceptible cells of cytotoxic T lymphocytes comprising the cytotoxic T lymphocytes according to [2] as an active ingredient;

[15] Human major histocompatibility antigens (HLA) in test cells, wherein the cytotoxic T lymphocytes described in [2] and the test cells are contacted in the presence of the HLA-A24 binding antigen peptide according to the above [1]. ) -A24 molecule detection method,

[16] a nucleic acid encoding the HLA-A24 binding antigen peptide described in [1] above,

[17] a method for producing target cells injured by cytotoxic T lymphocytes, the method comprising introducing a nucleic acid encoding a human major histocompatibility antigen and / or a nucleic acid encoding an antigenic protein into a cell;

[18] The method according to the above [17], wherein the introduction into the nucleic acid cell is carried out through an adenovirus vector.

[19] The production method according to the above [17] or [18], wherein the nucleic acid encoding a human major histocompatibility antigen is a nucleic acid encoding HLA-A24;

[20] The production method according to any one of [17] to [19], wherein the nucleic acid encoding the antigenic protein is the nucleic acid according to the above [16];

[21] A target cell that is injured by cytotoxic T lymphocytes obtained by the method according to any one of [17] to [20].

Embodiment of the invention

In the present invention, an antigen peptide was searched among a number of candidate peptides centered on a peptide having a HLA-A24 binding motif in a PSMA protein as a tumor antigen, and thus, among the candidate peptides, a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or 2 was selected. Using antigen-stimulated lymphocytes, based on the knowledge of the present inventors, it is possible to induce cytotoxic T lymphocytes (CTLs) that selectively injure these tumor antigen expressing cells from peripheral blood monocytes expressing HLA-A24. do.

In addition, in this specification, the terms "target cell" and "antigen presenting cell" are used as a term which shows the cell which presents the complex of HLA-A24 binding antigen peptide and HLA-A24 molecule on the cell surface.

In particular, the above-mentioned "target cell" refers to a cell that is injured by cytotoxic T lymphocytes (hereinafter also referred to as CTL) that specifically recognize the cell. Meanwhile, the "antigen-presenting cell" refers to a cell having a function of presenting an antigen together with an MHC molecule and causing activation of T lymphocytes, and unless otherwise specified, is a cell in which the MHC molecule is an HLA-A24 molecule.

The above-mentioned "cell with antigen-producing ability" means a cell capable of becoming an antigen-presenting cell by forming a complex of an MHC molecule and an antigen peptide present in the cell, and unless otherwise specified, the MHC molecule is an HLA-A24 molecule. It is a cell.

As used herein, cancer refers to a malignant tumor and includes any of carcinoma and internal tumor. In addition, the cancer of the present specification includes not only tumor cells but also tumor-related sites such as neovascularization within the tumor sites.

The HLA-A24 binding antigen peptides of the present invention are (a) or (b):

(a) the amino acid sequence as set forth in SEQ ID NO: 1 or 2 or

(b) the following ① to ③ in SEQ ID NO: 1 or 2:

① deletion of at least one residue of amino acid,

② substitution of another amino acid or amino acid analog of at least one residue

③ has an amino acid sequence of an amino acid sequence having one or more mutations selected from the group consisting of addition of at least one residue of other amino acids or amino acid analogs, and

The CTL having a T cell receptor capable of specifically recognizing a cell presenting the complex of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2 and the HLA-A24 molecule on the cell surface can be induced. There is one feature to have. In the present specification, the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2 may be referred to as a PSMA antigen peptide.

According to the HLA-A24 binding antigen peptide of the present invention, HLA-A24 binding CTL, i.e., a complex of the peptide and HLA-A24 molecule, useful for the treatment and diagnosis of a disease such as cancer caused by the expression of PSMA specifically on the cell surface It exerts an excellent effect of inducing CTL having a T cell receptor capable of specifically recognizing the presenting cell. In other words, since the HLA-A24-binding antigen peptide of the present invention is highly HLA-A24-binding to Asians, especially Japanese, tumor cells and / or tumor-associated tumors and / or tumors are particularly effective for the treatment of Asians, especially Japanese, in the treatment of diseases such as tumors. It exerts an excellent effect capable of specifically inhibiting a site, specifically a tumor cell and / or a tumor related site that specifically expresses PSMA, in particular a prostate cancer cell and / or a prostate cancer related site.

The present invention also includes a CTL having a T cell receptor capable of specifically recognizing cells presenting the complex of the HLA-A24 binding antigen peptide of the present invention and the HLA-A24 molecule on the cell surface.

As used herein, the term "antigenic peptide" refers to a peptide which is bound to an MHC class I molecule and has a complex-forming ability with this MHC class I molecule, and at the same time the formed complex has an antigenicity recognized by a specific CTL TCR.

In addition, in this specification, an "HLA binding antigen peptide" means that an MHC class I molecule is an antigen peptide which is an HLA molecule.

The HLA-A24 binding antigen peptide of the present invention may be a homomeric peptide consisting of only amino acids or a heteromeric peptide containing a non-amino acid component. The amino acid may be a natural amino acid or a chemically modified amino acid. In addition, the peptide may be a monomer or a multimer.

As the HLA-A24 binding antigen peptide of the present invention, for example, in the amino acid sequence shown in SEQ ID NO: 1 or 2, in the group consisting of deletion, substitution and addition for at least one residue amino acid, specifically one or several amino acids One or more selected mutations differ from the amino acid sequence shown in SEQ ID NO: 1 or 2, but have the ability to form a complex with the HLA-A24 molecule, while the complex formed forms the antigenic peptide represented by SEQ ID NO: 1 or 2 and HLA. And peptides recognized in CTL having a T cell receptor capable of specifically recognizing cells presenting a complex of -A24 molecules on the cell surface.

Specifically as the variation

① deletion of at least one residue of amino acid,

② substitution of at least one residue of an amino acid with another amino acid or an amino acid analog, and

③ addition of at least one residue of another amino acid or an amino acid analog. These variations may be alone or in combination.

The length of the amino acid sequence of the HLA-A24 binding antigen peptide of the present invention can specifically recognize a cell presenting the complex of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2 and the HLA-A24 molecule on the cell surface. The CTL having a T cell receptor may be used to exhibit a property capable of inducing CTL, and it is preferably 9 to 10 residues, but is not particularly limited. For example, in the case of a peptide consisting of an amino acid sequence having a mutation of? In the amino acid sequence shown in SEQ ID NO: 1 or 2, the amino acid sequence is preferably 9 to 10 residues, but is not particularly limited. In the amino acid sequence shown in SEQ ID NO: 1 or 2, in the case of a peptide consisting of an amino acid sequence having a mutation of ③, the length of the amino acid sequence is preferably 9 to 10 residues, but is not particularly limited.

Examples of the amino acid analogs include N-acylates, O-acylates, esterates, acidamides and alkylates of various amino acids.

In addition, the complex with the HLA-A24 molecule was added to a CTL having a T cell receptor capable of specifically recognizing a cell presenting the complex of the antigen peptide shown in SEQ ID NO: 1 or 2 and the HLA-A24 molecule on the cell surface. If recognized, a formyl group, an acetyl group, a t-butoxycarbonyl (t-Boc) group, or the like is bound to the N-terminal or free amino group of the antigen peptide, or a methyl group, an ethyl group, or the like to the C-terminal or free carboxyl group of the antigen peptide. t-butyl group, benzyl group, etc. may be combined.

In addition, the HLA-A24 binding antigen peptide of the present invention may be subjected to various modifications to facilitate the introduction into the body.

HLA-A24 binding antigen peptide of the present invention is a CTL having a T cell receptor capable of specifically recognizing cells presenting the complex of the antigen peptide shown in SEQ ID NO: 1 or 2 and HLA-A24 molecules on the cell surface It can select by using. The selection can be carried out, for example, by the following methods 1 to 3.

Method 1

After the candidate substance as HLA-A24 binding antigen peptide and HLA-A24 expressing cells of the present invention are mixed and washed to remove the candidate substance not bound to the HLA-A24 molecule, the obtained candidate-binding HLA-A24 expressing cells are reacted with CTL. . If candidate substance specific cytotoxicity, cytokine release or proliferative reaction is seen, the candidate may be determined as the HLA-A24 binding antigen peptide of the present invention.

Method 2

After contacting the cell with the antigen-presenting agent with the candidate, for a suitable time, for example, the antigen is inserted into the cell for processing, and then reacted for the required time because the antigen peptide and the HLA-A24 molecular complex are presented on the cell surface. React the reactant with CTL. If a candidate specific cytokine release or proliferation reaction is seen, the candidate may be determined as the HLA-A24 binding antigen peptide of the present invention.

