WO1999003972A1 - Cytotoxic t lymphocytes - Google Patents
Cytotoxic t lymphocytes Download PDFInfo
- Publication number
- WO1999003972A1 WO1999003972A1 PCT/JP1998/003143 JP9803143W WO9903972A1 WO 1999003972 A1 WO1999003972 A1 WO 1999003972A1 JP 9803143 W JP9803143 W JP 9803143W WO 9903972 A1 WO9903972 A1 WO 9903972A1
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- WIPO (PCT)
- Prior art keywords
- antigen
- hla
- peptide
- cells
- ctl
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
- G01N33/56972—White blood cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/46448—Cancer antigens from embryonic or fetal origin
- A61K39/464482—Carcinoembryonic antigen [CEA]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464484—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
- A61K39/464486—MAGE
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- the present invention relates to cytotoxic T lymphocytes (sometimes referred to as cytotoxic T lymphocytes) useful for the treatment and diagnosis of cancer (cytotoxic T lymphocytes; hereinafter abbreviated as CTL) and to induce the CTL
- CTL cytotoxic T lymphocytes
- the present invention relates to a CTL inducer that is useful in such cases.
- CTLs contain major histocompatibility gene complex (MHC), a major histocompatibility antigen complex (MHC), which is encoded by an antigenic peptide and a major histocompatibility gene complex (MHC).
- MHC major histocompatibility gene complex
- MHC major histocompatibility antigen complex
- MHC major histocompatibility gene complex
- HLA class I A complex that binds to a molecule is recognized by a specific T cell receptor (TCR), and the complex is recognized on the cell surface. Some can present and damage cells.
- TCR T cell receptor
- Such CTLs are called MHC class I molecule-restricted CTLs because they recognize and damage only target cells having the same MHC class I molecule as themselves.
- the “antigen peptide” in the present specification refers to an antigenicity that binds to an MHC class I molecule and forms a complex with the MHC class I molecule, and that the complex is recognized by a specific CCR TCR.
- the “peptide” in the present specification is not particularly limited to a homomeric peptide consisting of only amino acids, but also includes a heteromeric peptide containing a non-amino acid component. Amino acids are not particularly limited to natural forms, and may be chemically modified amino acids. Further, the peptide in the present specification is not limited to a monomer, and may be a multimer.
- antigen peptides are produced, for example, by the processing of antigens and the like synthesized in mammalian cells in the endoplasmic reticulum and decomposing them into small epitope peptides.
- MHC class I molecules associates with MHC class I molecules and is displayed on the cell surface. That is, in a proteosome complex composed of many subunits, proteins are decomposed into peptides composed of 8- to 15-amino acids, some of which are converted from the cytoplasm to the endoplasmic reticulum by the TAP transport enzyme. Carried. If these peptides can bind to class IZ ⁇ 2 microglobulin heterodimers in the endoplasmic reticulum, they will be stabilized as trimolecular complexes and transported to the cell surface through the Golgi apparatus. Tumor cells expressing tumor-associated antigens or tumor-specific antigen proteins should be able to present the MHC class I molecule-restricted antigen peptides recognized by T lymphocytes on the tumor cell surface.
- MAGE melanoma antigen E
- the antigen derived from the protein is HLA-A1-restricted, and the MAGE-1-specific CTL clone binds to HLA-A1 molecule, which is a kind of HLA class I molecule, and is represented by SEQ ID NO: 7. Recognized a peptide sequence consisting of an amino acid sequence (EADPTGHSY) [C. Trave rsari et al., Journal of Experimental of Medicine, Vol. 176, No. 1 4 5 3 to 1 4 57 pages (1992) 3
- CTL tumor antigens
- MAGE-1 a type of CD-8-positive CTL
- the MAGE family, gp100, tyrosine synthase, carcinoembryonic antigen (CEA), HER2 / neu, and the like are well known. Recognized by CTL is a peptide derived from the tumor antigen protein and presented as a complex with HLA class I molecules
- HLA class I molecules are mainly HLA-A, -B, and -C, and the antigenic peptide presented by binding to them consists of 9 to 0 amino acids, and has a certain structure that differs depending on each HLA molecule. It is known to have the above characteristics.
- the world's most frequent peptide that binds to the HLA-A2.1 molecule is a peptide consisting of 9 to 10 amino acids having Leu at the N-terminus and Leu or Val at the C-terminus. Are the best known.
- peptides that bind to HLA-A24 molecules which are common in Asian peoples including Japanese, are Tyr, Phe, Met, or Trp at the second position from the N-terminal, and Leu, He, Trp, or C-terminal at the C-terminal.
