JP2009035534A - Hla-a24-binding antigen peptide and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a HLA-A24-binding antigen peptide. <P>SOLUTION: The HLA-A24-binding antigen peptide is a CML66-derived peptide and comprises a WYQDSVYYI or YYIDTLGRI amino acid sequence. Since a CML66-specific CD8+T cell to be activated by the antigen peptide recognizes the antigen peptide associated with a HLA-A24 molecule on a cell and exhibits cytotoxicity, the cell attacks a malignant tumor cell expressing CML66. The peptide is useful in a medical field of a malignant tumor by cellular immunotherapy, vaccinotherapy etc. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an HLA-A24 restricted antigen peptide and use thereof.

  Cytotoxic T lymphocyte (CTL) having a function of eliminating cancer cells recognizes a complex of an HLA class I molecule expressed on the surface of a cancer cell and a peptide derived from a cancer-specific protein, Eliminate cancer cells. In recent years, a method has been studied in which a target peptide recognized by cytotoxic T cells is identified using this mechanism and artificially given to a host as an antigen peptide to induce specific antitumor immunity. Yes. And because of the mechanism, the antigenic peptide is also called a cancer vaccine. Until now, identification of antigen peptides specific to some cancer cells has been performed and application of cancer vaccines has been attempted, but since the effect is not sufficient, identification of new antigen peptides is required. .

CML66 is a protein having a molecular weight of 66 kD composed of 583 amino acids, and has been reported to be a leukemia-related antigen associated with graft-versus-leukemia effect (GVL). Further analysis revealed that (1) the coding gene is located in the long arm of chromosome 8 (8q23), (2) the function of the protein is still unknown, (3) chronic myeloid leukemia ( (CML) Overexpression of CML66 protein and CML66 mRNA is observed in cells derived from patients and cells derived from prostate cancer patients, and in cell lines derived from patients such as human leukemia, melanoma, and lung cancer. Except that it is produced in testis cells that do not express HLA molecules, expression is confirmed only slightly in the heart, (4) two splicing isoforms (CML66-L, CML66-S) are seen even in normal condition, CML66-S, which is a truncated isoform consisting of 554 amino acids, is mainly expressed in testis cells and is further expressed in the serum of cancer-bearing patients. 66 specific antibodies, etc. It is mostly directed against CML66-L are found (Non-Patent Documents 1 to 3).
Yang XF et al. Proc. Natl. Acad. Sci. USA 98: 7492-7497, 2001. Wu CJ et al. Clin. Cancer Res. 11: 4504-4511, 2005. Yan Y. et al. J. Immunol. 172: 651-660, 2004.

  Although it is unclear whether or not CML66 is involved in the characteristics of tumor cells, the above-mentioned information shows immunotherapy for various cancers as so-called cancer-related antigens (cancer cell-specific antigens) as differentiation antigens. It is considered that it can be a target antigen.

  Therefore, an object of the present invention is to provide a new antigen peptide that is a peptide derived from CML66 and can be used as a cancer vaccine.

In order to achieve the object, the peptide of the present invention is the peptide according to any one of (1) to (4) below.
(1) Peptide consisting of the amino acid sequence of SEQ ID NO: 1 WYQDSVYYI (SEQ ID NO: 1)
(2) Peptide consisting of the amino acid sequence of SEQ ID NO: 2 YYIDTLLGRI (SEQ ID NO: 2)
(3) HLA that has a homology of 80% or more with the peptide of (1) or (2) above and can be specifically recognized by CD8 + T cells by binding to HLA-A24 molecule -A24-restricted antigen peptide (4) In the amino acid sequence of the peptide of (1) or (2) above, a peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, An HLA-A24-restricted antigen peptide that can be specifically recognized by CD8 + T cells by binding to an HLA-A24 molecule.

  The activation inducer of the present invention is an activation inducer that induces cytotoxic activation of CD8 + T cells, and includes the following first and second activation inducers. The first activation inducer is an activation inducer containing the peptide of the present invention or a derivative thereof, and the second activation inducer contains a polynucleotide encoding the peptide of the present invention or a derivative thereof. Activation inducer.

The method for producing CD8 + cytotoxic T cells of the present invention comprises the following steps (A) and (B).
(A) Preparing patient lymphocytes (B) Incubating the lymphocytes with the peptide of the present invention or a derivative thereof

The method for producing an antigen-presenting cell of the present invention includes the following steps (a) and (b).
(A) Step of preparing a patient's dendritic cell (b) Step of incubating the dendritic cell and the peptide of the present invention or a derivative thereof

  The pharmaceutical composition of the present invention is a pharmaceutical composition used for cancer immunotherapy. The 1st pharmaceutical composition of this invention contains the peptide of this invention, or its derivative (s) as an antigen peptide. The second pharmaceutical composition of the present invention comprises a polynucleotide encoding the peptide of the present invention or a derivative thereof. The third pharmaceutical composition of the present invention comprises CD8 + cytotoxic T cells produced by the method for producing cytotoxic T cells of the present invention. Moreover, the 4th pharmaceutical composition of this invention contains the antigen presentation cell manufactured by the manufacturing method of the antigen presentation cell of this invention.

  In general, when an antigenic peptide is recognized by the T cell receptor (TCR), it is first recognized by the TCR by binding to a specific HLA class I molecule (human histocompatibility antigen; human leukocyte antigen). . For this reason, it is thought that the versatility can be secured if it is a peptide that can bind to a high-frequency HLA class I molecule such as HLA-A24 molecule, which is occupied by about 60% of Japanese. As a result of intensive studies, the present inventors have identified a peptide derived from CML66 that binds to HLA-A24 and arrived at the present invention. The peptides of the present invention are CML66-specific HLA-A24 restricted antigen peptides that can be recognized by CD8 + T cells.

  According to the peptide of the present invention, activation / proliferation of CML66-specific CD8 + T cells can be promoted. As a result of this activation, CML66-specific CD8 + T cells become cytotoxic T cells having cytotoxic activity, so that effective treatment by destruction of malignant tumors becomes possible. For this reason, the peptide of this invention can be used as a cancer vaccine (malignant tumor vaccine) etc., for example, and is useful for the vaccine therapy with respect to the malignant tumor containing leukemia.

  Also, CML66-specific CD8 + cytotoxic T cells can be produced by co-culturing the peptides of the present invention with lymphocytes. By returning the cytotoxic T cells thus produced, for example, into the patient's body, for example, cellular immunotherapy against malignant tumors including leukemia becomes possible.

  In addition, antigen-presenting cells that present the peptide of the present invention as an antigen can be produced by co-culturing the peptide of the present invention with dendritic cells. Dendritic cells are extremely powerful antigen-presenting cells that can co-express major histocompatibility antigens (MHC) and costimulatory molecules and can also stimulate naive T cells that have not been antigen-sensitized. Therefore, according to the antigen-presenting cell of the present invention, CML66-specific CD8 + T cells can be effectively activated to induce cytotoxic T cells. Therefore, by returning such antigen-presenting cells to the body of a patient, for example, more effective cellular immunotherapy for malignant tumors such as leukemia becomes possible.