Method 3

The antigen-presenting ability described below is obtained by inserting a nucleic acid encoding an amino acid sequence of a candidate into an expression vector capable of presenting a peptide on a HLA-A24 molecule on a cell, and having the antigen-forming ability transformed using a recombinant vector. React the cell with CTL. If a candidate specific cytokine release or proliferation reaction is seen, the candidate may be determined as the HLA-A24 binding antigen peptide of the present invention.

In the HLA-A24 binding antigen peptide of the present invention, the complex with the HLA-A24 molecule may specifically recognize a cell presenting the complex of the antigen peptide shown in SEQ ID NO: 1 or 2 with the HLA-A24 molecule on the cell surface. If recognized by a CTL having a T cell receptor capable of, for example, the second amino acid at the N-terminus is added to the HLA-A24 molecule to enhance binding to the HLA-A24 molecule at the amino acid sequence shown in SEQ ID NO: 1 or 2. Amino acids characteristic of peptides to be bound: amino acids characteristic of peptides which are substituted with an amino acid selected from the group consisting of Tyr, Phe, Met and Trp and / or whose C-terminal amino acids are bound to the HLA-A24 molecule: Leu, Ile, Trp and It may also be a peptide substituted with an amino acid selected from the group consisting of Phe. For example, the peptide which has the amino acid sequence of SEQ ID NO: 5 by which the amino acid (Phe) of the C terminal of the amino acid sequence of SEQ ID NO: 2 is substituted with Leu is mentioned.

In the amino acid sequence shown in SEQ ID NO: 1 or 2, the peptide having an amino acid sequence in which one or several amino acids are substituted is a peptide substituted with an amino acid or an amino acid analog in which a side chain is similar to each amino acid to be substituted, and T which is capable of binding to HLA-A24 molecules and which can specifically recognize cells presenting complexes of HLA-A24 molecules with the antigen peptides represented by SEQ ID NO: 1 or 2 on the cell surface. Peptides recognized by CTL having a cell receptor may also be mentioned.

Amino acids having similar side chains include, for example, glycine (Gly) and alanine (Ala); Valine, isoleucine (Ile), leucine (Leu) and methionine (Met); Asparagine (Asn) and glutamine (Gln); Aspartic acid (Asp) and glutamic acid (Glu); Serine and threonine (Thr); Lysine (Lys) and arginine (Arg); Phenylalanine (Phe) and tyrosine (Tyr) are mentioned.

Specifically, in the amino acid sequence shown in SEQ ID NO: 1 or 2, as a peptide having an amino acid sequence in which one or several amino acids are substituted, the sequence in which the N-terminal amino acid (Asn) is substituted with Gln in SEQ ID NO: 2 A peptide having the amino acid sequence shown as No.:6; And a peptide having the amino acid sequence shown in SEQ ID NO: 7 in which the fifth amino acid (Thr) is substituted with Ser at the N-terminus.

Also, in the amino acid sequence shown in SEQ ID NO: 1 or 2, a peptide having an amino acid sequence in which one or several amino acids are substituted has a cell surface complex of the antigen peptide shown in SEQ ID NO: 1 or 2 with the HLA-A24 molecule. Side chains may have substitutions with amino acids that are not similar if they are recognized in a CTL having a T cell receptor capable of specifically recognizing the cells presented in Fig. 2. For example, in the amino acid sequence shown in SEQ ID NO: 2, one or several amino acids are peptides having an amino acid sequence substituted with another amino acid or an amino acid analog such as Ala, and also have an ability to bind to HLA-A24 molecules and to HLA-A24 Peptides recognized by the CTL having a T cell receptor capable of specifically recognizing a cell whose complex with the molecule presents the complex of the antigen peptide shown in SEQ ID NO: 1 or 2 and the HLA-A24 molecule on the cell surface Can be.

For example, in the amino acid sequence shown in SEQ ID NO: 1 or 2, as a peptide having an amino acid sequence in which one or several amino acids are substituted, a peptide having an amino acid sequence of any one of SEQ ID NO: 8 or SEQ ID NO: 9 is mentioned. Can be. There is a possibility that the amino acid at the N-terminal position and the fifth-terminal position at the N-terminal that can be substituted with Ala in the peptide can be replaced with other amino acids or amino acid analogs different from the original amino acid.

In addition, the HLA-A24 binding antigen peptide of the present invention may be a peptide having an amino acid added to the N or C terminus of SEQ ID NO: 1 or 2, or the HLA-A24 molecule of the antigen peptide shown in SEQ ID NO: 1 or 2 What is necessary is just a peptide recognized by CTL which has a T cell receptor which can specifically recognize the cell which shows a complex on a cell surface. These are efficiently presented to the HLA-A24 molecule by the addition of the appropriate amino acid to be the peptide of SEQ ID NO: 1 or 2 in the antigen presenting cell. For example, a peptide fragment derived from PSMA and containing a amino acid sequence shown in SEQ ID NO: 1 or 2 and having more amino acid residues than the amino acid sequence, specifically, a peptide consisting of 15 to 25 amino acid residues, is preferably used.

The HLA-A24 binding antigen peptide of the present invention can be prepared by an organic chemical method by coupling liquid or solid amino acids. In addition, the HLA-A24 binding antigen peptide can also be prepared using recombinant DNA technology using a nucleic acid encoding the amino acid sequence of the HLA-A24 binding antigen peptide. Peptides obtained by recombinant DNA technology may be modified by using appropriate organic chemical or biochemical techniques, if necessary.

The present invention also includes a nucleic acid encoding the HLA-A24 binding antigen peptide. That is, the polypeptide encoded by the nucleic acid of the present invention is a CTL having a T cell receptor capable of specifically recognizing cells presenting the complex of the antigen peptide shown in SEQ ID NO: 1 or 2 and the HLA-A24 molecule on the cell surface. It may be a variant having various variations in a range recognized by. In addition, such nucleic acid may be a nucleic acid different from the nucleic acid through condensation.

Nucleic acids encoding HLA-A24 binding antigen peptides include, for example, the PSMA gene, which gene sequence is disclosed in R. S. Israeli et al., Cancer Research, Vol. 53, pp 227-230 (1993). The region encoding the peptide in the sequence can be selected and used.

Cytotoxic T lymphocytes of the present invention are characterized in that they have a T cell receptor capable of specifically recognizing cells presenting the complex of the HLA-A24 binding antigen peptide and the HLA-A24 molecule on the cell surface. do. According to the CTL of the present invention, only cells presenting the complex of the HLA-A24 binding antigen peptide and the HLA-A24 molecule of the present invention can be specifically injured. That is, according to the CTL of the present invention, it is possible to specifically injure tumor cells, particularly prostate cancer cells.

In addition, since the CTL of the present invention is bound by many HLA-A24 to Asians, especially Japanese, the CTL can specifically injure tumor cells, especially prostate cancer cells, particularly effectively against Asians, especially Japanese, as a tumor treatment. have.

In addition, the CTL of the present invention specifically causes cell lysis or cytokine free reaction to target cells. In addition, CTL of this specification recognizes the antigenicity of the antigenic peptide shown on the HLA-A24 molecule, has an injury activity, and is not limited by other antigenic recognition.

Since the CTL of the present invention has a T cell receptor capable of specifically recognizing the antigen peptide shown in SEQ ID NO: 1 or 2, that is, a complex of a PSMA antigen peptide and an HLA-A24 molecule on the cell surface, Both HLA-A24 and PSMA can produce cytokines specifically for positive tumor cells. Therefore, it can be used as a cellular medicine for tumors and the detection of these CTL sensitive cells. Cells positive for both HLA-A24 and PSMA resulted in the introduction of the HLA-A24 or PSMA gene.

CTL of the present invention can be obtained by a method characterized by inducing cytotoxic T lymphocytes by using HLA-A24 binding antigen peptide. The method of inducing such cytotoxic T lymphocytes is also included in the present invention.

Specifically, the method of inducing cytotoxic T lymphocytes of the present invention

(1) Obtaining antigen-presenting cells by contacting cells having one type of antigen-presenting ability selected from the group consisting of HLA-A24-binding antigen peptides, macrophages, B cells and dendritic cells of the present invention, using the antigen-presenting cells obtained To give effector cells by antigen stimulation to cells with antigen-presenting ability,

(2) contacting effector cells obtained in step (1) and target cells injured by cytotoxic T lymphocytes according to claim 3 to detect the presence of cytotoxicity, and

(3) A method including the step of selecting effector cells having cytotoxic activity as cytotoxic T lymphocytes in the step (2).