- Peptides consisting of 9 to 10 amino acids with any of the Phe are best known [A. A. Kondo et al., Journal of Immunology, Vol. 155, No. 4, 307-4312 (1995)].
- tumor antigens whose antigenic peptides have been identified include MAGE-1, MAGE-3 for HLA-A1, MAGE_3 for HLA-A2.1, MARTI, tyrosinase, gplOO, HE R2 / neu, CEA, etc.
- MAGE-3 for HLA-Cwl MAGE-3 for HLA-B44
- tyrosinase 8-cathenin for HLA-A24.
- CEA-derived antigenic peptide a peptide consisting of the nine amino acid sequences shown in SEQ ID NO: 34 has been identified as an HLA-A2.1-restricted antigenic peptide [Journal of National Cancer Institute] Ute (Journa 1 of National al Cancer Institute), Vol. 87, pp. 982-990 (1995)].
- HLA-A2.1-restricted antigen peptide a peptide consisting of the nine amino acid sequences shown in SEQ ID NO: 35 or 36 has been identified as an HLA-A2.1-restricted antigen peptide [Journal of Experimental Medicine, Volume 181, Volume 209-211 (1995), Cancer Research (Cancer Research), Volume 54, Volume 1071-1076 (1 994)]. Many of these cells first establish a class I-restricted CTL that recognizes tumor cells, identify tumor antigens that are recognized by these CTLs, and then use genetic engineering methods to construct the smallest unit of tumor antigen protein. And the peptide in the smallest unit has been found based on information on the binding motif to HLA class I molecules [Y.
- HLA class I molecules are classified into several subtypes, but the types of possessed subtypes vary greatly among races, with HLA-A2 being the most common worldwide and 45% of white races Occupy.
- the identification of this HLA-A2-restricted antigen peptide is the most advanced.
- HLA-A2 accounts for 40% of the Japanese population, but its subtype is HLA-A * 0201, which is the same as that of Caucasians, and the rest is A * 0206.
- the binding peptides to these subtypes are different, and the HLA-A2 studied primarily is HLA-A * 0201.
- HLA-A24 accounts for more than 60% of the Japanese, and this HLA-A24 is higher in Asian races than in other races.
- An object of the present invention is to provide the CTL. Disclosure of the invention
- a first aspect of the present invention relates to at least one antigen peptide selected from an HLA-A24-restricted antigen peptide represented by the amino acid sequence set forth in any one of SEQ ID NOS: 1 to 6 and a functional derivative thereof.
- HL A Relates to a CTL that recognizes cells that present a complex with the A24 molecule on the cell surface.
- a second aspect of the present invention relates to an anticancer agent comprising the CTL of the first aspect as an active ingredient.
- a third embodiment of the present invention is directed to the first embodiment, The present invention relates to a method for inducing CTL.
- a fourth embodiment of the present invention provides at least one selected from HLA-A24-restricted antigenic peptide represented by the amino acid sequence of any one of SEQ ID NOs: 1 to 6 and a functional derivative thereof.
- the present invention relates to a CTL inducer comprising an antigen peptide as an active ingredient.
- a fifth aspect of the present invention relates to an HLA-A24-restricted antigenic peptide represented by the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 6 and at least one antigenic receptor selected from functional derivatives thereof. It relates to an anticancer drug containing peptide as an active ingredient.
- a sixth aspect of the present invention relates to at least one antigen peptide selected from an HLA-A24-restricted antigenic peptide represented by the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 6 and a functional derivative thereof.
- an antigen presenting cell that presents a complex of HLA-A24 and HLA-A24 on the cell surface.
- target cell and the term “antigen-presenting cell” are used as cells that present a complex of an HLA-A24-restricted antigen peptide and an HLA-A24 molecule on the cell surface.
- the term “target cell” refers to a cell that is damaged by CTL that specifically recognizes the cell.
- the term “antigen-presenting cell” as used herein refers to a cell having a function of presenting an antigen together with an MHC molecule and inducing the activation of T lymphocytes.
- the MHC molecule is an HLA-A24 molecule.
- the term “cells having antigen-presenting ability” as used herein means cells that can become antigen-presenting cells by forming a complex between an MHC molecule and an antigen peptide present in the cells, and in particular, may be used in particular. Unless otherwise indicated, cells whose MHC molecules are HLA-A24 molecules are used.
- a 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 an anticancer agent 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 a cell that is sensitive to the CTL of the first aspect, using a change generated when the CTL is brought into contact with the test cell as an index.
- the “test cell” in the present specification is a human cell or a cell line derived from an extracorporeal sample such as peripheral blood lymphocytes and tissues.
