KR100480546B1 - Cytotoxic t lymphocytes - Google Patents

Abstract

A cell surface of a complex of one or more antigenic peptides selected from human major histocompatibility antigens (HLA) -A24 binding antigen peptides and functional derivatives thereof represented by the amino acid sequence of any one of SEQ ID NOs: 1-6 and HLA-A24 molecules Cytotoxic T lymphocytes (CTLs) that recognize cells present in the cell; Cancer inhibitors containing the CTL; Deriving the CTL; Inducers of said CTL; Cancer inhibitors containing one or more antigenic peptides selected from the above HLA-A24 binding antigen peptides and functional derivatives thereof; Antigen-presenting cells presenting a complex of one or more antigenic peptides selected from the HLA-A24 binding antigen peptide and functional derivatives thereof and a HLA-A24 molecule on a cell surface; CTL inducer containing the antigen-presenting cell; A cancer inhibitor containing the antigen presenting cell; A method for detecting cells susceptible to the CTL; A detection agent for cells susceptible to said CTL containing said CTL; And methods of detecting HLA molecules.

Description

Cytotoxic T lymphocytes {CYTOTOXIC T LYMPHOCYTES}

The present invention relates to cytotoxic T lymphocytes (sometimes referred to as cytotoxic T lymphocytes) (hereinafter referred to as CTLs) useful for the treatment and diagnosis of cancer, and CTL inducers useful for inducing such CTLs. will be.

CTLs include the major histocompatibility gene complex (MHC), which is encoded by the antigen peptide and the major histo-compatibility gene complex (MHC) in humans, called human leukocyte antigen class I (HLA class I). The complex, which is a combination with the molecule, may be recognized by a specific T cell receptor (hereinafter referred to as TCR), and some may damage cells presenting the complex on the cell surface. Such CTL is called MHC class I molecular binding CTL because it recognizes and injuries only target cells having the same MHC class I molecule. Thus, in order for this cytotoxic reaction to be established, 1) CTLs with specific TCRs must be present, and 2) binding to MHC class I antigen molecules to form antigenic peptides recognized in CTL that are present in HLA class I molecules. In addition, there is a need for an antigen peptide capable of forming a complex that is recognized by TCR.

That is, as used herein, the term "antigenic peptide" refers to a peptide that binds to an MHC class I molecule, has a complex-forming ability with an MHC class I molecule, and has an antigenicity where the complex is recognized by specific CTL TCR. In addition, in this specification, "HLA-binding antigen peptide" means that the MHC class I molecule is an antigen peptide which is an HLA molecule. In addition, the term "peptide" in the present specification includes a heteromeric peptide containing a non-amino acid component, without being particularly limited to a homomeric peptide consisting of only amino acids. The amino acid is not particularly limited to the natural form but may be a chemically modified amino acid. In addition, the peptide herein may be a multimer, not limited to the monomer.

Such antigenic peptides are generated by, for example, antigens synthesized in the cells of mammalian cells, acting on the endoplasmic reticulum, and cleaved into small epitope peptides, which are then 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 to 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 can bind to heterodimers of class I / β ₂ microglobulin in the endoplasmic reticulum and are transported to the cell surface through the Golgi apparatus. Tumor cells expressing tumor-associated antigens or tumor-specific antigen proteins may present MHC class I molecularly binding antigen peptides recognized on T lymphocytes on tumor cell surfaces.

As the first tumor specific antigen recognized by T lymphocytes, a gene called melanoma antigen E (hereinafter referred to as MAGE) was cloned and identified by T. Boon et al. (P. van der Bruggen et al. 254, pages 1643-1647 (1991). They isolate MAGE by expression cloning using a CTL clone from Melanoma patients, and a series of Melanoma cell lines from the same patient recognized for that CTL, and several of them immunoselected to be resistant to the CTL. . In other words, when a 5 kb DNA fragment prepared from a melanoma cell line recognized by CTL was transduced into a cell line that was not originally recognized, the CTL clone was recognized. This DNA fragment coded an estimated 26,000 proteins. The gene encoding this protein is called MAGE-1, and mRNA analysis revealed that not only metastasis but also 40% of cell lines derived from early melanoma express MAGE-1. This protein-derived antigen is HLA-A1 binding and the MAGE-1 specific CTL clone recognizes a peptide sequence (EADPTGHSY) consisting of the 9 amino acid sequence as set forth in SEQ ID NO: 7 bound to an HLA-A1 molecule, which is a type of HLA class I molecule. [C. Traversari et al., Journal of Experimental of Medicine, Vol. 176, pp. 1453-1457 (1992).

Many tumor antigens are known, but the MAGE family, gp 100, tyrosinase, and cancer fetus, which are known to be present in more than 10 species including MAGE-1, are currently recognized in CD-8 positive CTL. Carcinoembryonic antigens (hereinafter referred to as CEA) and HER2 / neu are well known. Recognized by CTL is a peptide derived from tumor antigen protein and is presented as a complex with HLA class I molecules.

HLA class I molecules are mainly HLA-A, -B, -C, and the antigen peptides presented by binding to these are composed of 9 to 10 amino acids, and also have certain structural characteristics that are different for each HLA molecule. This is known. For example, peptides consisting of 9 to 10 amino acids having a Leu at the N terminus and a Leu or Val at the C terminus are best known as peptides that bind the most frequently HLA-A2.1 molecules in the world. In addition, peptides that bind many HLA-A24 molecules to Asian races, including Japanese, are any one of Tyr, Phe, Met, and Trp at the N-terminus and any of Leu, Ile, Trp and Phe at the C-terminus. Peptides consisting of 9 to 10 amino acids having the most well known [A. Kondo et al., Journal of Immunology, Vol. 155, pp. 4307-4312 (1995).

Tumor antigens for which antigen peptides have been identified to date include MAGE-1, MAGE-3, HAGE-A, MAGE-3, MART1, tyrosinase, gp 100, HER2 / neu, and CEA against HLA-A1. Et al., MAGE-3 for HLA-Cw1, MAGE-3 for HLA-B44, and the like, tyrosinase, β-catenin, and the like for HLA-A24. Concerning antigen peptides derived from CEA, peptides consisting of nine amino acid sequences shown in SEQ ID NO: 34 have been identified as HLA-A2.1 binding antigen peptides [Journal of National Cancer Institute, Vol. 87, pp. 982 to 990 ( 1995). Similarly, with respect to antigen peptides derived from HER2 / neu, peptides consisting of nine amino acid sequences shown in SEQ ID NOs: 35 or 36 have been identified as HLA-A2.1 binding antigen peptides (Journal of Experimental of Medicine, Vol. 181, Pages 2109-2117 (1995), Cancer Research, Vol. 54, pages 1071-1076 (1994)). Most of these first identify the tumor antigens recognized by these CTLs by coining class I binding CTLs that recognize tumor cells, and then continue to find the smallest units of tumor antigen proteins by genetic engineering methods, furthermore the HLA class I molecules. Peptides in the smallest unit have been found based on information on binding motifs to Y. Kawakami et al., Proceedings of the National Academy of the Sciences of the USA, Vol. 91, pp. 3315-3519 (1994). In addition, 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 one that can induce CTL using antigen presenting cells was selected. Finally, antigenic peptides are determined according to whether or not CTLs with injuring tumor cells are induced (E. Celis et al., Proceedings of the National Academy of Sciences of the USA, Vol. 91, pp. 2105-2109). (1994), B. Gaugler et al., Journal of Experimental of Medicine, page 179, pages 921-930 (1994).

On the other hand, HLA class I molecules are classified into several subtypes, and the types of retained subtypes vary greatly among races, and HLA-A2 is the largest in the world, accounting for 45% of the white race. The identification of this HLA-A2 binding antigen peptide is the most advanced. In Japanese, HLA-A2 accounts for 40%, but in its subtype, HLA-A ^* 0201, which is the same as the white race, is 20% and most of the rest is A ^* 0206. The binding peptides to these subtypes are different and the HLA-A2 being mainly studied is HLA-A ^* 0201. On the other hand, HLA-A24 accounts for more than 60% in Japanese, and this HLA-A24 is higher in Asians than other races. However, the tyrosinase and β-catenin are the tumor antigens to which the HLA-A24 binding antigen peptide has been found so far, but the interpretation thereof is far behind. Therefore, the interpretation of CTLs that act on tumor cell-specific properties in Asians, especially Japanese, has been lagging behind, making it impossible to provide CTLs useful for tumor treatment. It is an object of the present invention to provide this CTL.

Disclosure of the Invention

 A first aspect of the present invention provides a cell surface complex of a HLA-A24 molecule and one or more antigenic peptides selected from HLA-A24 binding antigen peptides and functional derivatives thereof represented by the amino acid sequence of any one of SEQ ID NOs: 1-6. It relates to a CTL that recognizes the cells presented in. The second aspect of the present invention relates to a cancer inhibitor comprising the CTL of the first aspect as an active ingredient. A third aspect of the invention relates to a method of deriving CTL of the first aspect. A fourth aspect of the present invention relates to a CTL inducer comprising as an active ingredient one or more antigenic peptides selected from HLA-A24 binding antigen peptides and functional derivatives thereof represented by the amino acid sequence of any one of SEQ ID NOs: 1-6. will be. A fifth aspect of the present invention relates to a cancer inhibitor comprising at least one antigen peptide selected from HLA-A24 binding antigen peptides and functional derivatives thereof represented by the amino acid sequence of any one of SEQ ID NOs: 1 to 6 as an active ingredient. . In a sixth aspect of the present invention, a complex of an HLA-A24 molecule with one or more antigenic peptides selected from HLA-A24 binding antigen peptides and functional derivatives thereof represented by the amino acid sequence of any one of SEQ ID NOs: 1-6 It relates to an antigen presenting cell presented on the surface. In addition, in this specification, as a cell which presents the complex of a HLA-A24 binding antigen peptide and an HLA-A24 molecule on the cell surface, the term "target cell" and the term "antigen presenting cell" are used. As used herein, a "target cell" is a cell that is injured by a CTL that specifically recognizes this cell. Meanwhile, the term "antigen-presenting cell" as used herein refers to a cell having a function of presenting an antigen together with an MHC molecule to cause activation of T lymphocytes, and unless otherwise specified, a cell whose MHC molecule is an HLA-A24 molecule. In addition, in the present specification, "a cell having an antigen-presenting 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 HLA-A24. It is a cell that is a molecule. The seventh aspect of the present invention relates to a CTL inducer comprising the antigen-presenting cell of the sixth aspect as an active ingredient. An eighth aspect of the present invention relates to a cancer inhibitor comprising the antigen-presenting cell of the sixth aspect as an active ingredient. A ninth aspect of the present invention relates to a method for detecting cells susceptible to this CTL, using the change occurring when the CTL of the first aspect is brought into contact with a test cell. In addition, in this specification, a "test cell" is a human cell or coin cell derived from in vitro extraction samples such as peripheral blood lymphocytes and tissues. In addition, as used herein, "susceptible cells" refers to abnormal cells including tumor cells that are recognized by CTL and cause cytolysis or cytokine release. A tenth aspect of the present invention relates to a detection agent for cells susceptible to this CTL, comprising the CTL of the first aspect as an active ingredient. In addition, the eleventh aspect of the present invention is characterized by contacting a test cell with a CTL capable of recognizing a complex of the antigenic peptide and the HLA molecule in the presence of one or more antigenic peptides selected from HLA-binding antigen peptides and functional derivatives thereof. The present invention relates to a method for detecting HLA molecules.