Induction of this CTL in vitro can be induced by stimulating this ex vivo sample with, for example, an in vitro ex vivo sample expressing HLA-A24 and using the HLA-A24 binding antigen peptide of the present invention. Can be. Examples of the "external extraction sample" include lymph nodes, spleen, and other various organs extracted by blood, surgery, or the like. In the present invention, lymphocytes or infiltrating lymphocyte cells present in these samples are preferably used.

For example, when blood is used, CTLs can be induced by giving repeated antigenic stimulation to peripheral blood mononuclear cells (hereinafter referred to as PBMCs) made of HLA-A24 positive human blood.

The antigen stimulation is for example, a cell presenting the HLA-A24 binding antigen peptide of the present invention on the cell surface as a complex with an HLA-A24 molecule in a cell having antigen-producing ability prepared from the same blood producing lymphoblasts. cell); This can be carried out by using the HLA-A24 binding antigen peptide.

Induced CTLs can be further propagated by stimulation with an anti-CD3 antibody or the like or by the application of various stimuli in the presence of the HLA-A24 binding antigen peptide or antigen presenting cell of the present invention. For example, CTL induction can be performed by mixing at a ratio of 0.01-1 antigen-presenting cells to one lymphocyte made of PBMC in the presence of IL-7. In another embodiment, the HLA-A24-binding antigen peptide of the present invention is added to PBMC in the presence of IL-7 and Keyhole Limpet Hemocyanin (KLH) in a range of 1 ng / ml to 100 µg / ml per antigen peptide in cell culture. Preferably, 10 ng / ml-1 microgram / ml can add CTL.

Induced CTLs can also be maintained as lymphocytes with stable cytotoxicity by cloning. For example, they can be propagated by adding antigens, various cytokines, and anti-CD3 antibody stimuli to induced CTLs.

Induced specific CTLs are antigen specific increases in CTL proliferation or antigen specific from CTLs or target cells which can be measured according to injury or radioactive contamination to target cells labeled with CTL-induced antigen peptides and radioactive material. It can detect by measuring the amount of cytokines, such as GM-CSF and IFN- (gamma) which are liberated. For example, it can be evaluated by measuring the injury of CTL to the ⁵¹ Cr labeled target cells.

Specifically, an appropriate time period is obtained by mixing CTL with a cell mixture containing cells (e.g., K562 cells, etc.) for removing nonspecific injury by NK cells incorporated into ⁵¹ Cr labeled target cells in each well such as a 96 well plate. After incubation for a while, the amount of free ⁵¹ Cr is measured with a gamma counter or the like. Then, the following equation 1:

[In formula, the minimum release value is the amount of ⁵¹ Cr of the well containing only the target cell and the cell for removing the nonspecific injury activity, the natural free amount of ⁵¹ Cr from the target cell, and the maximum release value is surfactant or the like. And a free amount of ⁵¹ Cr when the target cells are destroyed.] Specific cytotoxic activity can be calculated.

In addition, it can also be directly confirmed by using an antigen peptide or a complex labeled with fluorescent dyes. In this case, for example, the CTL is contacted with a first fluorescent marker coupled with a CTL specific antibody and then the antigen peptide-MHC complex coupled with a second fluorescent marker is contacted, and the presence of double marker cells is monitored by FACS (fluorescence-). activated cell sorting) analysis.

On the other hand, CTL induction in vivo can be performed, for example, by administering antigen-presenting cells in which the complex of the HLA-A24 binding antigen peptide and the HLA-A24 molecule of the present invention is presented to the cell surface in vivo. For administration to humans, per adult dose is preferably normally 10 ^{4-10 9} individuals. Alternatively, the HLA-A24 binding antigen peptide of the present invention may be mixed with a suitable adjuvant and administered in vivo to induce CTL specific for this peptide.

Examples of the adjuvant include (1) Freund's complete adjuvant, (2) Freund's incomplete adjuvant, (3) inorganic gels such as aluminum hydroxide and alum, (4) surfactants such as rizolecithin and dimethyloctadecylammonium bromide, and (5) sulfuric acid. Dextran, polyanions such as polyIC, peptides such as (6) mulamyl peptides and taphtocin, and monophosphoryl lipids (MPL) A manufactured by (7) Ribi. In vivo CTL induction may be administered by mixing a MHC class II binding helper T cell antigen peptide in addition to a mixture of antigen peptide and adjuvant.

In addition, the HLA-A24 binding antigen peptide of the present invention may be covalently administered with a higher fatty acid or helper T cell antigen peptide through an appropriate linker for stabilization in vivo. When administered to humans, the dose per adult is HLA-A24 binding antigen peptide concentration, 0.1 µg / kg to 10 mg / kg, preferably 1 µg / kg to 1 mg / kg, more preferably per antigen peptide. Preferably 1 µg / kg to 100 µg / kg.

For the stimulation by the HLA-A24 binding antigen peptide of the present invention for inducing CTL, a complex of this HLA-A24 binding antigen peptide and HLA-A24 molecule can also be used. In this case, the HLA-A24 molecule need not be a native HLA-A24 molecule, but may be a fragment which essentially preserves the binding property with the HLA-A24 binding antigen peptide. These fragments can be produced, for example, by proteolytic or recombinant DNA techniques of native HLA-A24 molecules. This complex can be stabilized by co-existing β ₂ -microglobulin, in addition an antibody that recognizes the complex.

Since the CTL of the present invention is induced by the HLA-A24 binding antigen peptide of the present invention, it can be used as a CTL inducer for obtaining cytotoxic T lymphocytes (CTL) containing such a peptide as an active ingredient. Such CTL inducers are also included within the scope of the present invention.

CTL inducers of the invention can be supplied, for example, in the form of suspensions of HLA-A24 binding antigen peptides in physiological saline or phosphate buffered physiological saline alone or as a mixture with other molecules (helper T cell antigen peptides and / or adjuvant). The HLA-A24 binding antigen peptide may be used as a covalent conjugate with a higher fatty acid or helper T cell antigen peptide or as a complex with an HLA-A24 molecule.

The content of the HLA-A24 binding antigen peptide of the present invention in the CTL inducing agent of the present invention may be sufficient to induce CTL, for example, from 0.01 to 100% by weight, preferably 0.1 to 100%, of the antigen peptide per class of antigen peptide. It is preferable to contain 95 weight%. These contents are, for example, isolated from HLA-A24 antigen-positive human body, and HLA-A24 positive cells when cultured with stimulation by adding each of the agents having different content of HLA-A24 binding antigen peptide in vitro. Can be determined as the content of this HLA-A24 binding antigen peptide in a CTL-derived agent that specifically recognizes. Here, the presence or absence of CTL induction can be investigated by measuring the amount of various cytokines (eg, IFN-γ) produced by CTL in response to target cells, for example, by a common ELISA method. In addition, it can also be investigated by measuring the injury of CTL to a labeled target cell at ⁵¹ Cr.

The CTL inducer of the present invention can be used for additives to the medium for proliferating the CTL of the present invention in vitro, T lymphocyte proliferative activity, diagnosis of an immunosensitized condition whose index is a delayed skin response. For example, when used as an additive to the medium, the amount used is 1 ng / ml-100 µg / ml, preferably 10 ng / ml-1 µg / ml per antigen peptide as the peptide concentration in the media. Moreover, application as a cancer suppression agent mentioned later is also possible.

According to the HLA-A24 binding antigen peptide of the present invention, it is possible to induce the CTL of the present invention, and since this CTL is much HLA-A24 binding to Asians, especially Japanese, it is particularly useful for Asians, especially Japanese as a tumor treatment. Effectively, cancer suppressors capable of specifically injuring tumor cells, particularly prostate cancer cells, such as cancer suppressants based on the CTL or its induction are provided. Therefore, according to the present invention, (1) cancer inhibitors containing the HLA-A24 binding antigen peptide of the present invention as an active ingredient and (2) cancer inhibitors containing the CTL of the present invention as an active ingredient can be provided. Such cancer inhibitors are also included in the scope of the present invention.

Since the cancer suppressing agent of the present invention contains (1) the HLA-A24 binding antigen peptide of the present invention or (2) the CTL of the present invention as an active ingredient, according to the cancer suppressing agent, a prostate specific membrane antigen, PSMA hereinafter) exhibits an excellent property of specifically injuring tumor cells in a specifically expressed cancer, particularly prostate cancer.