Peptide and its use (1) Peptide The peptide of the present invention is an antigen peptide of CML66 that binds on the HLA-A24 molecule and can be specifically recognized by CD8 + T cells. The HLA genotype of the HLA-A24 molecule is preferably A ^* 2402.

  In the present invention, “antigenic peptide” refers to a peptide constituting an antigenic determinant (epitope). The antigen peptide does not need to be the minimum unit of an antigenic determinant, and examples thereof include peptides composed of amino acids in the range of 8 to 26, 9 to 24, 10 to 22, or 11 to 20. .

  In the present invention, “binding to HLA-A24 molecule and being specifically recognized by CD8 + T cells” means that, for example, a certain peptide is presented on HLA-A24 molecule on the surface of antigen-presenting cells such as dendritic cells. If so, it means that CD8 + T cells can be activated in a peptide antigen-specific manner together with an appropriate costimulatory factor (costimulatory factor).

  Binding of the peptide of the present invention to the HLA-A24 molecule is possible even when, for example, a peptide expressed inside the cell or a peptide taken into the cell by phagocytosis binds to the HLA-A24 molecule in the cell. Good. Moreover, the case where the peptide which exists outside a cell couple | bonds directly with the HLA-A24 molecule which already exists on the cell surface may be sufficient.

In the present invention, the HLA class I molecule is a molecule corresponding to a human MHC class I molecule, and the HLA-A24 type means that the alloantigen type of the HLA class I molecule is A24 type, that is, HLA- The gene product of the A24 gene means that the HLA-A ^* 2402 type means that the genotype of the allele of the HLA-A24 gene is A ^* 2402.

The peptide of the present invention includes a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In CML66 protein (for example, NCBI accession number AAK73017), the amino acid sequence of SEQ ID NO: 1 (WYQDSVYYI) corresponds to the 70th to 78th amino acid sequence, and the amino acid sequence of SEQ ID NO: 2 (YYIDTLLGRI) is from 76th to It corresponds to the 84th amino acid sequence. Hereinafter, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 is also referred to as “CML66 _70-78 ”, and the peptide consisting of the amino acid sequence of SEQ ID NO: 2 is also referred to as “CML66 _76-84 ”.

  Moreover, the peptide of this invention contains a peptide homologous to the peptide which consists of an amino acid sequence of sequence number 1 or sequence number 2. Here, that the amino acid sequences are “homologous” means that they are sufficiently similar to maintain their functions. In general, when two or more amino acid sequences show, for example, 80% or more, 84% or more, 88% or more, 92% or more, 96% or more, they are homologous. It can be said. The identity and similarity refer to a relationship determined by comparing these sequences between two or more base sequences or amino acid sequences. While identity indicates the percentage of identical residues when aligned, similarity includes residues that are conservatively substituted in addition to identical residues. Identity and similarity can be easily determined by various computer programs known in the art. In the peptide of the present invention, “function” means that it can be recognized specifically by a peptide antigen by CD8 + T cells by binding to an HLA-A24 molecule.

  The peptide of the present invention is a peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, Including those having The number of amino acid residues that differ is, for example, 1-6, 1-5, 1-4, 1-3, 1-2, or 1.

  The peptide of the present invention may be a natural product such as a natural CML66-derived peptide, a chemically synthesized peptide, or a peptide produced by genetic engineering. The method of production by chemical synthesis or genetic engineering is not particularly limited, and conventionally known methods can be employed. When genetic engineering is used, for example, a polynucleotide encoding the peptide of the present invention or an expression vector into which the polynucleotide is inserted is introduced into a suitable host such as E. coli, yeast, insect cell, etc. May be manufactured.

  The peptide of the present invention can be used as a cancer vaccine as described above. For example, after adopting CML66-specific cytotoxic T-cells outside a patient's body, they can be transferred into the body (for example, infusion). It can be used for immunotherapy (passive immunotherapy) and the like. In addition, for example, the present invention can be applied to a peptide / HLA tetramer diagnostic method capable of detecting CML66-specific cytotoxic T cells existing in the body with high sensitivity and speed.

(2) First activation inducer As described above, the first activation inducer of the present invention is an activation inducer that induces cytotoxic activation of CD8 + T cells, It includes at least one of a peptide and a derivative thereof. For example, the activation / proliferation of CML66-specific CD8 + T cells can be promoted by administering the first activation inducer of the present invention to, for example, a target cell or tissue and the vicinity thereof. This activation enables CML66-specific CD8 + T cells to be cytotoxic T cells having cytotoxic activity.

  The first activation inducer of the present invention may include only one or both of the peptide and derivative of the present invention, for example. Moreover, the peptide and derivative of this invention may be one type, and may contain two or more types. The first activation inducer of the present invention may be, for example, only the peptide or derivative of the present invention, or may further contain various additives. In addition, about a concrete usage method etc., it is the same as that of the 1st pharmaceutical composition mentioned later, for example.

(3) 1st pharmaceutical composition As mentioned above, the 1st pharmaceutical composition of this invention is a pharmaceutical composition used for cancer immunotherapy, Comprising: At least one of the peptide of this invention and its derivative (s) is included. It is characterized by. The first pharmaceutical composition of the present invention may be, for example, only the peptide or derivative of the present invention, as in the case of the first activation inducer described above. In addition, additives such as carriers or excipients may be included. The derivative of the peptide of the present invention is not particularly limited, and includes, for example, those modified by a conventionally known modification method, for example, salts, sugar chain-modified, lipid-modified, and the like. Hereinafter, the “peptide of the present invention” includes a derivative of the peptide of the present invention.

  The first pharmaceutical composition of the present invention can be used for vaccine therapy of cancer (for example, malignant tumor), for example, by administering an effective amount to a patient as a vaccine. When the vaccine is administered, for example, in the patient's body, the peptide of the present invention is taken up by antigen-presenting cells such as dendritic cells and bound to HLA class I molecules and presented on the antigen-presenting cells. . Then, CML66-specific CD8 + T cells are stimulated and induced (activated) together with the expressed costimulatory molecules. CML66-specific CD8 + cytotoxic T cells that have cytotoxic activity upon activation destroy malignant tumor cells that express CML66 in the patient's body. This makes it possible to treat malignant tumors. In addition, it is said that the HLA gene frequency of HLA-A24 accounts for about 60% in Japanese. Therefore, it can be said that the pharmaceutical composition containing the peptide of the present invention has extremely high clinical usefulness from the viewpoint of being able to bind to HLA-A24 with such a high frequency.