The present inventors searched for antigen peptides among many candidate peptides centered on peptides having HLA-A24 binding motif in MAGE-3 protein, which is a tumor antigen, and when using peptides consisting of amino acid sequences represented by SEQ ID NO: 1 or 2, It has been shown that CTLs that selectively injure these tumor antigen expressing cells can be induced from peripheral blood mononuclear cells of normal people expressing HLA-A24. Similarly, the HLA-A24 binding antigen peptide was searched among candidate peptides centered on peptides having HLA-A24 binding motifs among various proteins which are tumor antigens. A peptide comprising an amino acid sequence, a peptide consisting of an amino acid sequence represented by SEQ ID NO: 4 or 5 present in a CEA protein amino acid sequence, or a peptide consisting of an amino acid sequence represented by SEQ ID NO: 6 present in an HER2 / neu protein amino acid sequence The present invention was completed by demonstrating that CTLs that selectively injure these tumor antigen-expressing cells can be induced from peripheral blood mononuclear cells of normal people expressing HLA-A24.

1 shows specific cytotoxic activity of target cells of effector cells induced by stimulation of antigen peptide MA3-2.

2 shows specific cytotoxic activity of target cells of effector cells induced by stimulation of antigen peptide MA3-4.

FIG. 3 shows specific cytotoxic activity of various cancer cells of effector cells induced by stimulation of antigen peptide MA3-2.

4 shows specific cytotoxic activity of various cancer cells of effector cells induced by stimulation of antigen peptide MA3-4.

5 shows specific cytotoxic activity of various target cells of effector cells induced by stimulation of antigen peptide MA1-1.

Best Mode for Carrying Out the Invention

A first aspect of the present invention provides a cell surface complex of a HLA-A24 molecule and one or more antigenic peptides selected from HLA-A24 binding antigen peptides and functional derivatives thereof represented by the amino acid sequence of any one of SEQ ID NOs: 1-6. As a CTL for recognizing the cells to be presented, the CTL specifically causes cytolysis or cytokine free reaction to target cells. In addition, CTL in this specification refers to having the injury activity by recognizing the antigenicity of the antigen peptide shown on the HLA-A24 molecule, and is not limited to other antigenic recognition.

In the present specification, the functional derivative of the HLA-A24 binding antigen peptide refers to an antigen peptide and HLA-A24 in which a complex formed simultaneously with the ability to form a complex with the HLA-A24 molecule is represented by the amino acid sequence of any one of SEQ ID NOs: 1-6. Recognized by the CTL to recognize a complex of molecules. For example, in the amino acid sequence of a peptide represented by the amino acid according to any one of SEQ ID NOs: 1 to 6, one or several amino acids are deleted, substituted with another amino acid or amino acid analog, and / or one or several amino acids or amino acid analogs The amino acid sequence is different depending on the addition or combination thereof, but the complex formed simultaneously with the ability to form a complex with the HLA-A24 molecule is a peptide recognized by the CTL of the present invention, and dimers of the peptide by disulfide bond or the like are also included. The amino acid sequence length of the functional derivative is preferably 9 to 10, but is not particularly limited thereto. In addition, the amino acid analog in this specification means N-acylated compound, 0-acylated compound, esterified product, acidamide compound, alkylated compound, etc. of several amino acids.

In addition, when the complex with the HLA-A24 molecule is recognized by CTL, the N-terminal or free amino group of the antigen peptide may be bound to a formyl group, an acetyl group, a t-butoxycarbonyl (t-Boc) group, or the like. In addition, when the complex with the HLA-A24 molecule is recognized in the CTL, the C terminal or the free carboxyl group of the antigen peptide may be bonded to a methyl group, ethyl group, t-butyl group, benzyl group, and the like. Similarly, if the complex with the HLA-A24 molecule is recognized by CTL, the thiol group of the cysteine contained in the antigen peptide may be bound to an acetamide methyl group, a methoxybenzyl group, or the like.

Functional derivatives can be identified by using a CTL that recognizes a complex of the antigen peptide represented by the amino acid sequence of any one of SEQ ID NOS: 1 to 6 with the HLA-A24 molecule. For example, the following method may be mentioned.

The first method mixes a candidate substance as a functional derivative and HLA-A24 expressing cells, washes the unbound candidate substance to the HLA-A24 molecule, and then reacts with CTL. If a candidate specific cytotoxicity, cytokine release, or proliferative reaction is recognized, it can be determined to be a functional derivative.

The second method adds a candidate to a cell having antigen-presenting ability, reacts with the time necessary for the antigen peptide and the HLA molecular complex to be presented on the cell surface at the appropriate time, for example, by introducing the antigen into the cell to act. React with If a candidate specific cytokine release or proliferative reaction is recognized, the substance can be determined to be a functional derivative.

The third method binds a nucleic acid encoding an amino acid sequence of a candidate to an expression vector capable of presenting a peptide on an HLA-A24 molecule on a cell having an antigen-presenting ability to be described later, and the antigen transformed by the recombinant vector. React CTLs with cells with presentation capacity. If a candidate specific cytokine release or proliferative reaction is recognized, the substance can be determined to be a functional derivative.

As the functional derivative, for example, in the amino acid sequence of the peptide represented by the amino acid sequence of any one of SEQ ID NOs: 1 to 6, the second amino acid at the N-terminus is HLA-A24 to enhance the binding to the HLA-A24 molecule. Substitution with an amino acid selected from Tyr, Phe, Met, Trp, which is characteristic of a peptide that binds to the molecule, and / or selected from Leu, Ile, Trp, Phe, which is characteristic of a peptide where the C-terminal amino acid binds to an HLA-A24 molecule Of the peptides substituted with amino acids, the binding ability with the HLA-A24 molecule is recognized, and the complex with the HLA-A24 molecule is recognized by the CTL which recognizes the complex of the peptide according to any one of SEQ ID NOS: 1 to 6 and the HLA-A24 molecule. And a peptide to be mentioned.

Moreover, in the peptide represented by the amino acid sequence by which one or several amino acids of the amino acid sequence of any of SEQ ID NOS: 1-6 were substituted, it is the peptide substituted by the amino acid or amino acid analog which is similar in the side chain to each amino acid substituted. And peptides which have a binding capacity with HLA-A24 molecules and whose complexes with HLA-A24 molecules are recognized in the CTL of the present invention. 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 (Ser) and threonine (Thr); Lysine (Lys) and arginine (Arg); Phenylalanine (Phe) and tyrosine (Tyr) are mentioned.

For example, a functional derivative of an antigen peptide represented by SEQ ID NO: 4, which is a peptide represented by the amino acid sequence of SEQ ID No. 24 in which the sixth amino acid (Val) is substituted at the N-terminal with Ile, and the amino acid (Gly) is the eighth amino acid at the N-terminal And peptides represented by the amino acid sequence set forth in SEQ ID NO: 46 wherein Ala is substituted with Ala. On the other hand, as a functional derivative of the antigenic peptide represented by SEQ ID NO: 5, the peptide represented by the amino acid sequence of SEQ ID NOs: 53 and 55 in which the sixth amino acid (Val) is substituted at the N terminus with Ile and Met, or at the N terminus, Reaction of the fourth amino acid (Cys) with the thiol group of Cys and 4,5-dihydroxy-2-cyclopenten-l-one (hereinafter referred to as DHCP) The peptide described in SEQ ID NO: 33 substituted with the product obtained by the above is mentioned. The fourth amino acid at the N-terminus may be a product obtained by the reaction of a thiol group of Cys with N-ethylmaleimide, dithiothreitol, and 4-vinylpyridine. have.

Further, the functional derivative may be a substitution of amino acids having similar side chains, for example, an amino acid sequence of one or several amino acids in the amino acid sequence of the peptide represented by the amino acid sequence of any one of SEQ ID NOS: 1-6. Or an amino acid analogue, such as a peptide represented by an amino acid sequence substituted with Ala, having a binding capacity with an HLA-A24 molecule, and wherein the complex with the HLA-A24 molecule is a peptide according to any one of SEQ ID NOs: 1 to 6 and an HLA-A24 molecule. The peptide recognized by CTL which recognizes a complex with is mentioned.

For example, as a functional derivative of the antigenic peptide shown in SEQ ID NO: 4, a peptide represented by the amino acid sequence of any one of SEQ ID NOs: 39, 40, 42 to 45, 48, as a functional derivative of the antigenic peptide shown in SEQ ID NO: 5 The peptide represented by the amino acid sequence of No. 26, 27, 30, 32 is mentioned.

In addition, the amino acid at the position which can be substituted by Ala in the said peptide may be substituted by another amino acid different from an original amino acid, or an amino acid analog. For example, the fourth amino acid of the antigen peptide represented by the amino acid sequence set forth in SEQ ID NO: 5 may be substituted with an amino acid or an amino acid analog other than acidic amino acids such as Asp and Glu, in addition to Ala. For example, the peptide substituted by Ser or Gly represented by the amino acid sequence of sequence number 51 or 52 is mentioned.

Moreover, as a dimer of the peptide by disulfide bond etc., the dimer of the peptide represented by the amino acid sequence of sequence number 5 by the disulfide bond through the thiol group of cysteine can be illustrated.

In addition to the above, functional derivatives of peptides consisting of the amino acid sequence described in SEQ ID NO: 1 also include peptides derived from MAGE family proteins including MAGE-3 recognized in CTL derived using peptides consisting of the amino acid sequence described in SEQ ID NO: 1. . Equally, functional derivatives of the peptides represented by the amino acid sequence described in SEQ ID NO: 2 also include peptides derived from MAGE family proteins including MAGE-3 recognized in CTL derived by the peptides represented by the amino acid sequence described in SEQ ID NO: 2. do. Functional derivatives of peptides represented by the amino acid sequence described in SEQ ID NO: 3 also include peptides derived from MAGE family proteins including MAGE-1 recognized in CTL derived by the peptide represented by the amino acid sequence described in SEQ ID NO: 3.

Functional derivatives of the peptide consisting of the amino acid sequence of SEQ ID NO: 4 include CEA and non-specific cross-reacting antigen (NCA) recognized in CTL derived using the peptide consisting of the amino acid sequence of SEQ ID NO: 4. The derived peptide can also be mentioned. Examples of the functional derivative of the peptide represented by the amino acid sequence of SEQ ID NO: 5 include peptides derived from CEA and NCA recognized by CTL derived by the peptide represented by the amino acid sequence of SEQ ID NO: 5.

Hereinafter, in the present specification, a peptide represented by SEQ ID NO: 1 or 2 and a functional derivative thereof are collectively referred to as MAGE-3 antigen peptide, and a peptide represented by SEQ ID NO: 3 and a functional derivative thereof as MAGE-1 antigen peptide. Equally, the peptides represented by SEQ ID NO: 4 or 5 and functional derivatives thereof are collectively referred to as CEA antigen peptides, the peptides represented by SEQ ID NO: 6 and functional derivatives thereof as HER2 / neu antigen peptides. Hereinafter, "antigenic peptide" as used herein refers to a peptide optionally selected from MAGE-3 antigen peptides, MAGE-1 antigen peptides, CEA antigen peptides and / or HER2 / neu antigen peptides.

Antigenic peptides and derivatives thereof can be prepared by organic chemical methods by liquid or solid amino acid coupling. Moreover, it can also be prepared using the recombinant DNA technique using the nucleic acid which encoded the amino acid sequence of an antigen peptide. The peptide obtained by the recombinant DNA technique may be modified by using an appropriate organic chemical or biochemical technique, if necessary.

In the CTL of the present invention, CTLs derived from MAGE-3 antigen peptides were positive tumor cells of both HLA-A24 and MAGE-3, and CTLs derived from MAGE-1 antigen peptides were HLA-A24 and MAGE. -1 all positive tumor cells, CTL derived using CEA antigen peptides were positive for HLA-A24 and CEA both positive tumor cells, CTL derived using HER2 / neu antigen peptides were HLA-A24 and HER2 / neu Both positive tumor cells can be selectively used for the detection of these CTL-sensitive cells and the like as cell medicaments for tumors in that they can selectively injure each.