The cancer suppressor of the present invention comprises: 1) a HLA-A24 binding antigen peptide of the present invention or a CTL of the present invention alone, 2) a HLA-A24 binding antigen peptide of the present invention or a CTL and a pharmaceutically acceptable carrier of the present invention; and / Or a mixture of diluents or 3) further adjuvant if necessary in addition to adjuvant in 1) or 2) above. Moreover, as said carrier, human serum albumin is mentioned, for example. As the diluent, for example, a phosphate buffer solution, distilled water, saline solution, phosphate buffered saline solution, a medium suitable for CTL preservation of the present invention, and the like can be given. As a medium, media, such as RPMI and AIM-V, are mentioned generally. Moreover, the said adjuvant etc. which are pharmaceutically acceptable are mentioned as an adjuvant. When the cancer suppressing agent of the present invention is administered to a human body, it may be administered by a syringe or may be administered by transdermal absorption from the mucosa by a spraying method or the like. Moreover, when used as an injection, it may contain the solvent for injection (for example, water, ethanol, glycerin, etc.).

The effective amount of the cancer suppressor of the present invention is that the amount of CTL induced by the HLA-A24 binding antigen peptide of the present invention or the amount of CTL of the present invention is specifically expressed in prostate specific membrane antigen (hereinafter referred to as PSMA). The amount is sufficient to injure tumor cells of cancer, especially prostate cancer.

Said "amount sufficient to injure tumor cells" can be evaluated, for example, by measuring the injury of CTL to the ⁵¹ Cr labeled target cells.

Pharmacological action of the cancer suppression agent of the present invention to evaluate according to the injury of tumor cells as described above; Cancer inhibitors of the present invention can be administered to mice having tumors, and the change in tumor size can be evaluated as an index.

The formulation of the cancer suppressor of the present invention may be an injection or a formulation such as a liposome formulation, a particulate formulation bound to a pharmaceutically acceptable beads having a diameter of several micrometers in diameter, a formulation bound to a lipid, and the like for enhancing affinity to the body. .

The injection may be administered alone or in admixture with conventional adjuvants such as glucose, amino acids, or intramuscular, intradermal, subcutaneous or intraperitoneal administration as needed.

Administration methods include intradermal administration, subcutaneous administration, intravenous injection, oral administration, nasal administration and the like.

Moreover, the cancer suppression agent of this invention can also use the other cancer suppression agent which can be used together in immunotherapy.

In the cancer suppressor of (1), the HLA-A24 binding antigen peptide of the present invention is contained in an amount of 0.01 to 100% by weight, preferably 0.1 to 95% by weight, per antigen peptide. The dose per adult is a peptide concentration of 0.1 µg / kg to 10 mg / kg, preferably 1 µg / kg to 1 mg / kg, more preferably 1 µg / kg to 100 µg / kg, per antigen peptide. to be. Cancer inhibitors of the present invention are excellent in adaptability to the human body.

CTL content of this invention in said cancer suppression agent of said (2) is 10 <^4> -10 <^8> pieces / mL per CTL type, Preferably it is 5 * 10 <^{5> -5} * 10 <^7> pieces / mL.

When the anticancer agent of (2) is administered to a human body, it may be administered as a syringe as an injection, and the dosage per adult may be appropriately set according to the age, weight, disease state, etc. of the patient, for example, CTL The number may be 10 ⁶ to ¹⁰ 10 per CTL. In addition, the said range is a target and it is not limited to this. In the cancer suppression agent of the present invention, CTL, which is an active ingredient, is derived from the human body to be administered, and thus has excellent adaptability to the human body.

The cancer inhibitor of (2) may be administered to cancer patients including prostate cancer patients having tumor cells expressing antigen peptides recognizable by CTL as an active ingredient. Therefore, cancer inhibitors containing the CTL of the present invention as an active ingredient derived using the PSMA antigen peptide are useful for the immunotherapy of cancer such as prostate cancer in which both HLA-A24 and PSMA are positive tumor cells.

In addition, the present invention can provide an antigen-labeling cell for presenting a HLA-A24 binding antigen peptide, ie, a complex of a PSMA antigen peptide and an HLA-A24 molecule, on the cell surface. According to the antigen presenting cell of the present invention, it can be used to induce CTL of the present invention.

Antigen-presenting cells of the present invention can be produced by using cells having antigen-presenting ability.

Examples of cells having antigen-presenting ability include leukocytes including macrophages, B cells, dendritic cells (DCs) capable of presenting the complex of the HLA-A24 binding antigen peptide of the present invention and HLA-A24 molecules on the cell surface thereof. And the like. Since the DC has a large amount of antigen presentation per cell and also has a high expression level of cell surface molecules (co-stimulatrory signal molecules such as CD80, CD83, CD86, etc.) required for antigen presentation, It is especially suitable as a cell which has.

Cells having the antigen-presenting ability can be prepared with the in vitro extraction sample. For example, 1) DC is isolated and purified from PBMC based on several cell surface markers, 2) derived from GM-CSF and IL-4 from monocytes, 3) GM-CSF, TNF from CD34 positive cells It can be produced by a method of inducing cytokines such as -α and SCF.

In order to present the HLA-A24-binding antigen peptide of the present invention on the cell surface having the antigen-presenting ability, for example, after mixing the cell having the antigen-presenting ability with the HLA-A24-binding antigen peptide of the present invention, an excess amount is necessary if necessary. The method of washing | cleaning and loading the said antigen peptide on HLA-A24 molecule | numerator is mentioned. In addition, cells thought to have already presented antigenic peptides unrelated to the present invention on HLA-A24 molecules, such as cells prepared by the method 1) of the cells having the antigen-presenting ability, may be HLA-A24. Acid treatment or the like prior to loading the binding antigen peptide can also remove the peptide that cannot induce the CTL of the present invention present on the HLA-A24 molecule.

The antigen presenting cell of the present invention may be a cell loaded with several HLA-A24 binding antigen peptides per cell in addition to a cell loaded with a single HLA-A24 binding antigen peptide.

Moreover, the method of transforming the cell which has the said antigen-presenting ability by the expression vector which can show HLA-A24 binding antigen peptide to B cell or CD34 positive cell by another method is mentioned.

Examples of such vectors include expression plasmid vectors in which commercially available plasmids such as pcDNA3, pMQMneo, pCEP4 and the like are combined with genes encoding PSMA by recombinant DNA technology. In addition, the expression vector may include a gene encoding an appropriate amino acid sequence at both ends of the antigen peptide to be presented on the cell surface so that the protease is efficiently degraded in the cell. Cells or cell extracts transformed with an expression vector in which the HLA-A24 gene is introduced into LNCaP cells, which are high-expressing PSMA molecules, can also be used. Cells or cell extracts transformed at the same time using the HLA-A24 expression vector and the PSMA expression vector can also be used. Retroviral vectors and adenovirus vectors are also suitably used.

Antigen presenting cells are preferably non-proliferative. In order to make a cell non-proliferative, it is good to treat with irradiation, such as X-rays, or a drug, such as mitomycin.

Whether the cells obtained as described above are antigen presenting cells can be evaluated by examining the cytokine production of CTL.

Further, by introducing a nucleic acid encoding a major human histocompatibility antigen and / or a nucleic acid encoding an antigenic protein into a cell, the major human histocompatibility antigen that is injured by cytotoxic T lymphocytes and the antigen that is bound to the antigen Target cells can be produced that present a complex of protein-derived peptides on the cell surface. The present invention also includes a method for producing such target cells and target cells injured by cytotoxic T lymphocytes obtained by the method. According to the target cell of the present invention, it is possible to evaluate cytotoxic T lymphocytes which could not be evaluated because target cells have not been established until now. In addition, according to the method for producing a target cell of the present invention, since it is possible to express any human major histocompatibility antigen or antigen protein, it is possible to express human major histocompatibility antigen and antigen protein in a combination unknown in the existing target cells. The production of target cells is possible.

As the nucleic acid encoding the human major histocompatibility antigen, for example, a nucleic acid encoding HLA-A24. Such nucleic acid sequences are disclosed in GenBank's Action No. L47206. The nucleic acid can be obtained from a suitable sample based on the sequence information by a known method such as PCR.

By introducing a nucleic acid encoding a human major histocompatibility antigen and / or a nucleic acid encoding an antigenic protein into an appropriate cell, a cell expressing both thereof and a cell injured by cytotoxic T lymphocytes can be obtained.

Introduction into a nucleic acid cell can be carried out through a vector suitable for the cell to be introduced, and can be preferably carried out through an adenovirus vector from the viewpoint of obtaining a high introduction efficiency.

In the case of using an adenovirus vector, a nucleic acid encoding a major human histocompatibility antigen and / or a nucleic acid encoding an antigenic protein is introduced by conventional homologous recombination or the like, and the adenovirus containing the adenovirus vector into which the nucleic acid is introduced. Virus particles can be produced (e.g., DMGlover et al., DNA Cloning 4, Mammalian Systems Second Edition A Practical Approach (Japanese version: issued by Takarashuzo Co., Ltd.), such as the COS-TPC method [S.Miyake et al. , Proc. Natl. Acad. Sci. USA, Vol. 93, p1320 (1966).