  As a method for preparing the first pharmaceutical composition of the present invention, for example, a conventionally known method for preparing a vaccine can be used. The vaccine may be prepared, for example, as a solution or suspension for use as an injection from the peptide of the present invention and a pharmaceutically acceptable excipient. The vaccine may be prepared in a solid form. Examples of the excipient include water, salt water, dextrose, glycerol, ethanol and the like, and combinations thereof. If desired, the vaccine may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that increase the effectiveness of the vaccine. The adjuvant is not particularly limited, and conventionally known adjuvants can be used. Among them, those that promote the uptake of the peptide of the present invention by antigen-presenting cells such as dendritic cells in the patient's body are preferable. In addition, the peptide of the present invention may be formulated into a vaccine as a neutral or pharmaceutically acceptable salt.

  The method for administering the vaccine is not limited, but is administered parenterally, for example, by injection into the epidermis, intradermis, dermis or intramuscularly. Formulations suitable for other administration methods include, for example, suppositories, oral, buccal, sublingual, intraperitoneal, intrathecal, anal and intracranial formulations. The vaccine is preferably administered in a therapeutically effective amount for the patient and dosage form.

  In the present invention, the malignant tumor to be treated is not limited. Specific examples include malignant tumors expressing CML66, such as various leukemias and various solid tumors. Examples of the leukemia include acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), and acute lymphoblastic leukemia (ALL). Examples of the solid tumor include lung, prostate cancer, and black Although a tumor etc. are mentioned, it is not limited to these.

Polynucleotides and Vectors and Their Uses (1) Polynucleotides The polynucleotide of the present invention is a polynucleotide encoding the peptide of the present invention. For example, when the polynucleotide of the present invention is introduced into a target cell and the peptide of the present invention is expressed in the cell, activation / proliferation of CML66-specific CD8 + T cells can be promoted. By this activation, CML66-specific CD8 + T cells can be made into cytotoxic T cells having cytotoxic activity.

Examples of the polynucleotide include polynucleotides consisting of the following base sequences or their complementary sequences. The polynucleotide consisting of the base sequence shown in SEQ ID NO: 5 is an example of a polynucleotide encoding the peptide CML66 _{70-78 of the} present invention consisting of the amino acid sequence of SEQ ID NO: 1. The polynucleotide consisting of the base sequence shown in SEQ ID NO: 6 is an example of a polynucleotide encoding the peptide CML66 _{76-84 of the} present invention consisting of the amino acid sequence of SEQ ID NO: 2. The polynucleotide of the present invention may be a single-stranded polynucleotide or a double-stranded polynucleotide.
CML66 _70-78 5'-tggtatcaagacagtgtctactatatt-3 '(SEQ ID NO: 5)
CML66 _76-84 5'-tactatattgatacccttggaagaatt-3 '(SEQ ID NO: 6)

Furthermore, the polynucleotide of the present invention includes a polynucleotide comprising a base sequence homologous to the base sequence encoding the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a complementary base sequence thereof. The phrase “homologous” in the base sequence of a polynucleotide means that the peptide encoded by the polynucleotide is sufficiently similar to maintain the function as the peptide of the present invention. For example, even if there are differences in the base sequence due to point mutations, deletions or additions, they can be said to be homologous if they do not affect the function of the gene. The number of different bases is, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 1 to 2, or 1. Also, in two or more sequences, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or about 99% identity These are said to be homologous. In addition, when one of the two polynucleotides hybridizes with a polynucleotide comprising the other complementary sequence under stringent conditions, they can be said to be homologous. The stringent conditions are not particularly limited. For example, in a solution containing 6 × SSC, 0.5% SDS, 5 × Denhardt, 0.01% denatured salmon sperm nucleic acid, a temperature of “Tm−25 ° C.” For example, the conditions for keeping the temperature overnight. The Tm is obtained by the following formula, for example. In the following formula, N is the length of the polynucleotide probe, and% G + C is the content of guanine and cytosine residues in the polynucleotide probe primer.
Tm = 81.5-16.6 (log ₁₀ [Na ⁺ ]) + 0.41 (% G + C) − (600 / N)

  The method for producing the polynucleotide of the present invention is not particularly limited. For example, the polynucleotide may be produced by chemical synthesis, or produced by various gene amplification methods using primers designed based on the known CML66 gene. Alternatively, it may be cloned into an appropriate vector.

(2) Vector The vector of the present invention is a vector comprising the polynucleotide of the present invention. For example, when the vector of the present invention is introduced into a target cell and the peptide of the present invention is expressed in the cell, activation / proliferation of CML66-specific CD8 + T cells can be promoted. By this activation, CML66-specific CD8 + T cells can be made into cytotoxic T cells having cytotoxic activity.

  The vector of the present invention is a vector containing the polynucleotide of the present invention. The vector of the present invention is preferably an expression vector, and the polynucleotide of the present invention is preferably linked so that it can be expressed in a host cell into which the vector of the present invention is introduced. The type of the vector of the present invention is not particularly limited, and may be, for example, a viral vector or a non-viral vector. Examples of the viral vector include retrovirus vectors, lentiviral vectors such as immunodeficiency virus (HIV), adenovirus vectors, adeno-associated vectors (AAV vectors; adeno associated viruses), herpes viruses, vaccinia viruses, poxviruses, polioviruses, and the like. Examples thereof include DNA viruses and RNA viruses such as viruses, Shinbis viruses, Sendai viruses, and SV40. Examples of non-viral vectors include expression vectors such as pcDNA3.1 (Invitrogen), pZeoSV (Invitrogen), pBK-CMV (Stratagene), and pCAGGS (Gene108, 193-200 (1991)). Usually, the polynucleotide may be inserted downstream of the promoter of these vectors so as to allow expression.

(3) Second activation inducer As described above, the second activation inducer of the present invention is an activation inducer that induces cytotoxic activation of CD8 + T cells. It contains at least one of a polynucleotide and a vector. The activation / proliferation of CML66-specific CD8 + T cells can be promoted by introducing the second activation inducer of the present invention into, for example, a target cell and expressing the polypeptide or derivative of the present invention. This activation enables CML66-specific CD8 + T cells to be cytotoxic T cells having cytotoxic activity.

  The second activation inducer of the present invention may include, for example, only one or both of the polynucleotide and the vector of the present invention. Moreover, the polynucleotide and the vector of the present invention may be one kind or two or more kinds. The second activation inducer of the present invention may be, for example, only the polynucleotide or vector of the present invention, or may further contain various additives. In addition, about a concrete usage method etc., it can carry out similarly to the 2nd pharmaceutical composition mentioned later, for example.

(4) Second Pharmaceutical Composition The second pharmaceutical composition of the present invention is a pharmaceutical composition used for cancer immunotherapy, and comprises at least one of the polynucleotide and the vector of the present invention. The second pharmaceutical composition is, for example, a DNA that induces a CML66-specific immune response by inoculating a patient's body, preferably subcutaneous tissue, lymph nodes, muscle cells, and expressing the peptide of the present invention. (RNA) Can be used for vaccine therapy. The method for introducing the second pharmaceutical composition into the cell is not particularly limited, and can be appropriately determined according to the kind of the polynucleotide or vector. Specific examples include a method using a viral vector, a lipofection method, an electroporation method, a microinjection method, a cell fusion method, a DEAE dextran method, a calcium phosphate method, and the like.