The second aspect of the present invention relates to a cancer inhibitor comprising the CTL of the first aspect as an active ingredient. This cancer inhibitor is provided in a form in which this CTL is suspended in a pharmaceutically acceptable diluent. In addition, a diluent here is a medium suitable for preservation of this CTL, physiological saline, or phosphate buffered physiological saline. As a medium, media, such as RPMI and AIM-V, are mentioned generally. In addition, a pharmaceutically acceptable carrier may be added to this cancer inhibitor for stabilization purposes. The carrier herein is human serum albumin and the like. This cancer inhibitor contains CTLs of the first embodiment 10 ⁴ to 10 ⁸ / ml, preferably 5 x 10 ⁵ to 5 x 10 ⁷ / ml per CTL.

When the cancer inhibitor is administered to humans, it can be administered by syringe, and the dosage per adult is usually 10 ⁶ to 10 ¹⁰ CTLs per type of CTL. And the said range is not limited to this as a reference. In addition, the toxicity of this cancer inhibitor is not particularly recognized since the active ingredient CTL is derived from human to be administered.

The cancer inhibitor of the present invention is administered to cancer patients having tumor cells expressing antigenic peptides recognizable by CTL as an active ingredient. For example, MAGE is initially an antigen identified in malignant tumor cells of melanoma patients, It is confirmed that it expresses in the frequency of about 10-50% among cancer patients other than melanoma. As tumor cells expressing MAGE-1, MAGE-2 or MAGE-3, tumor cells derived from lung cancer, breast cancer, head and neck cancer, gastric cancer, esophageal cancer, bladder cancer and the like, in addition to melanoma, are mentioned [B. Gaugler et al., Journal of experimental of medicine, Vol. 179, pp. 921 to 930 (1994). Thus, cancer inhibitors containing CTLs of the first aspect of the invention, e.g., derived using MAGE-3 antigen peptides, can be used in patients with the cancers comprising both HLA-A24 and MAGE-3 positive tumor cells. It is useful for treatment.

And when using the said cancer inhibitor, although you may use together with another cancer inhibitor, the combination of the cancer inhibitor which acts immunosuppressively is not preferable.

A third aspect of the invention relates to a CTL derivation method of the first aspect. This CTL can be derived by using one or more antigenic peptides selected from HLA-A24 binding antigen peptides and functional derivatives thereof represented by the amino acid sequence of any one of SEQ ID NOs: 1-6.

When in vitro induces CTLs, it may be induced, for example, using ex vivo samples from living organisms expressing HLA-A24. The extracorporeal sample in the present specification includes, in addition to blood, lymph nodes, spleen, and other various organs extracted by surgery or the like, and lymphocytes or infiltrating lymphocyte cells present in these samples are preferably used.

For example, when blood is used, CTL can be induced by repeatedly applying antigenic stimulation to lymphocytes obtained from peripheral blood mononuclear cells (hereinafter referred to as PBMCs) prepared with HLA-A24 positive human blood. Antigen stimulation can be performed, for example, by using an antigen-presenting cell or the like that has been presented on the cell surface as an antigen peptide complexed with HLA-A24 molecules in a cell having antigen-producing ability prepared from the same blood prepared with lymphocytes. Induced CTLs can be further propagated by stimulation with CD-3 antibodies or the like, or by applying various stimuli in the presence of antigen peptides. For example, CTL induction can be performed by mixing antigen-presenting cells in a ratio of 0.01 to 1 with respect to lymphocytes 1 prepared with PBMC in the presence of IL-7. As an alternative embodiment, antigen peptides in the cell culture medium in the presence of IL-7 and Keyhole Limpet Hemocyanin (KLH) in PBMCs, from 1 ng / ml to 100 µg / ml per antigenic peptide, preferably CTL induction can be performed by adding 10 ng / ml to 1 µg / ml.

Induced CTLs may be maintained as lymphocytes with stable cytotoxicity by cloning. For example, they can be propagated by imparting antigen, various cytokines, and anti-CD 3 antibody stimulation to induced CTLs.

Induced specific CTL is antigen specific increase in CTL proliferation or antigen specific in CTL or target cells, which can be measured by the introduction of injury or radioactivity to target cells labeled with CTL-induced antigen peptides and radioactive materials. It can be detected by measuring the amount of cytokines such as GM-CSF, IFN-γ, and the like, which are freely released. It can also be confirmed directly by use of an antigen peptide or complex labeled with other fluorescent dyes. In this case, for example, CTL is contacted with a first fluorescent marker coupled with a CTL specific antibody, followed by contact with an antigenpeptide-MHC complex coupled with a second fluorescent marker, and the presence of double marker cells is monitored by FACS. activated cell sorting) analysis.

On the other hand, induction of CTL in vivo can be performed, for example, by administering antigen-presenting cells presented on the cell surface to a living body as a complex of an antigen peptide and a HLA-A24 molecule. When administered to humans, the dosage per adult is usually 10 ⁴ to 10 ⁹ . Alternatively, the antigen peptide may be mixed with a suitable adjuvant and administered to a living body to induce CTLs specific for that peptide. Adjuvant includes ① Freund's complete adjuvant, ② Freund's incomplete adjuvant, ③ Mineral gel such as aluminum hydroxide, alum, ④ Surfactant such as risorecithin, dimethyloctadecylammonium bromide, ⑤ Dextrin sulfate, Polyanions such as poly IC, peptides such as (6) muramyl peptide and taftocin, and monophosphoryl lipid (MPL) A manufactured by (7) Ribi, but are not particularly limited. Induction of CTL in a living body may be performed by mixing a combination of an antigen peptide and an adjuvant, as well as an MHC class II binding helper T cell antigen peptide. The antigen peptide may be covalently administered with higher fatty acid or helper T cell antigen peptide through a suitable linker for stabilization in vivo. When administered to humans, the dose per adult is as antigen peptide concentration 0.1 μg / kg to 10 mg / kg, preferably 1 μg / kg to 1 mg / kg, more preferably 1 per antigen peptide. Μg / kg to 100 μg / kg.

Complexes of HLA-A24 molecules with antigen peptides can also be used for stimulation with antigen peptides to induce CTLs. In this case, the HLA-A24 molecule need not be a natural HLA-A24 molecule, but may be a fragment which essentially preserves the binding property to the antigen peptide. These fragments can be prepared, for example, by proteolytic or recombinant DNA techniques of native HLA-A24 molecules. This complex can be stabilized by co-existing β ₂ -microglobulin, and also an antibody that recognizes the polymer.

A fourth aspect of the present invention relates to a CTL inducer comprising at least one antigen peptide selected from HLA-A24 binding antigen peptides and functional derivatives thereof represented by the amino acid sequence of any one of SEQ ID NOs: 1 to 6 as an active ingredient. . CTL inducers are supplied in a form suspended in physiological saline or phosphate buffered physiological saline either alone or as a mixture with other molecules (helper T cell antigen peptides and / or adjuvant). The antigen peptide may be a covalent conjugate with a higher fatty acid or helper T cell antigen peptide or a complex with an HLA-A24 molecule. This inducer contains an antigen peptide of 0.01 to 100% by weight, preferably 0.1 to 95% by weight per antigen peptide.

This CTL inducer can be used in vitro for the addition of an additive to a medium for propagating the CTL of the present invention, T-immune proliferative activity, and for the diagnosis of an immunosensitized condition based on a delayed skin response. When used as an additive to the medium, for example, the amount used is 1 ng / ml to 100 μg / ml, preferably 10 ng / ml to 1 μg / ml per antigen peptide as the peptide concentration in the medium.

A fifth aspect of the present invention relates to a cancer inhibitor comprising at least one antigen peptide selected from HLA-A24 binding antigen peptides and functional derivatives thereof represented by the amino acid sequence of any one of SEQ ID NOs: 1 to 6 as an active ingredient. . The cancer inhibitor is provided in the form of 1) an antigen peptide alone, 2) a mixture of the antigen peptide with a pharmaceutically acceptable carrier and / or diluent, or 3) an adjuvant added to 1) or 2) above if necessary. do. In addition, the carrier used here is, for example, human serum albumin, and the diluent is, for example, phosphate buffer, distilled water, saline solution and the like. In addition, the adjuvant includes the pharmaceutically acceptable adjuvant and the like. When administering this cancer inhibitor to a human, it may administer by syringe or you may administer by transdermal absorption from a mucosa, etc. by the method of spraying. The cancer inhibitor contains an antigen peptide of 0.01 to 100% by weight, preferably 0.1 to 95% by weight per antigen peptide. The dosage 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 / per antigen, as the peptide concentration. Kg. And when administered to humans, the toxicity of this peptide is not particularly recognized.

When using this cancer inhibitor, you may use together with another cancer inhibitor, but the combination of the cancer inhibitor which acts immunosuppressively is not preferable.

In a sixth aspect of the present invention, a complex of HLA-A24 molecule and one or more antigenic peptides selected from HLA-A24 binding antigen peptide and functional derivatives thereof represented by the amino acid sequence of any one of SEQ ID NOs: 1 to 6 can be obtained. It relates to an antigen presenting cell presented on the surface. Hereinafter, "antigen-presenting cells" in the present specification, unless otherwise indicated, one or more antigenic peptides selected from MAGE-3 antigen peptides, MAGE-1 antigen peptides, CEA antigen peptides and / or HER2 / neu antigen peptides are presented. Antigen presenting cells. These antigen presenting cells can be used for induction of CTL of the present invention.

This antigen presenting cell can be prepared by using a cell having an antigen presenting ability. Examples of cells having antigen-presenting ability include leukocytes including macrophages, B cells, and dendritic cells (DCs) capable of presenting complexes of antigen peptides and HLA-A24 molecules on the cell surface thereof. DC is particularly preferable as a cell having antigen-presenting ability because it has a large amount of antigen presentation per cell and a high expression level of cell surface molecules (co-stimulatory signal molecules such as CD 80, CD 86, etc.) required for antigen presentation. The cells having the antigen-presenting ability can be prepared by the ex vivo extraction sample in the present specification. For example, DC is 1) isolated from PBMC based on several cell surface markers, etc., 2) derived from GM-CSF and IL-4 from monocytes, 3) GM-CSF from CD-34 positive cells, It can be prepared by any method such as induced by cytokines such as TNF-α and SCF.

In order to present the antigen peptide on the cell surface having the antigen-presenting ability, for example, one or more of the antigen-presenting cells and the antigen-peptide are mixed, and the excess is washed as necessary, and the antigen peptide is prepared on the HLA-A24 molecule. The method of loading is mentioned. Like the cells prepared by the method 1) in the method for preparing a cell having the antigen-presenting ability, a cell that is already presenting an antigen peptide unrelated to the present invention on an HLA-A24 molecule is an antigen peptide. The acid treatment or the like may be performed before loading to remove the peptide that cannot induce the CTL of the present invention on the HLA-A24 molecule. A cell having this antigen-presenting ability may be loaded with several kinds of antigen peptides per cell, in addition to cells that are loaded with a single antigen peptide. As another method, a method of transforming these cells with an expression vector capable of presenting the antigenic peptide to B cells or CD34 positive cells is mentioned. As such a vector, the expression plasmid vector which inserted the gene which codes one or more of the antigen peptides which are intended to show on the cell surface by recombinant DNA technology to commercially available plasmids, such as pcDNA3, pMQMneo, pCEP4, is mentioned. 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 surface of the cell so as to be efficiently degraded by the protease in the cell. Retroviral vectors and adenovirus vectors are also suitably used.

Antigen-presenting cells are preferably non-proliferative. In order to make the cells non-proliferative, treatment with radiation such as X-ray or mitomycin may be used.

A seventh aspect of the present invention relates to a CTL inducer containing the antigen presenting cells of the sixth aspect as an active ingredient. The inducer is supplied in a form in which the antigen presenting cells are suspended in a medium suitable for preservation of cells, saline, or phosphate buffered saline. As a medium, media, such as RPMI, AIM-V, and X-VIVO 10, are generally used. In addition, serum albumin or the like may be added to this medium or buffer for stabilization purposes. This inducer contains 10 ³ to 5 x 10 ⁶ / ml, preferably 10 ⁴ to 10 ⁶ / ml per antigen presenting cell.