Nucleic acid introduction using an adenovirus vector can infect adenovirus particles containing the adenovirus vector prepared by a known method, such as the COS-TPC method, into cells to be introduced.

In addition, it is preferable that a suitable element is present in the vector when a nucleic acid encoding a human major histocompatibility antigen and / or a nucleic acid encoding an antigenic protein is expressed in a cell. For example, a recombinant vector capable of replicating and functioning in a cell can be prepared by adding a control sequence such as a promoter sequence for controlling transcription, a co-initiation codon, and the like, and adding a codon during translation downstream of the nucleic acid to be expressed. .

Specifically, when adenovirus vectors are used and introduced into animal cells, the promoters include SV40 gene promoter, adenovirus major late gene promoter, thymidine kinase gene promoter, metallothionein gene promoter, β-actin gene promoter, and CAG promoter. And SRα promoter, EF1α promoter, CMG promoter, PGK promoter and the like. In the present invention, for example, a CAG promoter is preferably used in view of expression intensity.

When expressing a nucleic acid encoding a human major histocompatibility antigen and / or a nucleic acid encoding an antigenic protein by the adenovirus vector, only one of the major human histocompatibility antigens and antigen proteins is expressed as the cells to be introduced or And both of which are not expressed.

The obtained transfect may be cultured in an appropriate medium depending on the cells used.

Whether the resulting transfects present the major human histocompatibility antigens and / or antigenic proteins can be assessed by cytokine production by CTL.

In addition, since the antigen-presenting cell of the present invention presents a complex of the HLA-A24-binding antigen peptide and the HLA-A24 molecule on the cell surface, the CTL of the present invention is induced, and thus, such antigen-presenting cell can be used as a CTL inducer. The CTL inducer is included in the present invention. These inducers contain the antigen presenting cells of the present invention as an active ingredient.

The CTL inducer of the present invention may be supplied in a formulation suspended in these antigen presenting cells in a medium suitable for cell preservation, saline or phosphate buffered saline. Examples of the medium include RPMI, AIM-V, and X-VIVO10. In addition, serum albumin or the like may be added to the medium or buffer for the purpose of stabilization.

The content of the antigen presentation in CTL inducer of the present invention if the cell is an amount sufficient to induce a CTL is 10 per one kinds of antigen presenting cells ³ ~5 × 10 ⁶ pieces / ㎖, preferably 10 ^{4-10 6} / It is preferable that it is ml. This content is specifically used to isolate peripheral blood lymphocytes from HLA-A24 antigen-positive human body and to specifically culture HLA-A24 positive cells when cultured with stimulation with the addition of a different antigen presenting cell in vitro. It can be determined as the content of antigen presenting cells in the agent from which CTL is recognized.

The CTL inducer of the present invention can be used for diagnosis of an immunosensitized condition whose T lymphocyte proliferative activity is in addition to the use as an additive to the medium for proliferating the CTL of the present invention in vitro. For example, when used as an additive to a medium, it can be normally used in 0.01-1 ratio with respect to 1 lymphocyte induced by CTL.

In addition, according to the antigen-presenting cells of the present invention, many HLA-A24-binding CTLs can be induced to Asian species, especially Japanese, and thus, these antigen-presenting cells themselves can be used as cancer suppressors for cancer, particularly prostate cancer. Cancer inhibitors of the present invention include (3) cancer inhibitors containing the antigen-presenting cells of the present invention as an active ingredient.

The cancer suppression agent of (3) is provided in a dosage form suspended in the pharmaceutically acceptable diluent of the antigen-presenting cells of the present invention. The diluent includes a medium suitable for cell preservation, a phosphate buffer solution, a saline solution, and the like. Examples of the medium include media such as RPMI, AIM-V, and X-VIVO10. In addition, the cancer suppressor of (3) may contain a pharmaceutically acceptable carrier for stabilization. Examples of the carrier include human serum albumin.

The content (effective amount) of the antigen presenting cells in the cancer suppressor of (3) is a cancer in which the amount of CTL induced by the antigen presenting cells is specifically expressed in prostate specific membrane antigen (hereinafter referred to as PSMA). In particular, a sufficient amount to injure tumor cells in prostate cancer. Such an effective amount can be determined as in the case of the cancer suppressor of (1) or (2) above. Specifically, the effective amount is preferably 10 ⁵ to 10 ⁸ / ml, preferably 5 x 10 ^{5 to} 5 x 10 ⁷ / ml per antigen-presenting cell in cancer suppressor.

When the anticancer agent of (3) is administered to a human body, it may be administered by a syringe, and the dose per adult may be appropriately set according to the age, weight, disease state, etc. of the patient, for example, as an antigen presenting cell number. presenting cells is preferably 1 usually 10 ^{4-10 9} individuals per category. In addition, the said range is a target to the last and is not limited to this. In addition, since the antigen-presenting cell as an active ingredient is derived from the human body to be administered, it is excellent in adaptability to the human body.

Such cancer inhibitors may also use other cancer inhibitors that may be used in immunotherapy.

According to the CTL of the present invention, cells that are sensitive to such CTL can be detected. The present invention also includes a method for detecting CTL sensitive cells.

In the method for detecting susceptible cells of cytotoxic T lymphocytes of the present invention, when a change occurs by contacting the cytotoxic T lymphocytes of the present invention with a test cell, the test cells are susceptible to the cytotoxic T lymphocytes of the present invention. It is characterized by setting it as an index called a cell (sensitive cell).

The above-mentioned "test cell" means human cells or coin cells derived from in vitro extraction samples such as peripheral blood lymphocytes and tissues. In addition, the term "sensitive cells" in the present specification means specific cells including tumor cells that are recognized by CTL and cause cell lysis or cytokine release.

Examples of the changes that occur due to the contact between CTL and susceptible cells in test cells include cell lysis by CTL, cytokine release, CTL proliferation, and the like.

Detection of cell lysis can be carried out by measuring the amount of radioisotope or fluorescent dye released from the test cells when the test cells are labeled with radioisotopes or fluorescent dyes and then mixed with CTL.

The detection of cytokine release can be carried out by measuring the amount of release of GM-CSF, TNF, IFN-γ, etc. from CTL or test cells.

CTL proliferation can be performed by measuring the combined amount of ³ H-thymidine into cells, the measurement of the number of CTL cells by microscopic observation, or the like.

According to this change, both HLA-A24 and PSMA present in the test cells can detect positive tumor cells.

Moreover, the detection agent of the sensitive cell is used suitably for CTL containing the CTL of this invention as an active ingredient in such a detection method. Such a detector is also included in the present invention.

The detecting agent of the present invention is provided in a formulation suspended in a medium, saline or phosphate buffered saline suitable for CTL preservation.

Examples of the medium include media such as RPMI, AIM-V, and X-VIVO10. In addition, serum albumin or the like may be added to the medium or buffer for the purpose of stabilizing CTL.

The CTL content (active ingredient amount) of the detection agent may be an amount sufficient to generate a change (for example, cell lysis by CTL, cytokine release, CTL proliferation, etc.) caused by contact between the CTL and susceptible cells in the test cells. . Specifically, the CTL content is preferably 10 ⁴ to 10 ⁸ / ml per CTL.

The detection agent of the present invention can also be used for typing HLA that identifies whether HLA expressed in test cells is HLA-A24 in addition to detection of CTL sensitive cells of the present invention in test cells.

The detection agent of the present invention may further suitably contain a reagent such as a label for detecting the change.

The present invention also provides a method for detecting human major histocompatibility antigen (HLA) -A24 molecules of test cells. The method for detecting human major histocompatibility antigen (HLA) -A24 molecule is characterized by contacting test cell with cytotoxic T lymphocytes of the present invention in the presence of HLA-A24 binding antigen peptide.

For example, the HLA-A24 molecule on the test cell surface can detect a change caused by contacting the CTL of the present invention with the test cell as an index. Examples of changes caused by contact between CTL and test cells include cell lysis by CTL, cytokine release or CTL proliferation. Such a change can be detected as described above.

According to the measurement result, not only the presence or absence of HLA-A24 molecule expression detection, but also the expression level of HLA-A24 molecule on a test cell can be measured.

In addition, when it is not clear whether PSMA is expressed in a test cell, HLA-A24 molecule can be detected by the following method. In this method, the HLA-A24 binding antigen peptide of the present invention and the CTL of the present invention, which recognizes the complex of the antigen peptide and the HLA-A24 molecule, coexist with the test cells and the final 0.01-50 µg / ml in the reaction solution. The CTL and the test cell may be contacted.