  The preparation method as a vaccine is not specifically limited, It can prepare similarly to the vaccine (1st pharmaceutical composition) containing the peptide of the above-mentioned this invention. In the second pharmaceutical composition of the present invention, the polynucleotide of the present invention may be in any form that can express the peptide of the present invention in the introduced cell. In this case, the second pharmaceutical composition may contain, for example, the polynucleotide of the present invention that is not linked to a vector together with an introduction agent. The introduction agent is not particularly limited, and examples include those used in non-vector gene introduction methods such as liposomes. Moreover, the 2nd pharmaceutical composition containing the polynucleotide of this invention can also be inoculated by the direct injection method using a particle gun etc., for example.

Method for Producing CD8 + Cytotoxic T Cell and Its Use (1) Method for Producing CD8 + Cytotoxic T Cell The method for producing CD8 + cytotoxic T cell of the present invention is as described above. It is a manufacturing method, Comprising: The following process (A) and (B) is included, It is characterized by the above-mentioned.
(A) Preparing patient lymphocytes (B) Incubating the lymphocytes with at least one of the peptide of the present invention and a derivative thereof.

  In the step (B), for example, the lymphocytes are preferably incubated with antigen-presenting cells that present the peptide of the present invention. The method for preparing the antigen-presenting cell is not particularly limited. As a specific example, antigen-presenting cells can be prepared in the step (B) by, for example, incubating the lymphocytes, dendritic cells, and the peptide of the present invention. The dendritic cell becomes, for example, an antigen-presenting cell that presents the peptide of the present invention by being pulsed with the peptide of the present invention. Since the antigen-presenting cells induce proliferation and activation of the lymphocytes, CD8 + cytotoxic T cells having cytotoxic activity can be obtained.

  The lymphocytes are usually collected from the patient's blood. As the antigen-presenting cells, for example, those collected from a patient from whom lymphocytes have been collected can be used. In addition to this, for example, autologous, allogeneic, and allogeneic antigen-presenting cells that match the patient for HLA class I can be used. The antigen-presenting cell is preferably one that presents the peptide of the present invention to CD8 + T cells and expresses HLA-A24 molecule and costimulatory molecule (costimulatory molecule) that can be activated by proliferation stimulation. And dendritic cells. The antigen-presenting cells are preferably collected or prepared from a patient from whom lymphocytes have been collected, for example.

  CML66-specific CD8 + cytotoxic T cells produced by the production method of the present invention have a characteristic function of exhibiting HLA-A24-restricted cytotoxicity against malignant tumor cells including leukemia cells, for example. Therefore, CML66-specific CD8 + cytotoxic T cells destroy malignant tumor cells that express CML66 in the body of the HLA-A24-positive patient, for example.

(2) Third Pharmaceutical Composition The third pharmaceutical composition of the present invention comprises CML66-specific CD8 + cytotoxic T cells produced by the method for producing CD8 + cytotoxic T cells of the present invention. By administering an effective amount of this pharmaceutical composition to a patient, for example, cellular immunotherapy for treating a malignant tumor becomes possible.

Method for producing antigen-presenting cell and use thereof (1) Method for producing antigen-presenting cell As described above, the method for producing an antigen-presenting cell includes the following steps (a) and (b): .
(A) Preparing a patient's dendritic cell (b) Incubating the dendritic cell with at least one of the peptide of the present invention and a derivative thereof

  In the step (a), the dendritic cell may be, for example, a cell present in patient blood or a dendritic cell prepared by artificially inducing differentiation. In the former case, for example, it is obtained by a method of collecting dendritic cells themselves from the patient's own blood using a blood component separation device. In the latter case, immature dendritic cells of the patient, for example, collect peripheral blood mononuclear cells (PBMC) and, for example, culture in the presence of GM-CSF, IL-4, etc., which are a kind of cytokine. Can be obtained. Furthermore, it is differentiated into mature dendritic cells with, for example, TNF-α. Then, by incubating the dendritic cells thus obtained with the peptide or derivative of the present invention, for example, dendritic cells with enhanced immunity-inducing ability against CML66-specific CD8 + T cells can be produced.

In the method for producing an antigen-presenting cell of the present invention, the following step (c) may be included instead of the step (b).
(C) Step of introducing the polynucleotide of the present invention or the vector of the present invention into the dendritic cell

  Thus, the peptide of the present invention is expressed in dendritic cells by introducing the polynucleotide of the present invention or the vector of the present invention into dendritic cells. As a result, an antigen-presenting cell is obtained by presenting the expressed peptide on the cell surface in the dendritic cell. This antigen-presenting cell is a dendritic cell with enhanced immunity-inducing ability against CML66-specific CD8 + T cells. The method for introducing a polynucleotide or vector into a gene is not particularly limited, and can be appropriately determined depending on the type. Specific examples include a method using a viral vector, a lipofection method, an electroporation method, a microinjection method, a cell fusion method, a DEAE dextran method, a calcium phosphate method, and the like.

  The cells produced by the method for producing antigen-presenting cells of the present invention can be used, for example, as antigen-presenting cells in the method for producing CML66-specific CD8 + T cells of the present invention, in addition to the following pharmaceutical composition.

(2) Fourth Pharmaceutical Composition The fourth pharmaceutical composition of the present invention is a pharmaceutical composition comprising antigen-presenting cells produced by the method for producing antigen-presenting cells of the present invention. Since the 4th pharmaceutical composition of this invention contains the dendritic cell which the immunity induction power improved specifically for CML66, it can be utilized for the dendritic cell therapy of a malignant tumor, for example. The fourth pharmaceutical composition of the present invention may further contain the aforementioned CML66-specific CD8 + cytotoxic T cells in addition to the dendritic cells.

  The present invention will be further described below using examples.

[Example 1]
In this example, HLA class I restricted antigen peptides were identified. HLA class I expression in tumor cells and PBMC was analyzed by flow cytometry using a FITC-labeled anti-HLA-A24 antibody. In the analysis by the flow cytometry method, FACScan (manufactured by BD Bioscience) and Cell Quest software (manufactured by BD Bioscience) were used.

(1) Synthesis of CML66 peptide A plurality of peptides derived from the amino acid sequence of CML66 were synthesized as peptides capable of binding to HLA-A24.