This CTL inducer can be used not only as an additive to a medium for propagating CTL of the present invention in vitro but also for diagnosing an immunosensitized condition based on T lymphocyte proliferation activity. For example, when used as an additive to the medium, it is usually used in a ratio of 0.01 to 1 relative to lymphocytes 1 induced by CTL.

An eighth aspect of the present invention is a cancer inhibitor containing the antigen-presenting cell of the sixth aspect as an active ingredient. The cancer inhibitor is provided in a form in which the antigen presenting cells are suspended in a pharmaceutically acceptable diluent. Diluents used herein are media, phosphate buffers, saline, and the like suitable for preservation of cells. As a medium, media, such as RPMI, AIM-V, X-VIVO 10, are mentioned generally. In addition, a pharmaceutically acceptable carrier may be added to the cancer inhibitor for stabilization. The carrier as used herein is human serum albumin or the like. The cancer inhibitor contains 10 ⁵ to 10 ⁸ / ml, preferably 5 x 10 ⁵ to 5 x 10 ⁷ / ml per antigen presenting cell. When the cancer inhibitor is administered to humans, it can be administered by syringe, and the dose per adult is usually 10 ⁴ to 10 ⁹ per antigen-presenting cell type as the number of antigen-presenting cells. In addition, the range is only a reference, but is not limited thereto. In addition, since the antigen-presenting cell as an active ingredient is derived from a human to be administered, the toxicity of this cancer inhibitor is not particularly recognized.

When using this cancer inhibitor, you may use together with another cancer inhibitor, but the combination of the cancer inhibitor which acts immunosuppressively is not preferable.

A ninth aspect of the present invention relates to a method for detecting susceptible cells of this CTL, using the change occurring when the CTL of the first aspect is brought into contact with a test cell. Changes caused by contact of CTL with susceptible cells in test cells include, for example, target cell lysis by CTL, cytokine release, or proliferation of CTL. Target cell lysis can be detected by, for example, labeling a test cell with radioisotopes or fluorescent dyes and measuring the amount of radioisotopes or fluorescent dyes released from the test cells when mixed with CTL. 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. Further growth of CTL can be carried out through measurement of the number of CTL cells due to the introduced amount measured and microscopic observation of the ³ H- thymidine in the cells. This change results in tumor cells positive for both HLA-A24 and MAGE-3 or positive for both HLA-A24 and MAGE-3 related antigens, positive for both HLA-A24 and MAGE-1 or HLA-A24 and MAGE- Tumor cells positive for all related antigens, positive for both HLA-A24 and CEA or positive for both HLA-A24 and CEA related antigens, and / or positive for both HLA-A24 and HER2 / neu or HLA-A24 and HER2 / neu All related antigens can detect positive tumor cells.

A tenth aspect of the present invention relates to a cell detection agent that is susceptible to CTL of the first aspect, which contains the CTL of the first aspect as an active ingredient. The detection agent is supplied in a form suspended in a medium suitable for the preservation of this CTL, physiological saline, or phosphate buffered physiological saline. As the medium, a medium such as RPMI, AIM-V or X-VIVO 10 is generally used. In addition, serum albumin or the like may be added to this medium or buffer for the purpose of CTL stabilization. This detection agent contains 10 ⁴ to 10 ⁸ / ml of CTL of the first embodiment per CTL type.

In addition to the detection of susceptible cells in the CTL of the present invention in test cells, the detection agent can be used for HLA typing to identify whether HLA expressed in test cells is HLA-A24.

An eleventh aspect of the present invention provides a method for preparing an HLA molecule comprising contacting a test cell with a CTL capable of recognizing a complex of the antigen peptide and the HLA molecule in the presence of at least one antigen peptide selected from the HLA-binding antigen peptide. It relates to a detection method.

For example, the HLA-A24 molecule on the test cell surface can detect, as an index, a change caused by contacting the test cell with the CTL of the first embodiment. Changes caused by contact of CTL with a test cell include, for example, target cell lysis by CTL, cytokine release, or proliferation of CTL. Target cell lysis can be detected by, for example, labeling a test cell with radioisotopes or fluorescent dyes and measuring the amount of radioisotopes or fluorescent dyes released from the test cells when mixed with CTL. 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. Further growth of CTL can be carried out through measurement of the number of CTL cells due to cell or the introduced amount measured by microscopic observation of the ³ H- thymidine.

The measurement value can be used to measure the expression level of HLA-A24 molecules on test cells as well as to detect the presence or absence of expression of HLA-A24 molecules. In addition, when it is not clear whether MAGE-3, MAGE-1, CEA or HER2 / neu are expressed in the test cells, HLA-A24 molecules can be detected by the following method. In the present method, at least one antigen peptide selected from an antigen peptide consisting of the test cell and the amino acid sequence of any one of SEQ ID NOs: 1 to 6 and a functional derivative thereof so as to be 0.01 to 50 µg / ml in the reaction solution, and furthermore, the antigen The CTL of the first embodiment that recognizes the complex of the peptide and the HLA-A24 molecule can coexist, and the CTL and the test cell may be contacted.

The present invention enables the detection of HLA-A24 molecules in tumor cells that could not be detected by the conventional method for detecting HLA molecules using a conventional anti-HLA antibody in which a complement activation step was essential.

The antigen peptide used in the present method is not particularly limited to one or more HLA-A24 binding antigen peptides selected from peptides represented by the amino acid sequences described in any one of SEQ ID NOs: 1 to 6 or functional derivatives thereof. By using a binding antigen peptide, detection of several HLA molecules can be performed. For example, detection of an HLA-A2 molecule can also be performed by using an HLA-A2 binding antigen peptide. Examples of HLA-A2 binding antigen peptides include peptides derived from MAGE-3 represented by SEQ ID NO: 37 and peptides derived from influenza matrix peptide represented by SEQ ID NO: 38.

In addition, each type of HLA further has a subtype. For example, HLA-A2 is classified into 10 or more types of subtypes including A ^* 0201, A ^* 0206, and A ^* 0207. Since HLA-binding antigen peptides are also different between each subtype, it is important to determine the subtype of HLA molecule when using CTL as a cancer inhibitor etc., for example. Classification of subtypes that were not possible in the method (typing) of the detection of HLA molecules using anti-HLA antibodies is possible in this method. For example, use of a peptide having the amino acid sequence set forth in SEQ ID NO: 37 can induce A ^* 0201 binding CTL, and the combination of this CTL and this peptide can also determine the surf type of the HLA-A2 molecule in a test cell. . The method is superior in that it detects HLA molecules expressed on the actual cell surface than the DNA typing method using DNA extracted from test cells.

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

Example 1 Preparation of MAGE-3 Antipeptide Specific CTL (1)

CTLs in this example were Staphylococcus aureus Cowan-I; It is prepared by an induction method using SAC-I.

(1) Selection and Synthesis of CTL Inducing Peptides

A sequence having a HLA-A24 binding motif structure with respect to the amino acid sequence of the MAGE-3 protein consisting of 314 amino acids (the second at the N-terminus is the amino acid of any of Tyr, Phe, Trp, Met, and the C-terminus is Leu, Ile (Peptide which is an amino acid of any one of Phe, Trp), and also searches for the kind of amino acid in other positions. As a result, it was found that there exist seven peptides shown in Table 1 as candidate peptides of the MAGE-3 antigen peptide.

SEQ ID NO: Location of MAGE-3 designation 8291011112 76-84113-121142-150142-151150-158195-203195-204 MA3-6MA3-4MA3-1MA3-5MA3-7MA3-2MA3-3

In addition, in Table 1, the number of the sequence number term represents the sequence number of the sequence table showing the amino acid sequence of each peptide. In addition, the number of the position term in MAGE-3 shows the number of amino acids from the N terminal of MAGE-3 protein. The symbol in the name section indicates the name of the peptide named by the inventors.

Seven peptides of MA3-1 to MA3-7 shown in Table 1 were produced by the solid state Fmoc method using a peptide synthesizer (PSSM-8: manufactured by Shimadzu Corporation).

(2) Preparation of PBMC

Blood collection is performed by component blood collection from a normal person having HLA-A24, and leukocyte fractions are collected to further separate PBMCs according to the following separation method. That is, after diluting the blood collection liquid about 2 times in RPMI 1640 medium, it superimposes on the Ficoll-Paque separation liquid (made by Palmacia), and centrifuged at 500 * g for 20 minutes at room temperature. The PBMCs of the intermediate layer are collected by pipettes, washed, and stored in liquid nitrogen while suspended in a preservation solution consisting of 90% fetal bovine serum (FCS, manufactured by Intergen) and 10% dimethyl sulfoxide (manufactured by Sigma).

(3) Preparation of effector cells

In the following manner, five types of effector cells individually stimulated by any one of MA3-1, MA3-2, MA3-4, MA3-6, and MA3-7 are prepared.

After melting the preservation PBMC prepared in (2), it is suspended in 5H-RPMI so that the cell concentration becomes 4 x 10 ⁶ cells / ml. 5H-RPMI was prepared by using a human AB-type serum (manufactured by Avain Science) at a final concentration of 5% (v / v) and a non-essential amino acid (manufactured by Gibco BRL) at a final concentration of 0.1 mM in sodium RPMI 1640 medium. (Gibco BRL Co., Ltd.) was added to a final concentration of 1 mM, L-glutamine (Gibco BRL Co., Ltd.) was added to a final concentration of 4 mM, and gentamicin sulfate (manufactured by Aine Science Co., Ltd.) to a final concentration of 10 µg / ml. It is a badge. 25 ml of the cell suspension was added to a T-150 flask (AIS MicroCELLector, manufactured by Apride Science, Inc.) bound to the anti-CD 4 antibody and allowed to react at room temperature for 1 hour. Then, non-adhesive cells were recovered and 2 × 10 ⁶ cells / ml were added. Suspend in 5H-RPMI if possible. This suspension was subjected to SAC-1 treatment prepared by the method described in (1) of Example 8 below using peptides of any of MA3-1, MA3-2, MA3-4, MA3-6, and MA3-7. The mixture was equally mixed with five kinds of antigen-presenting cells for the first time stimulation, IL-7 (manufactured by Genzyme) was added to a final concentration of 15 ng / ml, and 2 ml of each well of a 24-well culture plate was dispensed. Incubate in a 37 ° C. CO ₂ incubator. The next day, IL-10 (made by R & D) is added so that the final concentration may be 10 ng / ml. After 6 days, half the culture supernatant is removed and an equal amount of 5H-RPMI containing 20 ng / ml IL-7 is added.

After further incubation for 3 days, the cells are recovered by centrifugation and suspended in 5H-RPMI so that the cell concentration is 2 × 10 ⁶ cells / ml. The suspension was placed on a plate containing antigen-presenting cells prepared as in (2) of Example 8 below using any one of MA3-1, MA3-2, MA3-4, MA3-6 and MA3-7 peptides. 1 ml / well is added and restimulation with the same peptide is performed. The following day, IL-10 was added to a final concentration of 10 ng / ml, and after 2 days, half of the culture supernatant was removed and an equal amount of 5H-RPMI containing 20 IU / ml of rIL-2 (manufactured by Shionogi Seiyaku Co., Ltd.) was added. And incubate in a CO ₂ incubator for 1 week. After the same re-stimulation three more times, the cells are recovered and used as effector cells.

(4) Preparation of CTL Target Cells

As a target cell for measuring cytotoxic activity, TISI (WSNO 9042), an EBV transform B cell expressing HLA-A24, is used. First, five types of medium containing 10 μg / ml of peptides of any one of MA3-1, MA3-2, MA3-4, MA3-6, and MA3-7 prepared in (1) the day before measurement of TISI cells ( Hereinafter, TISI cultured in this medium is indicated by TISI (+), or a medium containing no peptide (hereinafter, TISI cultured in this medium is denoted by TISI (-)) is overnight. On the day of measurement, 5 x 10 ⁶ five types of TISI (+) and TISI (-) were respectively mixed in 200 μCi Na ₂ ⁵¹ CrO ₄ solution at 37 ° C. for 1 hour, and then cultured with RPMI 1640 containing 10% FCS. The cells were washed with ⁵¹ Cr labeled label target cells.