According to the present invention, it is possible to detect HLA-A24 molecules of tumor cells that were difficult to detect by HLA molecules using a conventional anti-HLA antibody in which a complement activation step was essential.

Example

Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited to these Examples.

Example 1 Preparation of PSMA Antipeptide Specific CTL

CTL of this Example is prepared by the induction method using antigen presenting dendritic cells (DC).

(1) Selection and Synthesis of CTL Inducing Peptides

Peptide having a sequence having a HLA-A24 binding motif structure with respect to the amino acid sequence of the PSMA protein consisting of 750 amino acids (the second residue at the N-terminal is an amino acid selected from the group consisting of Tyr, Phe, Trp and Met, C-terminal residue Is a peptide selected from the group consisting of Leu, Ile, Phe and Trp), and nine peptides shown in Table 1 are selected as candidate peptides of the PSMA antigen peptide.

SEQ ID NO: Location within PSMA designation 10 298-306 PSMA24-1 11 624-632 PSMA24-2 One 227-235 PSMA24-3 12 606-614 PSMA24-4 2 178-186 PSMA24-5 13 74-83 PSMA24-6 14 565-574 PSMA24-7 15 699-708 PSMA24-8 16 624-633 PSMA24-9

In Table 1, the number of the "SEQ ID NO" column shows the sequence number of the sequence list which shows the amino acid sequence of each peptide. In addition, the number of the term "position in PSMA" shows the number of amino acid residues from the N terminus of PSMA protein. In addition, the symbol of a "name" column represents the peptide name which the inventors named. These are manufactured by the solid state Fmoc method using a peptide synthesizer (PSSM-8: Shimadzu Corporation).

(2) Preparation of Effector Cells

For nine peptides of PSMA24-1 to PSMA24-9 prepared in the above (1), effector cells recognizing each peptide were induced from PBMCs of normal individuals having HLA-A24 to measure cytotoxic activity. Next, a manufacturing operation is shown.

① Manufacturing of PBMC

Blood is collected by component blood collection from normal individuals with HLA-A24. The collected blood was middle layered on a Ficoll-Paque separation liquid (manufactured by Pharmacia), and centrifuged at 500 x g for 20 minutes at room temperature. The PBMCs in the middle layer are recovered and washed and stored in liquid nitrogen in a state of being suspended in a stock solution consisting of 90% fetal bovine serum (FCS, manufactured by Intergen) and 10% dimethyl sulfoxide (manufactured by Sigma) (preserved PBMC).

② Preparation of target cell

Five types containing 10 μg / ml of any one of PSMA24-1 to PSMA24-9 prepared in (1) on TISI (WSNO9042), an EBV transform B cell expressing HLA-A24, on the day before measurement Incubated overnight in a medium of hereinafter [hereinafter referred to as TISI (+), or a medium containing no peptide [hereafter referred to as TISI (-)]. . On the day of measurement, each 5 x 10 ⁶ TISI (+) and TISI (-) are mixed for 1 hour at 37 ° C. in ₂₀₀ μCi of Na ₂ ⁵¹ CrO ₄ solution each, then rinsed in RPMI1640 culture containing 10% FCS and ⁵¹ Used as Cr labeled target cells.

③ Preparation of effector cells

After melting and washing the preserved PBMC prepared in ① above, 15 ml of the cell suspension was placed in a T-75 flask (manufactured by Corning Corporation) and left at 1.5 ° C. for 1.5 hours, followed by GM-CSF (manufactured by Schering). 25 ml of 5H-RPMI containing 1000 U / ml and 1000 U / ml of IL-4 (manufactured by Zenzyme Co., Ltd.) is added and cultured in a CO ₂ incubator at 37 ° C. to induce DC. One week later, cells were harvested and suspended at 3 × 10 ⁶ / ml in PBS containing 40 μg / ml peptide, 1 μg / ml β ₂ microglobulin and 1% bovine serum albumin and allowed to react at room temperature for 4 hours. . Next, X-rays (5500 Rad) are irradiated and diluted with 5 H-RPMI to be 1 × 10 ⁶ cells / ml to form antigen-presenting cells.

After thawing the preserved PBMC obtained in ① above, it is suspended in phosphate buffered saline containing 1% human AB serum (PBS: 1H-PBS hereinafter) at 4 ° C. so that the cell concentration is 2 × 10 ⁷ / ml. To obtain a PBMC suspension. Subsequently, 140 µl of beads (Dynabeads M450, manufactured by Dinar) which combined the anti-CD8 antibody washed with 1H-PBS was added to 2 × 10 ⁷ / ml PBMC and reacted at 4 ° C for 1 hour.

Subsequently, 0.1 ml of cell dissociation beads (DETACHaBEAD, manufactured by Dynasar) were added to the suspension obtained by suspending in 0.9 ml of 1H-PBS for 1 × 10 ⁸ cells used when preparing the PBMC suspension. Add. The resulting solution is then mixed for 1 hour at room temperature and the dissociated cells are recovered and washed with 1H-PBS.

The obtained washed cells are suspended in 5H-RPMI to be 2 x 10 ⁶ cells / ml. The obtained suspension is mixed in equal amounts with each of the DC suspensions incubated with the peptide of any of PSMA24-1 to PSMA24-9, and IL-7 is added again to a final concentration of 10 ng / ml. The resulting solution is poured into each well of a 48 well culture plate by 0.5 ml and incubated in a 37 ° C. CO ₂ incubator. The next day 50ul of 5H-RPMI containing 100ng / ml IL-10 is added to the culture.

After one week, the culture supernatant of each well is removed and the cells are suspended in 0.5 ml of 5H-RPMI to obtain a suspension. Next, adherent cells obtained from PBMCs irradiated with X-ray (5500 Rad) were subjected to 1 HSA- containing 20 μg / ml of any one of PSMA24-1 to PSMA24-9 and 1 μg / ml of β ₂ microglobulin. The suspension is added to each well containing PBS (phosphate buffered saline containing 1% human serum albumin) at room temperature for 2 hours and containing the restimulatory antigen presenting cells obtained by washing each well.

The following day 1 L-10 is added to each well to achieve a final concentration of 10 ng / ml. 5H-RPMI containing rIL-2 is added to each well again at a final concentration of 20 IU / mL every 2 days and incubated in a CO ₂ incubator for a week. After the same re-stimulation three times, cytotoxic activity against target cells is measured for effector cells in each well.

PSMA24-3 and PSMA24-5 effector cells exhibiting cytotoxic activity were individually grown using anti-CD3 antibodies. The proliferated cells are suspended in 5H-RPMI and cytotoxic activity against the target cells is measured by the following method (3).

(3) Measurement of cytotoxic activity by CTL

The effector cells obtained in the above (2) are diluted with 5 H-RPMI so as to be 3 x 10 ^{3 to} 1 x 10 ⁶ cells / ml. Pour the resulting dilution into 100 μl / well of each well of a 96 well culture plate. Next, 100 μl of cell suspension containing 1 × 10 ^{4 51} Cr labeled target cells and 3 × 10 ⁵ K562 cells is added to each well to obtain a cell mixture. In addition, the K562 cells are used to remove nonspecific injury activity by the NK cells incorporated.

The cell mixture is centrifuged at 400 × g for 1 minute and then left for 5 hours in a CO ₂ incubator at 37 ° C. Next, 100 µl of the culture supernatant of each well is collected, and the amount of free ⁵¹ Cr is measured using a gamma counter.

Specific cytotoxic activity is calculated according to the following equation (1).

[Equation 1]

In Equation 1, the minimum release value is ⁵¹ Cr of a well containing only target cells and K562 cells, and represents a natural free amount of ⁵¹ Cr from target cells. In addition, the maximum release value represents the amount of ⁵¹ Cr free flow when the cells were destroyed by adding the surfactant Triton X-100 to the target cells.

Specific cytotoxic activity curves at various E / Ts for TISI (−) and TISI (+) of effector cells induced by PSMA24-3 or PSMA24-5 are shown in FIGS. 1 and 2.

Figure 1 shows the results using effector cells induced by PSMA24-3. 2 shows the results of using effector cells induced by PSMA24-5. In addition, the white square (□) shows the result of using TISI (-) as a target cell and the black circle (●) using TISI (+). 1 and 2, the vertical axis represents specific cytotoxic activity (%), and the horizontal axis represents E / T ratio.

As a result, effector cells induced by PSMA24-3 and PSMA24-5 exhibit antigen peptide specific cytotoxic activity against TISI (+) to which each peptide was added.