(2) Measurement of binding ability of CML66 peptide to HLA class I molecule Since T2 cells are cells lacking TAP (transporter associated with antigen processing), which has an essential role in the antigen presentation system, Cannot be presented as HLA-peptide complexes on cell surface HLA. However, when the peptide added to the cell binds to HLA on the cell surface, an HLA-peptide complex can be formed on the cell surface. For this reason, it is known that it is useful as a cell used for the assay which analyzes the direct coupling | bonding of an antigen peptide and HLA. Therefore, first, T2-A24 cells in which HLA-A24 is expressed in T2 cells are seeded in a medium to 1 × 10 ⁶ cells, and 10 μmol / L of various synthesized CML66 peptides are added. Time culture was performed. As a medium for T2-A24 cells, RPMI 1640 medium containing 10% FCS supplemented with Geneticin (final concentration 800 μg / mL) was used. Then, the cultured cells were collected, and anti-HLA-A24 antibody labeled with FITC was added thereto. After reacting them at 4 ° C. for 20 minutes, the cells were washed twice with PBS (−) containing 0.1% FCS, and the fluorescence intensity was measured with a flow cytometer. The fluorescence index was calculated from the following formula.
Fluorescence index = (SB) / B
S: Average fluorescence intensity of each sample
B: Background average fluorescence intensity

  The results of examining the affinity for HLA class I for various CML66 peptides by flow cytometry are shown in Table 1 below. Table 1 below shows information on the CML66 peptide that showed affinity for HLA-A24 and the measurement results. In general, if the measured fluorescence index is 1.0 or more, it can be determined that the binding ability is sufficiently high, and if the measured value is higher, it can be determined that the binding ability is higher (high affinity).

As shown in Table 1, CML66 _217-225 (KYEIIIKRDI; SEQ ID NO: 3) as a peptide capable of binding to the HLA-A24 molecule (HLA-A ^* 2402) by flow cytometry using TAP-deficient cells, CML66 _22-30 (GYKLSLEPL; SEQ ID NO: 4), CML66 _70-78 (WYQDSVYYI: SEQ ID NO: 1), and CML66 _76-84 (YYIDTLLGRI; SEQ ID NO: 2) were respectively identified for the first time. CML66 _70-78 and CML66 _76-84 showed particularly high binding to the HLA-A24 molecule. In addition, the number attached | subjected to the name of the peptide is an amino acid site | part in CML66 protein, and shows the position of the N terminal amino acid residue and C terminal amino acid residue of each peptide.

[Example 2]
In this example, CML66 peptide-specific cytotoxic T cells were established using the peptides CML66 _70-78 and CML66 _76-84 identified in Example 1, and their cytotoxicity was confirmed.

(1) Establishment of CML66 peptide-specific cytotoxic T cells Peripheral blood mononuclear cells (PBMC) are collected from HLA-A24 (HLA-A ^* 2402) positive donors and used with MACS beads (Miltenyi Biotech). CD14 positive cells were collected. The collected CD14-positive cells were cultured for 5 days in RPMI 1640 medium containing 10% FCS supplemented with GM-CSF (75 ng / mL) and IL-4 (8.3 ng / mL). On the fifth day of culture, TNF-α (100 U / mL) was further added to induce and establish monocyte-derived mature dendritic cells. And the dendritic cell was collect | recovered on the 8th day of culture | cultivation, and this was used as an antigen presentation cell.

CD8 + T cells were recovered from PBMC of the same donor using MACS beads. Then, 10 μmol / L of CML66-derived peptide, that is, CML66 _70-78 (WYQDSVYYI) or CML66 _76-84 (YYIDTLGRI) is added to the dendritic cells, and the dendritic cells 1 × 10 6 are added to 100 μL of medium per well. ^Four were sprinkled, and about 1 × 10 ⁵ recovered CD8 + T cells were added to stimulate this. CD8 + T cells were cultured in RPMI 1640 medium containing 10% human serum. On the 8th day from the start of the culture, CML66-derived peptide (10 μmol / L) was further added to 1 × 10 ⁴ donor PBMCs treated with mitomycin C (MMC), and the second stimulation was performed. Two days later, IL-2 (10 U / mL) was further added, followed by continuous culture. On the 15th day from the start of the culture, 1 × 10 ⁵ self-PBMCs treated with mitomycin C (MMC) were subjected to the CML66-derived peptide (10 μmol / L) was added and the third stimulation was performed. Four to five days after the third stimulation, the degree of cytotoxicity was measured by a ⁵¹ Cr release test, and cytotoxic T cells showing a peptide-specific reaction were screened. Then, the selected cells were cultured while repeatedly stimulating with the CML66-derived peptide (10 μmol / L). Cells that showed specific reactivity to the CML66-derived peptide were continuously cultured in a medium supplemented with IL-2. Thereafter, autologous PBMC treated with MMC was used as antigen-presenting cells for the fourth and fifth stimulations, and autologous LCL (lymphoblastoid cells) treated with MMC were used as antigen-presenting cells after the sixth stimulation. LCL is a cell established by infecting peripheral blood B lymphocytes with EB virus and immortalizing them.

(2) Cytotoxicity measurement (2-1) CML66-derived peptide-specific cytotoxicity against autologous LCL cells The cytotoxic T cells established in (1) above were tested for cytotoxic activity by a conventionally known ⁵¹ Cr release test. confirmed. The target antigen-specific cytotoxic activity was examined by comparison with and without the addition of CML66-derived peptide.

First, target cells labeled with ⁵¹ Cr (Na ₂ ⁵¹ CrO ₄ ) were prepared. To a 96-well round-bottom plate, add 200 μL of 10% FCS-containing RPMI 1640 medium and 0.5 × 10 ⁴ target cells per well, and further add cytotoxic T cells of a predetermined number of cells, Culturing was performed for 5 hours. In addition, when adding the CML66-derived peptide, the target cells were cultured in the presence of the CML66-derived peptide for 2 hours in advance, and then cytotoxic T cells were added. The aforementioned LCL was used as a target cell. The ratio (E: T ratio) between cytotoxic T cells (effector; E) and target cells (target; T) was 5: 1. Then, after reaction with the target cells and cytotoxic T cells, was measured the supernatant were harvested and ^{a 51 Cr} release amount of the culture solution. The degree of cytotoxicity was calculated from the following formula.
Cytotoxicity = (S−N) / (M−N)
S: Each sample ⁵¹ Cr release amount
N: Natural ⁵¹ Cr release amount
M: Maximum ⁵¹ Cr release

These results are shown in FIG. (A) is a graph showing the cytotoxicity of cytotoxic T cells induced by CML66 _70-78 , and (B) shows cells by cytotoxic T cells induced by CML66 _76-84. It is a graph which shows an injury degree. In both figures, the horizontal axis represents the concentration of each CML66-derived peptide, and the vertical axis represents the degree of cytotoxicity (%) by CML66-derived peptide-specific cytotoxic T cells. Moreover, in both figures, ○ is added CML66 derived peptides self LCL cells, ● was added CML66 derived peptides ^HLA-A * 2402-negative healthy individual derived LCL ^cells, 3 rd: party (third party negative control ) Shows the results of cytotoxic T cell assay. As shown in FIG. 5A, the established cytotoxic T cells showed cytotoxicity against HLA-A ^* 2402-positive autologous LCL cells when CML66 _70-78 (WYQDSVYYI) was added. Although no data is shown, it has been confirmed that even when autologous LCL cells are targeted, no cytotoxicity is observed in the absence of CML66 peptide. From this, it was proved that the cytotoxicity of the cytotoxic T cells established in this Example shows CML66 _70-78 specificity. Furthermore, this cytotoxic T cell did not show cytotoxicity against HCL-A ^* 2402-negative healthy LCL cells (3 ^rd party) supplemented with CML66 _70-78 . In addition, as shown in FIG. 5B, similar results were obtained when CML66 _76-84 was used. From the above, it was considered that the established cytotoxic T cells recognize the CML66-derived peptide specifically bound to HLA-A ^* 2402 and damage target cells.