(5) Measurement of cytotoxic activity by CTL

After diluting the effector cells of (3) with 5H-RPMI so as to be 5 x 10 ⁵ to 1 x 10 ⁶ cells / ml, 100 μl / well was dispensed into each well of a 96 well culture plate, and (4 100 μl of the cell suspension containing 1 × 10 ^{4 51} Cr marker target cells and 3 × 10 ⁵ K562 cells prepared in the above) is added. K562 cells are also used to eliminate nonspecific injury activity by incorporating NK cells.

After centrifuging the cell suspension at 400 x g for 1 minute, the cell suspension is left to stand in a CO ₂ incubator at 37 ° C for 5 hours. Thereafter, 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 formula (formula 1).

Specific cytotoxic activity (%) = [(measured value of each well-minimum release value) / (maximum emission value-minimum release value)] × 100 (Equation 1)

In the above formula, the minimum emission value is the amount of ⁵¹ Cr of the well containing only target cells and K562 cells, and represents the natural free amount of ⁵¹ Cr from target cells. In addition, the maximum release value represents the amount of ⁵¹ Cr free when the cell was destroyed by adding surfactant Toliton X-100 to the target cell. The results are shown in Table 2.

Peptide Name E / T Specific cytotoxic activity TISI (-) TISI (+) MA3-1MA3-2MA3-4MA3-6MA3-7 101055.810 1.66.63.10.817.0 0.865.63.40.013.7

In Table 2, E / T represents the ratio of effector cells to target cells.

As a result, effector cells obtained by induction with MA3-2 exhibit antigen-peptide specific cytotoxicity against TISI (+) to which MA3-2 peptide was added.

Example 2 Preparation of MAGE-3 Antipeptide-Specific CTLs (2)

CTL in this example is prepared by an induction method using KLH. And the antigen peptide uses five peptides of MA3-1, MA3-2, MA3-4, MA3-6, and MA3-7 prepared in Example (1).

(1) Preparation of PBMC

Blood samples are collected from normal individuals with HLA-A24 and PBMCs are isolated according to the following separation method. That is, the blood sample is suspended in the same amount of RPMI 1640 medium and layered on the picol-pack separation solution, and centrifuged at 500 xg for 25 minutes at room temperature. The PBMCs of the middle layer are collected by pipette and placed in a 15 ml centrifuge tube and washed three times in RPMI 1640 medium. Subsequently, it is suspended in 5H-RPMI such that the cell final concentration is 4 × 10 ⁶ cells / ml.

(2) Preparation of effector cells

Five kinds of effector cells individually stimulated with any one of MA3-1, MA3-2, MA3-4, MA3-6 and MA3-7 are prepared by the following method.

After (1) mixing an equivalent amount of a 5H-RPMI was added to peptide 20 ㎍ / ㎖ the PBMC prepared in, and allowed to stand from 2 to 3 hours in a CO ₂ incubator for 37 ℃. To this was added a KLH (Carbiochem Co., Ltd.) solution and an IL-7 solution so as to have a final concentration of 5 µg / ml and 25 ng / ml, respectively. This cell suspension is dispensed in 2 ml portions of 2 wells of a 24 well culture plate and incubated in a 37 ° C CO ₂ incubator. After 3 days, 60 μl (final rIL-2 concentration 30 IU / ml) of 5H-RPMI containing 1000 IU / ml rIL-2 is added.

After 1 week incubation, the cells are collected by centrifugation and suspended in 5H-RPMI so that the cell concentration is 5 x 10 ⁵ cells / ml. Any of the antigen-presenting cells prepared using the suspension according to the method described in (4) of Example 8 below using peptides of any one of MA3-1, MA3-2, MA3-4, MA3-6, and MA3-7. 1 ml / well is added to five kinds of plates including one species and re-stimulated. The next day, 60 μl of rIL-2 solution (1000 IU / ml) is added and incubated in a CO ₂ incubator for 1 week. After the same restimulation four more times, the cells are recovered and used as effector cells.

Five effector cells obtained by the above method are suspended in 5H-RPMI so as to be 4 × 10 ⁶ cells / ml.

(3) Preparation of CTL Target Cells

As a marker target cell, WiDr (colon cancer cell line), TE11 (esophageal cancer cell line), which are positive for both MAGE-3 and HLA-A24, in addition to TISI (-) and five types of TISI (+) prepared in the same manner as in Example 4 (4), MRKnul (breast cancer cell line) is used. In addition, KATO-III (gastric cancer cell line), which is a cancer cell line of MAGE-3 positive and HLA-A24 negative, is also used. WiDr, TE11, MRKnul, and KATO-III mixed each 5 × 10 ⁶ cells in 200 μCi of Na ₂ ⁵¹ CrO ₄ for 1 hour at 37 ° C. and then washed with 10% FCS-containing RPMI 1640 culture. ⁵¹ Cr.

(4) Measurement of Cytotoxic Activity by CTL

The cytotoxic activity is measured using the effector cells prepared in (2) and the target cells prepared in (3). When TISI (-) and TISI (+) were used as target cells, it measures by the method similar to (5) of Example 1. The reaction time between the effector cells and the target cells is 4.5 hours. When four other types of marker cancer cell lines were used as target cells, 100 μl / well (4 × 10 ⁵ cells / well) of the effector cell suspension of (2) was dispensed into each well of a 96 well culture plate. 100 μl of cell suspension containing 5 × 10 ^{3 51} Cr marker target cells and 1.5 × 10 ⁵ K562 cells is added. After centrifuging for 1 minute at 400 × g, the mixture is left to stand in a 37 ° C CO ₂ incubator for 4.5 hours. Thereafter, 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 | required by the same calculation as (5) of Example 1. The results are shown in Table 3.

Peptide Name E / T Specific cytotoxic activity TISI (-) TISI (+) WiDr TE11 MRKnul MA3-1MA3-2MA3-4MA3-6MA3-7 8080808080 27.67.45.48.721.8 51.760.539.414.839.4 ND51.548.7NDND ND39.029.1NDND ND20.927.6NDND

In Table 3, E / T indicates the ratio of effector cells to target cells, and ND indicates unmeasured.

In addition, specific cytotoxicity curves in various E / Ts for TISI (-) and TISI (+) of effector cells induced by MA3-2 or MA3-4 are shown in FIGS. 1 and 2, and WiDr and TE11. 3 and 4 show specific cytotoxicity curves in various E / Ts against MRKnul cells.

FIG. 1 shows the results of using effector cells induced by MA3-2 and FIG. 2 using effector cells induced by MA3-4. Also, a square mark ( ) Shows the result of using TISI (+) as a target cell and the diamond notation (◇) using TISI (-).

Fig. 3 shows the results of using effector cells induced by MA3-2 and Fig. 4 using effector cells induced by MA3-4. Also, a square mark ( ) Indicates WiDr as target cell, rhombic (◇) indicates TE-11, and white circle ( ) Shows the results using MRKnul.

The cytotoxic activity shown in FIGS. 3 and 4 is inhibited by the coexistence of class I antibodies (W6 / 32) in the reaction system. In addition, effector cells induced by MA3-2 or MA3-4 did not show cytotoxicity to KATO-III, a cancer cell line of MAGE-3 positive and HLA-A24 negative.

From the above results, effector cells induced by MA3-2 and MA3-4 peptides were antigen-peptide-specific cytotoxicity, while both HLA-A24 and MAGE-3 were CTLs that showed cytotoxicity against positive cancer cell lines. .

Example 3 Preparation of MAGE-1 Antipeptide Specific CTL

CTL in this example is prepared by the CTL derivation method using KLH.

(1) Selection and Synthesis of CTL Inducing Peptides

A sequence having the HLA-A24 binding motif structure in the same manner as in (1) of Example 1 with respect to the amino acid sequence of the MAGE-1 protein consisting of 309 amino acids (Molecular Immunology, Vol. 31, pages 1423 to 1430 (1994)) Search for other types of amino acids based on the search. As a result, five peptides shown in Table 4 were selected as candidate antigen peptides.

SEQ ID NO: Location in MAGE-1 designation 314131516 135-143135-144188-196188-197275-284 MA1-1MA1-3MA1-2MA1-4MA1-5

In addition, in Table 4, the number of the sequence number term represents the sequence number of the sequence table showing the amino acid sequence of each peptide. In addition, the number of the position term in MAGE-1 shows the number of amino acids from the N terminal of MAGE-1 protein. The symbol in the name section indicates the name of the peptide named by the inventors.

Five peptides of MA1-1 to MA1-5 shown in Table 4 were produced using a peptide synthesizer.

(2) Preparation of MAGE-1 antigen peptide specific CTL by induction method using KLH

The peptide of any of MA1-1, MA1-2, MA1-3, MA1-4, and MA1-5 prepared in (1) of Example 3, and MA1-1, MA1-2, MA1-3, MA1-4 , Effector cells were prepared in the same manner as in Example 2 (2) using the antigen-presenting cells prepared by the method described in (4) below using the peptide of any one of MA1-5. The cytotoxic activity of various target cells is measured. The target cells used were MAGE- in addition to the five types of TISI (+) cultured in a medium containing any one of TISI (-), MA1-1, MA1-2, MA1-3, MA1-4, and MA1-5. NUGC-3 (gastric cancer cell line) positive for both 1 and HLA-A24, Raji (linformer) negative for both TE-11, MAGE-1 positive and KATO-III, MAGE-1 and HLA-A24 negative. Specific cytotoxicity curves in various E / Ts of the effector cells induced by MA1-1 on various target cells are shown in FIG. 5.

Black circle in FIG. ) Represents NUGC-3 as a target cell, a black square ( ) Shows the result of using TE11, the inverted triangle display (▽) using KATO III, the lozenge display (◇) using Raji, and the triangular display (△) using TISI (-).

In addition, cytotoxic activity against TISI (+), the NUGC-3 cell line and TE-11 cell line shown using MA1-1 addition medium is inhibited by coexistence of class I antibody (W6 / 32) in the reaction system.

Thus, it was found that effector cells induced by MA1-1 were CTLs for both cancer cells lines positive for HLA-A24 and MAGE-1.

Example 4 Preparation of CEA Antigenpeptide Specific CTL (1)

CTL in this embodiment is prepared by the CTL derivation method using SAC-I in the same manner as in the first embodiment.

(1) Selection and Synthesis of CTL Inducing Peptides

The amino acid sequence of the CEA protein consisting of 464 amino acids is searched in consideration of the amino acid sequence at other positions centering on the sequence having the HLA-A24 binding motif structure. As a result, it was found that five peptides shown in Table 5 exist.

SEQ ID NO: Position in the CEA Protein designation 17184195 101-109234-242268-277318-326652-660 CE-1CE-5CE-2CE-4CE-3

In Table 5, the number of the sequence number term indicates the sequence number of the sequence table showing the amino acid sequence of each peptide. In addition, the number of the position term in CEA protein shows the number of amino acids from the N terminal in CEA protein. The symbol in the name section indicates the name of the peptide named by the inventors.

Five peptides shown in Table 5 were produced using a peptide synthesizer.

(2) Preparation of effector cells

The method as described in (1) of following Example 8 using the peptide of any one of preservation PBMC, CE-1, CE-3, CE-4, CE-5 prepared by the method similar to (2) of Example 1 In the following Example 8 (2) using an antigen-presenting cell for the first stimulation by SAC-I treatment prepared by the present invention and a peptide of any one of CE-1, CE-3, CE-4 and CE-5 Any of CE-1, CE-3, CE-4 and CE-5 in the same manner as in Example 1 (3) using four kinds of plates containing any one of the antigen presenting cells prepared by the described method. Four kinds of effector cells individually stimulated by one peptide are prepared.