From the above results, it can be seen that the effector cells induced by PSMA24-3 (SEQ ID NO: 1) and PSMA24-5 (SEQ ID NO: 2) are CTLs showing antigen peptide specific cytotoxicity.

Example 2 Preparation of PSMA Antigenpeptide Functional Derivatives

(1) Synthesis of PSMA25-5 Modifier

Seven derivatives shown in Table 2 were designed according to PSMA25-5 synthesized in Example (1).

SEQ ID NO: designation 8 PSMA24-5-1A 17 PSMA24-5-4A 9 PSMA24-5-5A 18 PSMA24-5-6A 19 PSMA24-5-7A 20 PSMA24-5-8A 5 PSMA24-5-9L

Seven peptides of PSMA24-5-1A to PSMA24-5-9L shown in Table 2 were prepared using a peptide synthesizer.

(2) Identification of functional derivatives of PSMA24-5

Whether or not the CTL obtained in Example 1, which recognizes the complex of PSMA24-5 and HLA-A24 molecules, recognizes the complex of the peptides shown in Table 2 and the HLA-A24 molecule.

First, the CTL recognizing PSMA24-5 prepared in the same manner as in Example (2) was selected from the group consisting of ⁵¹ Cr labeled TISI (-) cells and PSMA24-5-1A to PSMA24-5-9L peptides. Using peptides, it was confirmed whether or not cytotoxicity was observed for seven kinds of ⁵¹ Cr-labeled TISI (+) cells prepared in the same manner as in Example (2).

As a result, the CTL for recognizing PSMA24-5 was ⁵¹ Cr-labeled TISI (+) obtained by culturing in a medium containing one selected from the group consisting of PSMA24-5-1A, PSMA24-5-5A and PSMA24-5-9L. It can be seen that it shows clear cytotoxicity against). This CTL does not exhibit cytotoxicity to ⁵¹ Cr-labeled TISI (-). Thus, it was found that PSMA24-5-1A (SEQ ID NO: 8), PSMA24-5-5A (SEQ ID NO: 9) and PSMA24-5-9L (SEQ ID NO: 5) are functional derivatives of PSMA24-5.

Example 3 Evaluation of CTL Using Adenovirus

(1) Preparation of target cell for transduction by adenovirus

Tumor cells expressing HLA-A24 and PSMA are prepared as target cells that inherently present antigen. That is, the LNCaP cells, which are PSMA expressing and non-expressing HLA-A24 prostate cancer cell lines, are introduced into the cells by HLA-A24 transgenic adenovirus vectors. HMA-A24 expression, PSMA non-expressing cancer cells MKN45, 888mel, SW480, T.T to the PSMA gene is introduced into the cell as adenovirus vectors.

The transgenic adenovirus vector is produced by the COS-TPC method.

First, 4 ml of 5 × 10 ⁵ cells / ml of the cell suspension are placed in a 6-well cell culture plate and incubated overnight. After culturing, HLA-A24 transgenic adenovirus or PSMA transgenic adenovirus or adenovirus without genes is infected with MOI = 20-30, and cultured for another 2 days. Adenovirus infected cells are harvested and washed with 10% FCS-containing RPMI1640 culture to prepare target cells.

(2) Evaluation of CTL function using transgenic target cells

It is confirmed by the cytokine production capacity of CTL whether the transgenic cells by adenovirus function as target cells for evaluating the antigen specific response of CTL.

After dilution of effector cells induced in the same manner as in Example 1 (2) with ⁵ H-RPMI to be 1 × 10 ⁵ / ml, the resulting dilution was diluted 100 l into each well of a 96 well U-shaped bottom culture plate. Pour well. Next, 100 μl of the cell suspension containing 1 × 10 ⁴ target cells prepared in (1) was added to each well.

The culture plate is left overnight in a CO ₂ incubator at 37 ° C. Next, 100 µl of the culture supernatant of each well is collected, and IFN-γ is measured by an ELISA kit (manufactured by Genzyme Techno Co., Ltd.) for human IFN-γ. The IFN-γ production is calculated from the calibration curve of the IFN-γ standard measured at the same time.

CTLs induced by PSMA24-3 or PSMA24-5 produce IFN-γ for LNCaP prostate cancer cell lines into which the HLA-A24 gene has been introduced, but not IFN-γ for LNCaP without the HLA-A24 gene. can not do it.

In addition, CTL induced by PSMA24-3 produces IFN-γ against HLA-A24 expressing cell lines 888mel and SW480 into which the PSMA gene was introduced. In addition, CTL induced by PSMA24-5 produces IFN-γ against HLA-A24 expressing cell lines MKN45 and T.T into which the PSMA gene was introduced. Neither CTL produces IFN-γ against the HLA-A24 nonexpressing cell lines MKN28 and 526mel.

In the above results, PSMA24-3 specific CTL and PSMA24-5 specific CTL produce adenosine in that HLA-A24 gene introduction or SMA gene-induced HLA-A24 expression and IFN-γ are produced for PSMA expressing target cells. It has been found that transgenic cells using viruses can be used for CTL function evaluation, and these transgenic cells can be used as antigen presenting cells in presenting the antigenic peptides of the present invention on HLA-A24 molecules.

Sequence Listing Free Text

SEQ ID NO: 1 is the sequence of the synthesized HLA-A24 binding antigen peptide (PSMA24-3).

SEQ ID NO: 2 is the sequence of the synthesized HLA-A24 binding antigen peptide (PSMA24-5).

SEQ ID NO: 3 is the sequence of HLA-A2.1 restrictive antigen peptide (PSM-P1).

SEQ ID NO: 4 is the sequence of HLA-A2.1 restrictive antigen peptide (PSM-P2).

SEQ ID NO: 5 is the sequence of a derivative peptide of the peptide of SEQ ID NO: 2 (PSMA24-5-9L).

SEQ ID NO: 6 is the sequence of a derivative peptide of the peptide of SEQ ID NO: 2 (PSMA24-5-1Q).

SEQ ID NO: 7 is a sequence of a derivative peptide of the peptide of SEQ ID NO: 2 (PSMA24-5-5S).

SEQ ID NO: 8 is the sequence of a derivative peptide of the peptide of SEQ ID NO: 2 (PSMA24-5-1A).

SEQ ID NO: 9 is the sequence of a derivative peptide of the peptide of SEQ ID NO: 2 (PSMA24-5-5A).

SEQ ID NO: 10 is the sequence of a synthetic peptide (PSMA24-1) with an HLA-A24 binding motif.

SEQ ID NO: 11 is the sequence of a synthetic peptide (PSMA24-2) having a HLA-A24 binding motif.

SEQ ID NO: 12 is the sequence of a synthetic peptide (PSMA24-4) with an HLA-A24 binding motif.

SEQ ID NO: 13 is the sequence of a synthetic peptide (PSMA24-6) with an HLA-A24 binding motif.

SEQ ID NO: 14 is the sequence of a synthetic peptide (PSMA24-7) having a HLA-A24 binding motif.

SEQ ID NO: 15 is the sequence of a synthetic peptide (PSMA24-8) with an HLA-A24 binding motif.

SEQ ID NO: 16 is the sequence of a synthetic peptide (PSMA24-9) with a HLA-A24 binding motif.

SEQ ID NO: 17 is the sequence of a derivative peptide of the peptide of SEQ ID NO: 2 (PSMA24-5-4A).

SEQ ID NO: 18 is the sequence of a derivative peptide of the peptide of SEQ ID NO: 2 (PSMA24-5-6A).

SEQ ID NO: 19 is the sequence of a derivative peptide of the peptide of SEQ ID NO: 2 (PSMA24-5-7A).

SEQ ID NO: 20 is the sequence of a derivative peptide of the peptide of SEQ ID NO: 2 (PSMA24-5-8A).