(2-2) for HLA-A24 that ^(HLA-A * 2402) cytotoxicity by positive cell CML66 derived peptide-specific cytotoxicity confirms to be bound by ^{HLA-A * 2402, HLA-} A * 2402 the positive self-LCL cells, ^HLA-a * 2402-positive non-self LCL, ^HLA-a * 2402-negative non-self LCL, ^C1R-a * 2402 cells in C1R cell a leukemia patient-derived cell lines was expressed ^HLA-a * 2402 A cytotoxicity assay was performed using as a target cell. The experimental conditions were the same as (2-1) of this example except that the concentration of CML66 _76-84 at the time of cell injury was 10 μmol / L. Moreover, it confirmed also about the cytotoxicity at the time of adding various antibodies. This is _because the concentration of CML66 _76-84 is 10 μmol / L, and 10 μg / mL anti-HLA class I monoclonal antibody (w6 / 32) or anti-HLA-DR monoclonal antibody (L243) is added for 30 minutes immediately before adding cytotoxic T cells. ) Was carried out in the same manner as in (2-1) except that.

These results are shown in FIG. (A) shows the results of cytotoxicity assay using cytotoxic T cells (CTL) in the presence or absence of CML66 _76-84 peptide, and (B) shows the results in the presence of various antibodies. It is a result which shows the cytotoxicity degree in. In both figures, “*” indicates a value below the background. As shown in the figure (A), it was found that cytotoxicity by CML66 _76-84 occurs only in the presence of HLA-A ^* 2402 of the target cells. In addition, as shown in FIG. 5B, the cytotoxicity was inhibited by the anti-HLA class I antibody that recognizes HLA-A ^* 2402, whereas the anti-HLA-DR antibody that does not recognize HLA-A ^* 2402 No inhibitory effect was observed. From the above results, it was confirmed that CML66-derived peptide-specific cytotoxicity is restricted by HLA-A ^* 2402.

(3) Further confirmation of HLA-A24 restriction of established cytotoxic T cells In order to further confirm the HLA-A24 restriction of cytotoxic T cells, various cells were used as target cells. In the same manner as above, cytotoxicity of cytotoxic T cells was examined. Target cells are HLA-A24 (HLA-A ^* 2402) positive cells, such as KH88 (chronic myelogenous leukemia acute inversion human cell line), KCL22 (chronic myelogenous leukemia acute inversion human cell line), KAZZ ( Chronic myelogenous leukemia acute inversion human cell line), SAS527 (chronic myelogenous leukemia acute inversion human cell line), OUN-1 (chronic myelogenous leukemia acute inversion human cell line) and MEG01 (chronic myelogenous cell line) Leukemia acute inversion type human cell line), and as HLA-A24 (HLA-A ^* 2402) negative cells, K562 (chronic myelogenous leukemia acute inversion type human cell line), KT-1 (chronic myelogenous leukemia) Acute inversion human cell line) and KG-1 (acute myeloid leukemia M1 human cell line) were used. Healthy PBMC (peripheral blood mononuclear cells), CB (human umbilical cord blood cells) and CBMC (human umbilical cord blood mononuclear cells) were used as controls. For the culture of various cells, RPMI1640 medium containing 10% FCS was used. The ratio of cytotoxic T cells (effector; E) to target cells (target; T) (E: T ratio) was 40: 1 in experiments using CML66 _70-78 specific cytotoxic T cells. 20: 1, 10: 1, or 5: 1, and 10: 1, 5: 1, or 2.5: 1 in experiments using CML66 _76-84 specific cytotoxic T cells. These results are shown in the graphs of FIGS. In both figures, the horizontal axis represents the degree of cytotoxicity (%) caused by CML66-specific cytotoxic T cells (CTL).

  Moreover, about some target cells, it confirmed also about the cytotoxicity at the time of adding various antibodies. This is because anti-HLA-class I antibody (w6 / 32) or anti-HLA-DR antibody (L243) is added to the target cell at a final concentration of 10 μg / mL before adding cytotoxic T cells as effectors. And then cultured for 1 hour, and cytotoxic T cells were added. The E: T ratio was 10: 1, and PBS was added instead of the antibody as a control. These results are shown in the graph of FIG. In the figure, the horizontal axis represents the degree of cytotoxicity (%) caused by CML66-specific cytotoxic T cells (CTL).

  The expression level of CML66 in the various target cells was confirmed in advance by a real-time PCR method. First, total RNA was extracted from each tumor cell, and cDNA was prepared by a known method. Then, using this cDNA as a template, real-time PCR was performed using a commercially available kit (TaqMan ™ Gene Expression Assays, CML66 primer, GAPDH primer, Applied Biosystems), and the number of CML66 mRNA copies was measured. Analysis was performed with ABI Prism 7700 (manufactured by Applied Biosystems). Then, using the Comparative Ct method, the relative value of expression in various cells was calculated when the expression level of CML66 in the K562 cell line was 1. The results are shown in the bar graph of FIG. In the figure, the vertical axis represents the relative expression level of CML66 mRNA. As shown, K562 cells, KH88 cells, KCL22 cells, KAZZ cells, SAS527 cells, KT-1 cells, OUN-1 cells, MEG01 cells, and acute bone marrow, which are cell lines derived from patients with chronic myelogenous leukemia (CML) All of KG-1 cells, which are cell lines derived from sexual leukemia (AML), expressed CML66 mRNA, and in particular, it was confirmed that KCL22 and KT-1 highly express CML66 mRNA.

FIG. 3 is a graph showing the degree of cytotoxicity against leukemia cells by CML66 _70-78 (WYQDSVYYI) -specific cytotoxic T cells, and FIG. 4 is a graph showing CMLCML66 _76-84 (YYIDTLGRI) -specific cytotoxic T cells. It is a graph which shows the cytotoxic degree with respect to a leukemia cell. In the figure, “*” indicates a value below the background. In the case of CML66 _70-78 (WYQDSVYYI) -specific cytotoxic T cells, as shown in the upper part of the figure, CML66 _70-78- specific cytotoxic T cells are HLA-A24 positive leukemias that strongly express CML66. It was confirmed that even when CML66 _{70-78 was} not added to cells (KH88, KCL22, KAZZ, SAS527), high cytotoxicity was exhibited. On the other hand, as shown in the middle of the figure, even cells that strongly express CML66 showed cytotoxicity against HLA-A24 negative leukemia cells (KT-1, KG-1, K562). There wasn't. Furthermore, as shown in the lower part of the figure, cytotoxicity was not exhibited even for cells derived from HLA-A24 positive healthy individuals. In the case of CMLCML66 _76-84 (YYIDTLGRI) -specific cytotoxic T cells, as shown in FIG. 4, CML66 _76-84- specific cytotoxic T cells are HLA-A24 positive leukemia cells (OUN-1, Only MEG01) was confirmed to exhibit high cytotoxicity.