(3) Preparation of CTL Target Cells

Five kinds of cells obtained by culturing in a medium containing any one of the peptides CE-1, CE-3, CE-4, CE-5 in the same manner as in Example (4) as target cells for measuring cytotoxic activity TISI (+) and gastric cancer cell line MKN-45 (HLA-A24 and CEA positive) were used. In addition, each cell is labeled with ⁵¹ Cr in the same manner as in Example 1 (4) on the day of cytotoxic activity measurement.

(4) Measurement of Cytotoxic Activity by CTL

Using the effector cells of (2) and the target cells of (3), specific cytotoxic activity (%) was calculated in the same manner as in Example (5).

The results are shown in Table 6.

Peptide Name E / T TISI (-) TISI (+) MKN-45 CE-1CE-3CE-4CE-5 10101010 24.81.97.54.8 86.792.55.94.6 0.09.20.20.0

In Table 6, E / T represents the ratio of effector cells to target cells.

As a result, effector cells obtained by induction of CE-3 showed antigen-peptide specific cytotoxicity against TISI (+) cultured in a medium containing CE-3 peptide and cytotoxicity against MKN-45. Activity.

Example 5 Preparation of CEA Antipeptide-Specific CTLs (2)

CTL in this example is prepared by an induction method using antigen-presenting dendritic cells (DC).

Two kinds of peptides of CE-2 and CE-3 prepared in Example 4 (1) were induced by effector cells recognizing each peptide in PBMCs of normal individuals to measure cytotoxic activity.

(1) Preparation of effector cells

In the following manner, two kinds of effector cells individually stimulated with a peptide of either CE-2 or CE-3 are prepared.

After melting the PBMC prepared in Example (2), it was suspended in PBS containing 1% human AB serum at 4 ° C (hereinafter referred to as 1H-PBS) so that the cell concentration was 2 x 10 ⁷ cells / ml. do. 14 μl of beads (Dynabeads M450, manufactured by Dynal Corporation) bound to 2 × 10 ⁷ / ml PBMC with anti-CD 8 antibody washed with 1H-PBS were added, and reacted at 4 ° C. for 1 hour. Remove it. After removing the non-adhesive cells, 0.1 ml of cell dissociation beads (DETACHaBEAD, manufactured by Dynal Corporation) were added to 1 × 10 ⁸ cells used for the first time and suspended in 0.9 ml of 1H-PBS. After mixing for 1 hour at room temperature, the dissociated cells are collected and washed with 1H-PBS. The washed cells were suspended in 5H-RPMI so as to be 2 x 10 ⁶ cells / ml, and the equivalent amount to the DC suspension prepared by the method described in (3) of Example 8 using the peptide of either CE-2 or CE-3. Mixed up. IL-7 is added to this suspension to a final concentration of 10 ng / ml, 0.5 ml is dispensed into each well of a 48 well culture plate and incubated in a 37 ° C CO ₂ incubator. The next day, 50 μl of 5H-RPMI containing 100 ng / ml IL-10 is added.

After one week, the culture supernatant of each well is removed and the cells are suspended in 0.5 ml of 5H-RPMI. This suspension is added to a plate containing any one of antigen presenting cells prepared by the method described in (2) of Example 8 using a peptide of either CE-2 or CE-3, and re-stimulated. The following day, IL-10 was added to a final concentration of 10 ng / ml, and after 2 days, half of the culture supernatant was removed and an equivalent amount of 5H-RPMI containing 20 IU / ml of rIL-2 was added in a 1 week CO ₂ incubator. Incubate. After the same restimulation was performed three more times, cytotoxic activity against TISI (+) and TISI (-) was measured for effector cells in each well. Effector cells exhibiting cytotoxic activity are individually propagated using the anti-CD 3 antibody or each peptide used for antigen stimulation. The proliferated cells are suspended in 5H-RPMI and cytotoxic activity is measured by the following method (3).

(2) Preparation of CTL Target Cells

As the target cells, two kinds of TISI (+) cultured in a medium to which TISI (-), CE-2 or CE-3 was added were prepared in the same manner as in Example (3). MKN-45 was also treated at 37 ° C. for 48 hours at 100 U / ml of IFN-γ. The four target cells are labeled with ⁵¹ Cr in the same manner as in Example 4 (3) on the day of cytotoxic activity measurement.

(3) Measurement of Cytotoxic Activity by CTL

Using the effector cells prepared in (1) and the labeled target cells prepared in (2), cytotoxic activity was measured in the same manner as described in (5) of Example 1. The results are shown in Table 7.

Peptide Name E / T TISI (-) TISI (+) MKN-45 CE-2CE-3 1010 0.91.9 89.192.0 25.867.9

In Table 7, E / T represents the ratio of effector cells to target cells.

As a result, it was found that the effector cells obtained by induction by the peptide of either CE-2 or CE-3 showed antigen-peptide specific cytotoxicity against TISI (+) and MKN-45, and were CEA-specific CTLs. .

Example 6 Preparation of HER2 / neu Antipeptide Specific CTL (1)

CTL in this embodiment is prepared by the CTL derivation method using SAC-I in the same manner as in the first embodiment.

(1) Selection and Synthesis of CTL Inducing Peptides

The amino acid sequence of the HER2 / neu protein consisting of 1255 amino acids is searched based on the sequence having the HLA-A24 binding motif structure, taking into consideration the kind of amino acids at other positions, and the peptides are selected by selecting five peptides shown in Table 8. Made using a synthesizer.

SEQ ID NO: Position in HER2 / neu Protein designation 620212223 8-16440-448780-788907-915951-959 HE-1HE-4HE-2HE-5HE-3

In addition, in Table 8, the number of the sequence number term represents the sequence number of the sequence table showing the amino acid sequence of each peptide. The number of the position term in the HER2 / neu protein also indicates the number of amino acids from the N terminus in the HER2 / neu protein. The symbol in the name section indicates the name of the peptide named by the inventors.

(2) Preparation of effector cells

In the following manner, five kinds of effector cells individually stimulated with a peptide of any of HE-1 to HE-5 are prepared.

For first time stimulation by SAC-I treatment prepared by the method described in (1) of Example 8 below using the peptide of any one of the storage PBMCs and HE-1 to HE-5 prepared in Example (2) Five kinds of plates containing any one of antigen presenting cells and antigen presenting cells prepared by the method described in (2) of Example 8 using peptides of any of HE-1 to HE-5 are used. 5 effector cells stimulated with any one of HE-1 to HE-5 peptides were prepared in the same manner as in Example 3 (3).

(3) Preparation of CTL Target Cells

Five kinds of TISI (+) and ovarian cancer cell lines cultured in a medium containing any one of HE-1 to HE-5 peptides in the same manner as in Example 4 (4) as target cells for measuring cytotoxic activity SKOV 3 (HLA-A3 and HER2 / neu positive) and its HLA-A24 transformant SKOV-A24 (HLA-A24 and HER2 / neu positive) are used. In addition, the cells are labeled with ⁵¹ Cr in the same manner as in Example 1 (4) on the day of cytotoxic activity measurement.

(4) Measurement of Cytotoxic Activity by CTL

Using the effector cells of (2) and the target cells of (3), specific cytotoxic activity (%) is calculated in the same manner as in Example (5). The results are shown in Table 9.

Peptide Name E / T TISI (-) TISI (+) SKOV3-A24 HE-1HE-2HE-3HE-4HE-5 1010101010 5.31.468.30.40.0 41.90.067.20.79.4 4.30.40.00.00.9

In Table 9, E / T represents the ratio of effector cells to target cells.

As a result, effector cells obtained by inducing HE-1 exhibit antigen-peptide specific cytotoxicity to TISI (+) cultured in the medium to which the HE-1 peptide was added.

Example 7 Preparation of HER2 / neu Specific CTLs (2)

CTL in this example is prepared by an induction method using antigen-presenting dendritic cells (DC).

With respect to the HE-1 peptide prepared in Example 6 (1), effector cells recognizing this peptide are induced from PBMCs of normal individuals, and cytotoxic activity is measured.

(1) Preparation of effector cells

Example 2 of Example 8 (2) using DC prepared in the method described in (3) of Example 8 below using PBMC and HE-1 peptide prepared in Example 1 (2), and (2) A plate containing antigen-presenting cells prepared by the method described in) is used to prepare effector cells stimulated by the HE-1 peptide in the same manner as in Example (1).

(2) Preparation of CTL Target Cells

As target cells, TISI (+) cultured in a medium to which TISI (-) and HE-1 were added in the same manner as in Example (3) was prepared. In addition, SKOV3 and SKOV3-A24 were treated at 37 ° C. for 48 hours at 100 U / ml of IFN-γ. The four target cells were labeled with ⁵¹ Cr on the day of cytotoxic activity measurement in the same manner as in Example (4).

(3) Measurement of Cytotoxic Activity by CTL

Using the effector cells prepared in (1) and the labeled target cells prepared in (2), cytotoxic activity was measured in the same manner as described in (5) of Example 1. The results are shown in Table 10.

E / T TISI (-) TISI (+) SKOV3-A24 SKOV3 45155 8.43.03.6 86.274.649.1 14.410.46.8 6.25.63.5

In Table 10, E / T represents the ratio of effector cells to target cells.

As a result, the effector cells obtained by induction with the HE-1 peptide showed antigen-peptide specific cytotoxicity against TISI (+) and SKOV3-A24, and proved to be HER2 / neu specific CTL.

Example 8 Preparation of Nonproliferative Antigen Presenting Cells

(1) Preparation of antigen presenting cells by SAC-I treatment

After melting the preserved PBMC prepared in Example (2), it was suspended in 5H-RPMI so that the cell concentration was 2 x 10 ⁶ cells / ml. In the cell suspension, the final concentration of SAC-I (Pansorbin cells, manufactured by Carbiochem Co., Ltd.) was 0.005%, Immunobeads (Rabbit anti-Human IgM, manufactured by Biorad, Inc.) was 20 µg / ml, and IL-4 (Genzyme, Inc.) was added. ) Is added to a final concentration of 20 ng / ml, 5 ml is dispensed into each well of a 6 well culture plate and incubated for 4 days in a 37 ° C. CO ₂ incubator. Cells cultured for 4 days were washed with physiological saline containing a final serum concentration of 1% bovine serum albumin (BSA), followed by 1% BSA and 3 μg / ml β so that the cell concentration was 1 × 10 ⁷ cells / ml. Suspension with citric acid buffer (pH 3.0) containing ₂ microglobulins and then treated for 2 minutes in ice. Subsequently, the cells were washed with a phosphate buffer (pH 7.5) containing 1% BSA, 3 µg / ml of β ₂ microglobulin and 10 µl / ml of peptide, 5 times the cell suspension, followed by 1% BSA and 3 µg. It is suspended in phosphate buffer containing / ml of β ₂ microglobulin and 50 μg / ml of peptide and reacted at 20 ° C. for 4 hours. After the reaction, X-ray irradiation (5500 Rad) is carried out and suspended in 5H-RPMI so as to have a cell concentration of 1 × 10 ⁶ cells / ml to prepare antigen-presenting cells for first stimulation.

(2) Preparation of antigen presenting cell from adherent cell

After melting the PBMC prepared in Example (2), X-ray irradiation (5500 Rad) is performed. Subsequently, 1 ml / well of the cell suspension suspended in 5H-RPMI so as to have a cell concentration of 4 × 10 ⁶ cells / ml is dispensed in a 24 well culture plate and incubated for 1.5 hours in a CO ₂ incubator. Thereafter, the non-adhesive cells are aspirated off, and each well is washed with RPMI 1640 to remove the non-adhesive cells. Subsequently, 5 H-RPMI containing 3 μg / ml of β ₂ microglobulin and 20 μg / ml of peptide are individually dispensed into each well of 0.5 ml and incubated in a CO ₂ incubator. After 2 hours, the supernatant was aspirated and washed once with 5H-RPMI, and the cells remaining in each well of the plate were used as antigen-presenting cells for restimulation.