According to the present invention, Asians, in particular Japanese, may be provided with a technique useful for the treatment and diagnosis of cancer, specifically cancer in which PSMA is specifically expressed, including prostate cancer. Specifically, according to the CTL of the present invention, it is possible to treat and diagnose cancer, specifically, cancer in which PSMA is specifically expressed, including prostate cancer, and both HLA-A24 and PSMA can selectively injure benign tumor cells. In addition, it can be used as a cell medicament (cancer inhibitor) for cancer treatment, and both HLA-A24 and PSMA in ex vivo samples can detect the presence or absence of positive tumor cells. According to the HLA-A24 binding antigen peptide of the present invention, such CTL can be selectively induced. Therefore, the HLA-A24 binding antigen peptides of the present invention are useful as CTL inducers and cancer suppressors. In addition, the antigen-presenting cells of the present invention are useful as a method for producing this CTL or as an cancer inhibitor. In addition, according to the CTL derivation method of the present invention has an excellent effect that the CTL of the present invention can be selectively and efficiently obtained. In addition, the CTL inducer of the present invention can efficiently induce the CTL of the present invention efficiently, and excellent in that it is useful for treating cancers that specifically express PSMA, including cancer, specifically prostate cancer, in Asians, especially Japanese. Express the nature. According to the method and detection agent for detecting CTL sensitive cells of the present invention, it is possible to efficiently detect CTL sensitive cells efficiently. Therefore, according to the CTL sensitive cell detection method and the detection agent of the present invention, it is possible to apply the HLA to typing, diagnosis of cancer, specifically prostate cancer and the like. In addition, according to the nucleic acid of the present invention, it is possible to efficiently supply the HLA-A24 binding antigen peptide of the present invention. In addition, according to the method for producing target cells injured by cytotoxic T lymphocytes of the present invention, it is possible to prepare target cells expressing major human tissue compatible antigens and antigen proteins in combinations unknown to existing target cells. Excellent effect. In addition, according to the target cells injured by the cytotoxic T lymphocytes of the present invention, it is possible to evaluate the cytotoxic T lymphocytes which could not be evaluated because target cells have not been established until now.

<110> Takara Shuzo Co. Ltd. <120> Cytotoxic T lymphocyte <130> Y02P-2771 <150> JP2001-104693 <151> 2001-04-03 <160> 20 <170> KopatentIn 1.71 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A sequence for HLA-A24 restricted antigen peptide which is          synthesized. <400> 1 Leu Tyr Ser Asp Pro Ala Asp Tyr Phe   1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A sequence for HLA-A24 restricted antigen peptide which is          synthesized. <400> 2 Asn Tyr Ala Arg Thr Glu Asp Phe Phe   1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> A sequence for HLA-A2.1 restricted antigen peptide. <400> 3 Leu Leu His Glu Thr Asp Ser Ala Val   1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> A sequence for HLA-A2.1 restricted antigen peptide. <400> 4 Ala Leu Phe Asp Ile Glu Ser Lys Val   1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A sequence for synthesized peptide which is a derivative of the          peptide of SEQ ID NO: 2. <400> 5 Asn Tyr Ala Arg Thr Glu Asp Phe Leu   1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A sequence for synthesized peptide which is a derivative of the          peptide of SEQ ID NO: 2. <400> 6 Gln Tyr Ala Arg Thr Glu Asp Phe Phe   1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A sequence for synthesized peptide which is a derivative of the          peptide of SEQ ID NO: 2. <400> 7 Asn Tyr Ala Arg Ser Glu Asp Phe Phe   1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A sequence for synthesized peptide which is a derivative of the          peptide of SEQ ID NO: 2. <400> 8 Ala Tyr Ala Arg Thr Glu Asp Phe Phe   1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A sequence for synthesized peptide which is a derivative of the          peptide of SEQ ID NO: 2. <400> 9 Asn Tyr Ala Arg Ala Glu Asp Phe Phe   1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A sequence for synthesized peptide having HLA-A24 binding motif. <400> 10 Gly Tyr Tyr Asp Ala Gln Lys Leu Leu   1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A sequence for synthesized peptide having HLA-A24 binding motif. <400> 11 Thr Tyr Ser Val Ser Phe Asp Ser Leu   1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A sequence for synthesized peptide having HLA-A24 binding motif. <400> 12 Lys Tyr Ala Asp Lys Ile Tyr Ser Ile   1 5 <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> A sequence for synthesized peptide having HLA-A24 binding motif. <400> 13 Leu Tyr Asn Phe Thr Gln Ile Pro His Leu   1 5 10 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> A sequence for synthesized peptide having HLA-A24 binding motif. <400> 14 Phe Tyr Asp Pro Met Phe Lys Tyr His Leu   1 5 10 <210> 15 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> A sequence for synthesized peptide having HLA-A24 binding motif. <400> 15 Lys Tyr Ala Gly Glu Ser Phe Pro Gly Ile   1 5 10 <210> 16 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> A sequence for synthesized peptide having HLA-A24 binding motif. <400> 16 Thr Tyr Ser Val Ser Phe Asp Ser Leu Phe   1 5 10 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A sequence for synthesized peptide which is a derivative of the          peptide of SEQ ID NO: 2. <400> 17 Asn Tyr Ala Ala Thr Glu Asp Phe Phe   1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A sequence for synthesized peptide which is a derivative of the          peptide of SEQ ID NO: 2. <400> 18 Asn Tyr Ala Arg Thr Ala Asp Phe Phe   1 5 <210> 19 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A sequence for synthesized peptide which is a derivative of the          peptide of SEQ ID NO: 2. <400> 19 Asn Tyr Ala Arg Thr Glu Ala Phe Phe   1 5 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A sequence for synthesized peptide which is a derivative of the          peptide of SEQ ID NO: 2. <400> 20 Asn Tyr Ala Arg Thr Glu Asp Ala Phe   1 5

Claims (21)

A human major histocompatibility antigen (HLA) -A24 binding antigen peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 5, 8, 9. A cytotoxic T lymphocyte having a T cell receptor capable of specifically recognizing a cell presenting the complex of the HLA-A24 binding antigen peptide and the HLA-A24 molecule according to claim 1 on a cell surface. A method of inducing cytotoxic T lymphocytes, wherein the cytotoxic T lymphocytes are induced in vitro by using the HLA-A24 binding antigen peptide according to claim 1. 4. The antigen-presenting cell of claim 3, wherein (1) the antigen-presenting cell is contacted by contacting a cell having one type of antigen-presenting ability selected from the group consisting of the HLA-A24-binding antigen peptide, macrophages, B cells and dendritic cells of claim 1. Obtaining and effector cells by giving antigen stimulation to the cells having antigen-presenting ability using the obtained antigen-presenting cells, (2) contacting effector cells obtained in step (1) and target cells injured by the cytotoxic T lymphocytes according to claim 2 to detect the presence or absence of cytotoxicity; (3) An induction method comprising the step of selecting effector cells having cytotoxic activity as cytotoxic T lymphocytes in the step (2). The induction method according to claim 4, wherein in the presence of IL-7, one lymphocyte is contacted at a ratio of 0.01 to 1 antigen-presenting cells. The method according to claim 4, wherein in the presence of IL-7 and keyhole limpet hemocyanin, 1 ng to 100 μg of the HLA-A24 binding antigen peptide according to claim 1 is added to 1 ml of the solution containing the peripheral blood monocytes. Induction method. CTL inducer for obtaining cytotoxic T lymphocytes (CTL) of Claim 2 which contain the HLA-A24 binding antigen peptide of Claim 1 as an active ingredient. An anticancer agent comprising the HLA-A24 binding antigen peptide of claim 1 as an active ingredient. Cancer inhibitor containing the cytotoxic T lymphocytes of Claim 2 as an active ingredient. An antigen presenting cell which presents a complex of the HLA-A24 binding antigen peptide and the HLA-A24 molecule according to claim 1 on a cell surface. CTL inducer for obtaining cytotoxic T lymphocytes (CTL) of Claim 2 which contain the antigen presenting cell of Claim 10 as an active ingredient. Cancer inhibitor containing the antigen presenting cell of Claim 10 as an active ingredient. When the test cells are contacted with the cytotoxic T lymphocytes according to claim 2, when a change selected from the group consisting of cell lysis, cytokine release and CTL proliferation by CTL occurs, the test cells are treated with cytotoxicity according to claim 2. A method for detecting susceptible cells of cytotoxic T lymphocytes, characterized by being an indicator of cells (sensitive cells) susceptible to sexual T lymphocytes. An agent for detecting susceptible cells of cytotoxic T lymphocytes comprising the cytotoxic T lymphocytes according to claim 2 as an active ingredient. The human histocompatibility antigen (HLA) -A24 molecule in a test cell characterized by contacting the test cell with the cytotoxic T lymphocytes according to claim 2 in the presence of the HLA-A24 binding antigen peptide according to claim 1. Detection method. A nucleic acid encoding the HLA-A24 binding antigen peptide of claim 1. Cytotoxicity comprising the step of introducing a nucleic acid encoding the HLA-A24 binding antigenic peptide according to claim 1, or a nucleic acid encoding the HLA-A24 binding antigen peptide described in claim 1 and a nucleic acid encoding HLA-A24 into the cell A method for producing target cells injured by sex T lymphocytes. 18. The method of claim 17, wherein the introduction into the nucleic acid cell is carried out via an adenovirus vector. delete delete A target cell injured by cytotoxic T lymphocytes obtained by the method according to claim 17 or 18.

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