FIG. 5 is a graph showing the degree of cytotoxicity in the presence of various antibodies. FIG. 5A shows the results of CML66 _70-78- specific cytotoxic T cells, and FIG. Results of CML66 _76-84 specific cytotoxic T cells. As shown in both figures, cytotoxicity of cytotoxic T cells was inhibited by adding anti-HLA-class I antibody. This confirmed that cytotoxic T cells exhibit HLA-A24-restricted cytotoxicity. These results imply that CML66 is fragmented within tumor cells and the peptide antigens CML66 _70-78 and CML66 _76-84 are presented in association with HLA-A24 molecules on the tumor cell surface. Yes. At the same time, it means that target-specific cytotoxic T cells can be induced in HLA-A24 positive individuals by targeting the CML66 antigen peptide.

[Example 3]
In this example, in order to further verify that CML66 can be a target molecule for cancer immunotherapy, the presence of CML66-specific cytotoxic T cells in the peripheral blood of leukemia patients was examined.

(1) Quantification of CML66 expression in leukemia cells HLA-A24 positive acute myeloid leukemia patients, chronic myeloid leukemia patients (chronic phase and blast crisis), various cells derived from acute lymphocytic leukemia patients, and chronic myeloid The expression of CML66 mRNA in the leukemia patient bone marrow derived CD34 positive cells was quantified by the same real-time PCR method as described above. Note that, in the recovery of CD34 positive cells, MACS beads (Miltenyi Biotech) were used as in Example 2.

  These results are shown in the graphs of FIGS. FIG. 7 shows cells from patients with acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), advanced chronic myelomonocytic leukemia (CMMoL), acute lymphocytic leukemia (ALL), and as a control from CML patients. It is a figure which shows the result of the K562 cell which is a cell, the peripheral blood mononuclear cell (PBMC) derived from a healthy subject, and the human umbilical cord blood cell (CB) derived from a healthy subject. FIG. 8 is a diagram showing the results of cells derived from patients of various subtypes (M0, M1, M2, M3, M4, M5b) of acute myeloid leukemia (AML) and K562. FIG. 9 is a diagram showing the results of cells derived from patients in the blast crisis (CML-BC) and chronic phase (CML-CP), which are clinical stages of CML, and K562. FIG. 10 shows the results for CD562 positive cells isolated from CML chronic stage (CML-CP) patients or healthy subjects, and K562. CD34 positive cells of CML patients are believed to contain CML leukemic stem cells. 7 to 10, the vertical axis represents the relative expression level of CML66 mRNA. As shown, acute myeloid leukemia cells (AML), acute myelogenous chronic myeloid leukemia cells (CML-BC), acute lymphocytic leukemia cells (ALL) and CD34 positive chronic myeloid leukemia cells (CML-CP CD34 +) In both cases, high expression of CML66 mRNA was confirmed. The above results indicate that CML66 is not only promising as a target for treatment of AML, CML, CMMoL, and ALL, but also a more aggressive malignant transformation that is difficult for conventional molecular targeted drugs such as Imatinib to succeed. It has also been shown to be effective against stage CML and CML leukemic stem cells.

(2) Measurement of CML66-specific cytotoxic T cell frequency First, HLA-A24-positive acute myeloid and lymphocytic leukemia patients (after chemotherapy and after allogeneic bone marrow transplantation), and from healthy individuals, peripheral blood mononuclear cells ( PBMC) was separated and seeded to 1 × 10 ⁶ to 2 × 10 ⁶ , and CML66 _70-78 or CML66 _76-84 (10 μmol / L) was further added. The PBMC were cultured in 10% human serum-containing RPMI1640 medium containing IL-7 (5 ng / mL) and IL-12 (100 pg / mL), and once a week, CML66 _70-78 or CML66 _76-84 ( 10 μmol / L) was added. From the 4th day of culture, IL-2 (10 U / mL) was added and the culture was continued. Around the 16th day of culture, the frequency of cytotoxic T cells specific for CML66 was measured by the ELISPOT method. In addition, the culture group without CML66-derived peptide was used as a negative control.

The ELISPOT method was performed as follows. First, an anti-IFN-γ monoclonal antibody (10 μg / mL) was added to a 96-well MultiScreen-HA plate whose bottom was covered with a nitrocellulose membrane, and left at 4 ° C. for 18 hours to bind the antibody to the membrane. I let you. The membrane was washed with PBS (−), coated with 10% FCS • RPMI1640 at 37 ° C. for 1 hour, and then coated with 5 × 10 ⁴ T2-A24 cells as described above. CML66 _70-78 Alternatively, CML66 _76-84 (10 μmol / L) was added and cultured for 1 hour. The T2-24 cells were cultured with RPMI1640 containing 10% FCS, and then PBMCs of each patient were spread and reacted for 18 hours. The plate was washed with PBS (-) containing 0.1% Tween 20, and added with rabbit anti-human IFN-γ antibody for 90 minutes, followed by addition of goat anti-rabbit IgG antibody for 90 minutes. Finally, 100 μL of the mixed solution (0.1 mol / L sodium acetate (pH 5.0) / 3-amino-9-Ethylcarbazole / N, N dimethylformamide / 0.015% H ₂ O ₂ ) was added and reacted for 40 minutes. Allowed to develop color. The plate was washed thoroughly with water and dried, and then the spots were confirmed with an inverted microscope, and the frequency of CML66-specific cytotoxic T cells was measured.

FIG. 11 shows the measurement results of CML66 _76-84 specific cytotoxic T cell frequency by the ELISPOT method. Fig. (A) is a graph showing the result of analysis by ELISPOT showing the frequency of CML66 _76-84 specific cytotoxic T cells present in the peripheral blood of a patient, and Fig. (B) shows two patients. Is a photograph of a spot obtained by the ELISPOT method. In both figures, “+” is the result of addition of CML66 _76-84 , and “−” is the result of addition of CML66 _76-84 . As shown in FIG. _5A , there were almost no cytotoxic T cells that increased in response to CML66 _76-84 in the peripheral blood of healthy subjects (◯, Δ, □). On the other hand, in peripheral blood of patients with acute lymphocytic leukemia (ALL) (▲, ×, ●) and patients with acute myeloid leukemia (AML) (◆, ■, ▼), increase in response to CML66 _76-84 It was confirmed that cytotoxic T cells were present. Further, as shown in FIG. 5B, in the case of adding CML66 _76-84 peptide, the increase in spots was more noticeable than in the case of no addition. Although not shown, it was confirmed that almost the same results were obtained when CML66 _70-78 peptide was used.