(3) Preparation of DC-rich antigen-presenting cells

After melting the preserved PBMC prepared in Example 1 (1), the suspension was suspended in 5H-RPMI so that the cell concentration was 2 x 10 ⁶ cells / ml, and 10 ml of the cell suspension was T-25 flask (manufactured by Genk). Put it in. After standing at 37 DEG C for 1.5 hours, the non-adhesive cells were removed, and the remaining adherent cells contained 5 U-mL containing 1000 U / mL of GM-CSF (manufactured by Genzyme) and 2000 U / mL of IL-4 (manufactured by Genzyme). It was added to RPMI 10 ㎖ by culturing in a CO ₂ incubator for 37 ℃ to induce DC cells. After 7 days, the floating cells are collected. Adhesion cells are collected in a cell dissociation buffer (produced by Gibco BRL), washed with 5H-PRMI, and combined with floating cells. It was suspended such that a number of cell peptide 40 ㎍ / ㎖, β ₂ microglobulin 3 ㎍ / ㎖ and 1% 3 × 10 ⁶ gae / ㎖ in physiological saline was added to human serum albumin, then reacted for 4 hours in a constant temperature bath at 20 ℃ . After the reaction, X-ray irradiation (5500 Rad) is carried out, and then diluted with physiological saline added with 1% human serum albumin to 1 x 10 ⁶ / ml to be used as antigen presenting cells.

(4) Preparation of antigen presenting cells from all PBMCs

PBMCs isolated in the same manner as in Example (1) were treated with mitomycin to prepare 200 mg / mL of mitomycin C (manufactured by ICN Biomedical Co., Ltd.) in a suspension of PBMC 1 × 10 ⁷ / ml. Added, left for 30 minutes], washed three times in RPMI 1640 medium, and then mixed in an equal amount of 5H-RPMI added to 20 μg / ml of peptide, and then aliquoted in 1 ml / well in a 24-well culture plate Used as a presentation cell.

Example 9 Preparation of CEA Antigenpeptide Functional Derivatives

(1) Synthesis of CE-2 and CE-3 Modifiers

Based on CE-2 synthesized in (1) of Example 5, 12 modified variants shown in Table 11 were designed.

SEQ ID NO: designation 242539404142434445464748 CE-201CE-202CE-203CE-204CE-205CE-206CE-207CE-208CE-209CE-210CE-211CE-212

Similarly, 17 modified variants shown in Table 12 are designed based on CE-3.

SEQ ID NO: designation 26272829303132495051525354555633 CE-301CE-302CE-303CE-304CE-305CE-306CE-307CE-308CE-309CE-310CE-311CE-312CE-313CE-314CE-315CE-316CE-317

27 peptides of CE-201 to CE-212 and CE-301 to CE-315 shown in Tables 11 and 12 were prepared using a peptide synthesizer.

And CE-316 in Table 12 added 100 microliters of aqueous solutions of DHCP (made by Takarashu Irradiation) (10 mg / mL) to CE-3 (2.85 micromol) produced by (1) of Example 5, and 37 It reacts by 19 degreeC and is produced by refine | purifying by HPLC.

In the amino acid sequence of CE-3 represented by SEQ ID NO: 5, CE-317 is two of CE-3 to which two molecules of CE-3 are bound by disulfide bond via a thiol residue of the fourth cysteine at the N-terminus. It is a monomer. This peptide is prepared by reacting with CE-3 in 0.02 M NH ₄ HCO ₃ aqueous solution dissolved at 0.3 mM for 24 hours at 37 ° C. and purifying by HPLC.

(2) Identification of functional derivatives of CE-2 or CE-3

Confirm that the CTL obtained in Example 4, which recognizes a complex of CE-2 or CE-3 and HLA-A24 molecules, recognizes a complex of HLA-A24 molecules with peptides or peptide analogs shown in Tables 11 and 12.

First, the CTL that recognizes CE-2 prepared in the same manner as in Example (2) was prepared by using one of ⁵¹ Cr-labeled TISI (-) cells and CE-201 to CE-212 peptides. 12 kinds of ⁵¹ Cr-labeled TISI (+) cells prepared in the same manner as in 3) were checked for injury. As a result, CTLs that recognize CE-2 were found in medium containing CE-201, CE-203, CE-204, CE-206, CE-207, CE-208, CE-209, CE-210, and CE-212. Clear cytotoxicity was observed against ⁵¹ Cr labeled TISI (+) obtained by culturing. In addition, this CTL showed no cytotoxicity against ⁵¹ Cr labeled TISI (-) cells, and therefore, CE-201, CE-203, CE-204, CE-206, CE-207, CE-208, CE-209, CE -210 and CE-212 were found to be functional derivatives of CE-2.

Four kinds of CTLs having different starting materials for recognizing CE-3 obtained by preparing in the same manner as in Example 4 (2), using four normal PBMCs as starting materials, ⁵¹ Cr-labeled TISI (-) cells and Table 12 Using any one of the 17 peptides shown below, it was confirmed whether or not the 17 Cr ⁵¹ Cr-labeled TISI (+) cells prepared in the same manner as in Example 4 (3) showed injury. As a result, CTLs that recognize CE-3 are among CE-301, CE-302, CE-305, CE-307, CE-310, CE-311, CE-312, CE-314, CE-316, and CE-317. Clear cytotoxicity is shown against 10 ⁵¹ Cr labeled TISI (+) obtained by culturing in a medium to which any one peptide is added individually. In addition, this CTL showed no cytotoxicity against ⁵¹ Cr labeled TISI (-) cells, and thus, CE-301, CE-302, CE-305, CE-307, CE-310, CE-311, CE-312, CE -314, CE-316, CE-317 were found to be functional derivatives of CE-3.

Example 10 Detection of HLA-A24 Molecules Using CTL

The human-derived PBMC is examined as a test cell whether HLA-A24 molecule can be detected by CTL of the present invention.

PBMCs were prepared in the same manner as in Example 1 (2) from eight persons whose HLA types expressed using conventional anti-HLA antibodies were found to be PBMC 8 samples for test cells. Each sample was divided into two and suspended in 5H-RPMI in which CE-3 was dissolved so that one side was 10 µg / ml. The other is suspended in 5H-RPMI, respectively, to form a CE-3 free sample. After leaving each group at 37 ° C. for 2 hours, the cells are collected by centrifugation and the supernatant is removed. By this operation, excess CE-3 is removed from the CE-3 addition group. Subsequently, the 16 cells were suspended in 5H-RPMI, and then 1 × 10 ⁴ cells were dispensed into each well of a 96 well microplate. Subsequently, 3 x 10 ⁴ CTLs obtained by using CE-3 in Example 4 were added to each well and allowed to stand in a CO ₂ incubator at 37 ° C for 22 hours, and then the amount of IFN-γ in the supernatant was added to each well. It is measured by Duo set (manufactured by Genzyme). As a result, the presence of IFN-γ was confirmed only in wells dispensed with PBMC 5 samples prepared from humans expressing HLA-A24 molecules in the CE-3 addition group. The amount of IFN-γ in any well of the CE-3 addition group used for the control was below the detection limit. Thus, it has been found that the HLA-A24 molecule can be detected by using the CTL of the present invention.

On the other hand, among the PBMC samples in which the presence of IFN-γ was recognized in the CE-3 addition group, the amount of IFN-γ in the wells in which the PBMC samples prepared from HLA-A24 homo were dispensed from the human HLA-A24 hetero There is about twice the amount of IFN-γ in the wells dispensed with PBMC samples. Therefore, it has been found that the amount of HLA-A24 expression can be estimated by using the CTL of the present invention.

Example 11 Detection of HLA-A2 Molecules Using CTL and Identification of HLA-A2 Subtypes

From PBMCs derived from normal individuals having HLA-A2 (A ^* 0201) in the same manner as in Example 1 (3) using FLWGPRALV, a HLA-A2 binding peptide derived from MAGE 3, represented by the amino acid sequence set forth in SEQ ID NO: 37 Prepare effector cells. Medium containing .221 (A2.1) cells, which are HLA-A2 cells as target cells, (.221 (A2.1) (+)) or medium containing 10 μg / ml of the peptide on the day before cytotoxicity measurement ( Incubate in .221 (A2.1) (-)). On the day of measurement, ⁵¹ Cr-labeled .221 (A2.1) (+) or .221 (A2.1) (-) cells and effector cells are mixed, and after 5 hours, the amount of ⁵¹ Cr released in the medium is measured. . As a result, the prepared effector cells were found to be CTL showing peptide specific injury. In addition, IFN-γ in supernatant after 1 day after mixing CTL with .221 (A2.1) (+) or .221 (A2.1) (-) cells was measured. It has been found to favor IFN-γ. After increasing this CTL, the presence or absence of peptide specific IFN-γ free action against various HLA-A2 cells is examined.

1) Reactivity against chemotactic cells.221 (A2.1) (HLA-A ^* 0201), CLA (HLA-A ^* 0206), FUN (HLA-A ^* 0203), p815 (HLA-A ^* 0202)

The peptide FLWGPRALV is added to each said cell so that it may become 10 micrograms / mL, and it incubates at 37 degreeC for 4 hours. Excess peptide was washed off, and then 1 × 10 ⁴ cells / 0.1 mL / well was put into a 96 well microplate, 1 × 10 ⁵ cells / 0.1 mL / well was added thereto, and reacted for 20 hours. Peptide-free cells are prepared and reacted with CTL cells identically for control. The supernatant is taken from the reaction solution, diluted appropriately, and the amount of IFN-γ is quantified. As a result, this CTL caused the release of peptide specific IFN-γ only for 221 (A2.1) (HLA-A ^* 0201).

2) Reactivity to Human PBMCs

To three PBMCs of normal volunteers (A: HLA-A ^* 0207 / A11, B: A ^* 0206 / A24, C: A ^* 0201 / A ^* 0206), the peptide FLWGPRALV is added to 10 占 // ml, Incubate at 37 ° C. for 2 hours. After removal of excess clean the peptide, 1 × 10 ⁴ /0.1 ㎖ gae added / well into, 1 × 10 ⁴ /0.1 ㎖ gae / well of CTL cells in this to a 96-well microplate, and reacted for 20 hours. Peptide-free cells are prepared and reacted with CTL cells identically for control. The supernatant is taken from the reaction solution, diluted appropriately, and the amount of IFN-γ is quantified. As a result, this CTL caused the release of peptide specific IFN-γ only to CB's PBMC having HLA-A ^* 0201.

According to the results of 1) and 2) above, the HLA-A2 binding peptide and the CTL derived from the PBMC of HLA-A ^* 0201 by the peptide were used to subclass HLA-A2 without performing DNA typing on the test cells. It has been found that it can be identified by type.

Sequence Table Free Text

The fourth amino acid of the amino acid sequence of SEQ ID 33 is 2-hydroxy-4 (R, S) -L-cysteine-S-yl-2-cyclopenten-l-one.

CTLs of the present invention selectively injure tumor cells positive for both HLA-A24 and MAGE-3, positive for both HLA-A24 and MAGE-1, positive for both HLA-A24 and CEA, or positive for both HLA-A24 and HER2 / neu As a cell medicament for cancer treatment, both HLA-A24 and MAGE-3 are positive tumor cells, HLA-A24 and MAGE-1 are positive, HLA-A24 and CEA are both positive, Alternatively, HLA-A24 and HER2 / neu are both useful for detecting the presence or absence of tumor cells that are positive, and for use in combination with antigen peptides for HLA typing of cells in in vitro samples. The antigen presenting cells of the present invention are also useful for preparing this CTL or as cancer inhibitors. In addition, the pharmaceutical composition comprising the antigen peptide of the present invention as an active ingredient is useful as an inducer of CTL and also a cancer inhibitor.

<110> Takara Shuzo Co., Ltd. <120> Cytotoxic T Lymphocytes <150> JP 97-203900 <151> 1997-07-15 <150> JP 97-203917 <151> 1997-07-15 <150> JP 98-14736 <151> 1998-01-12 <160> 56 <170> KOPATIN 1.5 <210> 1 <211> 9 <212> PRT <213> Homo sapiens <400> 1 Ile Met Pro Lys Ala Gly Leu Leu Ile 1 5 <210> 2 <211> 9 <212> PRT <213> Homo sapiens <400> 2 Val Ala Glu Leu Val His Phe Leu Leu 1 5 <210> 3 <211> 9 <212> PRT <213> Homo sapiens <400> 3 Asn Tyr Lys His Cys Phe Pro Glu Ile 1 5 <210> 4 <211> 10 <212> PRT <213> Homo sapiens <400> 4 Gln Tyr Ser Trp Phe Val Asn Gly Thr Phe 1 5 10 <210> 5 <211> 9 <212> PRT <213> Homo sapiens <400> 5 Thr Tyr Ala Cys Phe Val Ser Asn Leu 1 5 <210> 6 <211> 9 <212> PRT <213> Homo sapiens <400> 6 Arg Trp Gly Leu Leu Leu Ala Leu Leu 1 5 <210> 7 <211> 9 <212> PRT <213> Homo sapiens <400> 7 Glu Ala Asp Pro Thr Gly His Ser Tyr 1 5 <210> 8 <211> 9 <212> PRT <213> Homo sapiens <400> 8 Asn Tyr Pro Leu Trp Ser Gln Ser Tyr 1 5 <210> 9 <211> 9 <212> PRT <213> Homo sapiens <400> 9 Asn Trp Gln Tyr Phe Phe Pro Val Ile 1 5 <210> 10 <211> 10 <212> PRT <213> Homo sapiens <400> 10 Asn Trp Gln Tyr Phe Phe Pro Val Ile Phe 1 5 10 <210> 11 <211> 9 <212> PRT <213> Homo sapiens <400> 11 Ile Phe Ser Lys Ala Ser Ser Ser Leu 1 5 <210> 12 <211> 10 <212> PRT <213> Homo sapiens <400> 12 Ile Met Pro Lys Ala Gly Leu Leu Ile Ile 1 5 10 <210> 13 <211> 9 <212> PRT <213> Homo sapiens <400> 13 Ile Met Pro Lys Thr Gly Phe Leu Ile 1 5 <210> 14 <211> 10 <212> PRT <213> Homo sapiens <400> 14 Asn Tyr Lys His Cys Phe Pro Glu Ile Phe 1 5 10 <210> 15 <211> 10 <212> PRT <213> Homo sapiens <400> 15 Ile Met Pro Lys Thr Gly Phe Leu Ile Ile 1 5 10 <210> 16 <211> 10 <212> PRT <213> Homo sapiens <400> 16 Ser Tyr Val Lys Val Leu Glu Tyr Val Ile 1 5 10 <210> 17 <211> 9 <212> PRT <213> Homo sapiens <400> 17 Ile Tyr Pro Asn Ala Ser Leu Leu Ile 1 5 <210> 18 <211> 9 <212> PRT <213> Homo sapiens <400> 18 Leu Tyr Gly Pro Asp Ala Pro Thr Ile 1 5 <210> 19 <211> 9 <212> PRT <213> Homo sapiens <400> 19 Val Tyr Ala Glu Pro Pro Lys Pro Phe 1 5 <210> 20 <211> 9 <212> PRT <213> Homo sapiens <400> 20 Pro Tyr Val Ser Arg Leu Leu Gly Ile 1 5 <210> 21 <211> 9 <212> PRT <213> Homo sapiens <400> 21 Val Tyr Met Ile Met Val Lys Cys Trp 1 5 <210> 22 <211> 9 <212> PRT <213> Homo sapiens <400> 22 Ala Tyr Ser Leu Thr Leu Gln Gly Leu 1 5 <210> 23 <211> 9 <212> PRT <213> Homo sapiens <400> 23 Ser Tyr Gly Val Thr Val Trp Glu Leu 1 5 <210> 24 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 24 Gln Tyr Ser Trp Phe Ile Asn Gly Thr Phe 1 5 10 <210> 25 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 25 Gln Tyr Ser Trp Leu Ile Asn Gly Thr Phe 1 5 10 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 26 Ala Tyr Ala Cys Phe Val Ser Asn Leu 1 5 <210> 27 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 27 Thr Tyr Ala Ala Phe Val Ser Asn Leu 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 28 Thr Tyr Ala Cys Ala Val Ser Asn Leu 1 5 <210> 29 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 29 Thr Tyr Ala Cys Phe Ala Ser Asn Leu 1 5 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 30 Thr Tyr Ala Cys Phe Val Ala Asn Leu 1 5 <210> 31 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 31 Thr Tyr Ala Cys Phe Val Ser Ala Leu 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 32 Thr Tyr Ala Cys Phe Val Ser Asn Ala 1 5 <210> 33 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> MOD-RES <220> <221> MOD_RES (222) (4) .. (4) Xaa is 2-hydroxy-4 (R, S) -L-cystein-S-yl-2-cyclopenten-1-one. <400> 33 Thr Tyr Ala Xaa Phe Val Ser Asn Leu 1 5 <210> 34 <211> 9 <212> PRT <213> Homo sapiens <400> 34 Tyr Leu Ser Gly Ala Asn Leu Asn Leu 1 5 <210> 35 <211> 9 <212> PRT <213> Homo sapiens <400> 35 Lys Ile Phe Gly Ser Leu Ala Phe Leu 1 5 <210> 36 <211> 9 <212> PRT <213> Homo sapiens <400> 36 His Leu Tyr Gln Gly Cys Gln Val Val 1 5 <210> 37 <211> 9 <212> PRT <213> Homo sapiens <400> 37 Phe Leu Trp Gly Pro Arg Ala Leu Val 1 5 <210> 38 <211> 9 <212> PRT <213> Influenza virus <400> 38 Gly Ile Leu Gly Phe Val Phe Thr Leu 1 5 <210> 39 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 39 Ala Tyr Ser Trp Phe Val Asn Gly Thr Phe 1 5 10 <210> 40 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 40 Gln Ala Ser Trp Phe Val Asn Gly Thr Phe 1 5 10 <210> 41 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 41 Gln Tyr Ala Trp Phe Val Asn Gly Thr Phe 1 5 10 <210> 42 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 42 Gln Tyr Ser Ala Phe Val Asn Gly Thr Phe 1 5 10 <210> 43 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 43 Gln Tyr Ser Trp Ala Val Asn Gly Thr Phe 1 5 10 <210> 44 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 44 Gln Tyr Ser Trp Phe Ala Asn Gly Thr Phe 1 5 10 <210> 45 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 45 Gln Tyr Ser Trp Phe Val Ala Gly Thr Phe 1 5 10 <210> 46 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 46 Gln Tyr Ser Trp Phe Val Asn Ala Thr Phe 1 5 10 <210> 47 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 47 Gln Tyr Ser Trp Phe Val Asn Gly Ala Phe 1 5 10 <210> 48 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 48 Gln Tyr Ser Trp Phe Val Asn Gly Thr Ala 1 5 10 <210> 49 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 49 Thr Tyr Ala Asp Phe Val Ser Asn Leu 1 5 <210> 50 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 50 Thr Tyr Ala Pro Phe Val Ser Asn Leu 1 5 <210> 51 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 51 Thr Tyr Ala Ser Phe Val Ser Asn Leu 1 5 <210> 52 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 52 Thr Tyr Ala Gly Phe Val Ser Asn Leu 1 5 <210> 53 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 53 Thr Tyr Ala Cys Phe Ile Ser Asn Leu 1 5 <210> 54 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 54 Thr Tyr Ala Cys Phe Asp Ser Asn Leu 1 5 <210> 55 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 55 Thr Tyr Ala Cys Phe Met Ser Asn Leu 1 5 <210> 56 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VARIANT <400> 56 Thr Tyr Ala Cys Phe Lys Ser Asn Leu 1 5

Claims (11)

(1) and (2) below: (1) a human major histocompatibility antigen (HLA) -A24 binding antigen peptide represented by the amino acid sequence of any one of SEQ ID NOs: 1, 2, 4 to 6 , and (2) the functional derivative of (1) above selected from the group consisting of SEQ ID NOs: 24, 26, 27, 30, 32, 33, 42 to 46, 48, 51 to 53 and 55 Cytotoxic T lymphocytes that recognize cells presenting a complex of one or more antigenic peptides selected from HLA-A24 molecules on the cell surface. Cancer inhibitor containing the cytotoxic T lymphocytes of Claim 1 as an active ingredient. (1) and (2) below: (1) a human major histocompatibility antigen (HLA) -A24 binding antigen peptide represented by the amino acid sequence of any one of SEQ ID NOs: 1, 2, 4 to 6 , and (2) the functional derivative of (1) above selected from the group consisting of SEQ ID NOs: 24, 26, 27, 30, 32, 33, 42 to 46, 48, 51 to 53 and 55 A method for inducing cytotoxic T lymphocytes according to claim 1, wherein at least one antigen peptide selected from is used. (1) and (2) below: (1) a human major histocompatibility antigen (HLA) -A24 binding antigen peptide represented by the amino acid sequence of any one of SEQ ID NOs: 1, 2, 4 to 6 , and (2) the functional derivative of (1) above selected from the group consisting of SEQ ID NOs: 24, 26, 27, 30, 32, 33, 42 to 46, 48, 51 to 53 and 55 An agent for inducing cytotoxic T lymphocytes according to claim 1, comprising at least one antigenic peptide selected from the above as an active ingredient. (1) and (2) below: (1) a human major histocompatibility antigen (HLA) -A24 binding antigen peptide represented by the amino acid sequence of any one of SEQ ID NOs: 1, 2, 4 to 6 , and (2) the functional derivative of (1) above selected from the group consisting of SEQ ID NOs: 24, 26, 27, 30, 32, 33, 42 to 46, 48, 51 to 53 and 55 A cancer inhibitor comprising one or more antigenic peptides selected from the above as an active ingredient. (1) and (2) below: (1) a human major histocompatibility antigen (HLA) -A24 binding antigen peptide represented by the amino acid sequence of any one of SEQ ID NOs: 1, 2, 4 to 6 , and (2) the functional derivative of (1) above selected from the group consisting of SEQ ID NOs: 24, 26, 27, 30, 32, 33, 42 to 46, 48, 51 to 53 and 55 An antigen presenting cell comprising a complex of one or more antigenic peptides selected from HLA-A24 molecules on a cell surface. An inducer of cytotoxic T lymphocytes comprising the antigen-presenting cell of claim 6 as an active ingredient. A cancer inhibitor comprising the antigen-presenting cell of claim 6 as an active ingredient. A method for detecting cells susceptible to cytotoxic T lymphocytes according to claim 1, wherein the change occurs when the cytotoxic T lymphocytes according to claim 1 are contacted with a test cell. A detection agent for cells susceptible to cytotoxic T lymphocytes according to claim 1, comprising the cytotoxic T lymphocytes according to claim 1 as an active ingredient. (1) and (2) below: (1) a human major histocompatibility antigen (HLA) -A24 binding antigen peptide represented by the amino acid sequence of any one of SEQ ID NOs: 1, 2, 4 to 6 , and (2) the functional derivative of (1) above selected from the group consisting of SEQ ID NOs: 24, 26, 27, 30, 32, 33, 42 to 46, 48, 51 to 53 and 55 Determination of a subtype of human major histocompatibility antigen (HLA) characterized by contacting a test cell with cytotoxic T lymphocytes that recognize a complex of the antigenic peptide and HLA molecule in the presence of at least one antigenic peptide selected from Way.