As shown in FIGS. 7 to 10 described above, high expression of CML66 was confirmed in cells derived from leukemia patients. Moreover, as described above, it was confirmed by the ELISPOT assay that cytotoxic T cells specifically recognizing CML66 _70-78 or CML66 _76-84 were present in the peripheral blood of each patient. This result shows that the CML-derived 66 peptide actually functions as a target antigen for cytotoxic T cells in leukemia patients. Therefore, according to the peptide vaccines CML66 _70-78 and CML66 _76-84 , it can be said that there is a very high possibility that these cytotoxic T cells can be activated and tumor cells can be eliminated. From the above results, it can be said that CML66 _70-78 and CML66 _76-84 were clinically proven to be useful as cancer vaccines.

  The peptide of the present invention functions as a tumor antigen peptide for cells derived from various leukemia patients including chronic myelomonocytic leukemia and acute lymphoblastic leukemia in addition to myeloid leukemia. It can also function as a tumor antigen peptide for melanoma cell lines, various solid cancer cell lines, and cells derived from prostate cancer patients.

  As described above, the present invention is useful, for example, in the medical field of malignant tumors including leukemia, particularly in the field of immunotherapy including cellular immunotherapy, vaccine therapy, gene therapy and the like.

FIG. 1 is a graph showing the cytotoxicity of cytotoxic T cells induced with a CML-derived peptide in Example 2 of the present invention, and (A) shows the results of using CML66 _70-78. B) is the result of using CML66 _76-84 . FIG. 2 (A) is a graph showing the degree of cytotoxicity caused by cytotoxic T cells (CTL) in the presence or absence of CML66 _76-84 in Example 2, and FIG. It is a graph which shows the cytotoxic degree in presence of various HLA inhibitory antibodies. FIG. 3 is a graph showing the degree of cytotoxicity of CML66 _70-78- specific cytotoxic T cells (CTL) against cells derived from various leukemia patients in Example 2. FIG. 4 is a graph showing the degree of cytotoxicity of CML66 _76-84- specific cytotoxic T cells (CTLs) against cells derived from various leukemia patients in Example 2. FIG. 5 is a graph showing the cytotoxicity of cytotoxic T cells induced by CML66-derived peptides by the addition of various antibodies in Example 2, and (A) shows the results induced by CML66 _70-78. (B) is the result induced by CML66 _76-84 . FIG. 6 is a graph showing CML66 mRNA expression levels of various target cells in Example 2. FIG. 7 is a graph showing the expression level of CML66 mRNA in cells derived from various leukemia patients in Example 3 of the present invention. FIG. 8 is a graph showing the expression level of CML66 mRNA in patient leukemia cells for each clinical disease type in patients with acute myeloid leukemia (AML) in Example 3. FIG. 9 is a graph showing the expression level of CML66 mRNA in patient bone marrow-derived leukemia cells in the blast crisis phase (CML-BC) and chronic phase (CML-CP) of chronic myelogenous leukemia in Example 3. FIG. 10 is a graph showing the CML66 mRNA expression level of CD34 positive cells isolated from CML chronic phase (CML-CP) patients or healthy subjects in Example 3. FIG. 11 (A) is a graph showing the frequency of CML66 _76-84 specific cytotoxic T cells present in the peripheral blood of various leukemia patients in Example 3, and FIG. 11 (B) is a representative. It is the photograph which shows the spot of the said CML66 _76-84 specific cytotoxic T cell in two positive examples.

Claims (15)

The peptide according to any one of (1) to (4) below.
(1) Peptide comprising the amino acid sequence of SEQ ID NO: 1 (2) Peptide comprising the amino acid sequence of SEQ ID NO: 2 (3) Peptide having an amino acid sequence homology of 80% or more with the peptide of (1) or (2) An HLA-A24-restricted antigen peptide that can be specifically recognized by CD8 + T cells by binding to an HLA-A24 molecule (4) one or more in the amino acid sequence of the peptide of (1) or (2) An HLA-A24-restricted antigen peptide consisting of an amino acid sequence in which several amino acids are deleted, substituted or added, and which can be specifically recognized by CD8 + T cells by binding to an HLA-A24 molecule. The peptide according to claim 1, wherein the HLA-A24 molecule has a genotype of A ^* 2402. An activation inducer that induces cytotoxic activation of CD8 + T cells, comprising:
An activation inducer comprising at least one selected from the group consisting of the peptide according to claim 1 and 2 and derivatives thereof. An activation inducer that induces cytotoxic activation of CD8 + T cells, comprising:
An activation inducer comprising a polynucleotide encoding at least one selected from the group consisting of the peptide according to claim 1 and 2 and a derivative thereof.   The activation inducer according to claim 4, further comprising a vector, wherein the polynucleotide is inserted into the vector. A pharmaceutical composition for use in cancer immunotherapy,
A pharmaceutical composition comprising, as an antigen peptide, at least one selected from the group consisting of the peptide according to claim 1 and 2 and a derivative thereof. A pharmaceutical composition for use in cancer immunotherapy,
A pharmaceutical composition comprising a polynucleotide encoding at least one selected from the group consisting of the peptide according to claim 1 and 2 and derivatives thereof.   The pharmaceutical composition according to claim 7, further comprising a vector, wherein the polynucleotide is inserted into the vector. A method for producing CD8 + cytotoxic T cells, comprising:
A method for producing cytotoxic T cells comprising the following steps (A) and (B).
(A) Preparing patient lymphocytes (B) Incubating the lymphocytes with at least one selected from the group consisting of the peptide of claim 1 and 2 and derivatives thereof.   The method for producing cytotoxic T cells according to claim 9, wherein in the step (B), the lymphocytes are incubated with antigen-presenting cells presenting at least one of the peptide and the derivative. A method for producing an antigen-presenting cell, comprising the following steps (a) and (b).
(A) preparing a patient's dendritic cell (b) incubating the dendritic cell with at least one selected from the group consisting of the peptide of claim 1 and 2 and a derivative thereof.   12. The method for producing antigen-presenting cells according to claim 11, wherein in the step (a), dendritic cells are prepared by preparing patient monocytes and inducing differentiation of the monocytes. The method for producing an antigen-presenting cell according to claim 11 or 12, comprising the following step (c) instead of the step (b).
(C) introducing into the dendritic cell a polynucleotide encoding at least one selected from the group consisting of the peptide according to claim 1 and 2 and a derivative thereof.   The method for producing an antigen-presenting cell according to claim 13, wherein in the step (c), a vector in which the polynucleotide is inserted is introduced into the dendritic cell. A pharmaceutical composition for use in cancer immunotherapy,
A CD8 + cytotoxic T cell produced by the production method according to claim 9 or 10, and at least one of antigen-presenting cells produced by the production method according to any one of claims 11 to 14. Pharmaceutical composition.