- the term “sensitive cell” as used herein refers to a tumor that is recognized by CTL and causes cell lysis or cytokine release. Abnormal cells, including cells.
- a tenth aspect of the present invention relates to an agent for detecting a cell sensitive to CTL, comprising the CTL of the first aspect as an active ingredient. Furthermore, the first embodiment of the present invention is capable of recognizing a complex between the antigen peptide and an HLA molecule in the presence of at least one antigen peptide selected from an HLA-restricted antigen peptide and a functional derivative thereof.
- the present invention relates to a method for detecting an HLA molecule, which comprises contacting a CTL with a test cell.
- the present inventors searched for an antigen peptide from many candidate peptides centering on the peptide having the HLA-A24 binding motif in the MAGE-3 protein, which is a tumor antigen, and found that SEQ ID NO: 1 or The use of a peptide consisting of the amino acid sequence represented by 2 can induce CTLs that selectively damage the tumor antigen-expressing cells from peripheral blood mononuclear cells of healthy humans expressing HLA-A24. Revealed.
- SEQ ID NO: 6 present in the 2 / neu protein amino acid sequence
- the tumor antigen is obtained from peripheral blood mononuclear cells of a healthy human expressing HLA-A24.
- the present inventors have clarified that it is possible to induce CTLs that selectively damage expression cells, and have completed the present invention.
- FIG. 1 is a diagram showing the specific cytotoxic activity of effector cells on target cells induced by stimulation of antigen peptide MA321.
- FIG. 2 is a graph showing specific cytotoxic activity of target cells on efjucuta cells induced by stimulation of antigen peptide MA314.
- FIG. 3 is a view showing the specific cytotoxic activity of effector cells induced by stimulation of the antigen peptide MA3-2 against various cancer cells.
- FIG. 4 is a view showing specific cytotoxic activity of the effect cells induced by the stimulation of the antigen peptide MA3-4 on various cancer cells.
- FIG. 5 is a graph showing specific cytotoxic activity of effector cells induced by stimulation of antigen peptide MA1-1 against various target cells.
- a first aspect of the present invention relates to at least one antigen selected from an HLA-A24-restricted antigen peptide represented by the amino acid sequence shown in any one of SEQ ID NOs: 1 to 6 and a functional derivative thereof.
- a CTL that recognizes a cell that presents a complex of a peptide and an HLA-A24 molecule on the cell surface, and the CTL specifically causes cell lysis or cytoforce release reaction on a target cell.
- CTL refers to one that recognizes the antigenicity of the antigenic peptide presented on the HLA-A24 molecule and has a damaging activity, and is not limited by other antigenicity recognition or the like.
- the functional derivative of the HLA-A24-restricted antigen peptide has the ability to form a complex with an HLA-A24 molecule, and the formed complex has a sequence number of 1 to 6 or It means the one recognized by CTL which recognizes the complex of the antigen peptide represented by the amino acid sequence and the HLA-A24 molecule.
- the amino acid sequence of the peptide represented by any one of SEQ ID NOs: 1 to 6 one or several amino acids are deleted or replaced with another amino acid or an amino acid analog.
- amino acid sequence length of the functional derivative is preferably 9 to 10, but is not particularly limited thereto. Absent.
- amino acid analog as used herein means N-acylated, 0-acylated, esterified, acid amide, alkylated, and the like of various amino acids.
- the N-terminus and free amino group of the antigen peptide will be changed to formyl group, acetyl group, t-butoxycarbon (t-Boc) A group or the like may be bonded.
- the C-terminus of the antigen peptide and the free carboxyl group are bound by methyl group, ethyl group, t-butyl group, benzyl group, etc. Is also good.
- the thiol group of cysteine contained in the antigen peptide may be bound to an acetamidomethyl group, a methoxybenzyl group, or the like.
- a functional derivative can be identified by using a CTL that recognizes a complex of the antigenic peptide represented by any one of SEQ ID NOs: 1 to 6 and the HLA-A24 molecule. The following method is mentioned.
- a candidate substance as a functional derivative is mixed with HLA-A24-expressing cells, the candidate substance not bound to the HLA-A24 molecule is washed, and then reacted with CTL. If a candidate substance-specific cytotoxicity, cytokine release, or proliferative response is observed, it can be determined that the compound is a functional derivative.
- a candidate substance is added to cells capable of presenting antigen, and the antigen is incorporated into the cells and processed for an appropriate period of time, and the antigen peptide and HLA molecule complex are displayed on the cell surface. After reacting for the required time, react with CTL. If a candidate substance-specific cytokine release or proliferation reaction is observed, it can be determined that the substance is a functional derivative.
- a nucleic acid encoding an amino acid sequence of a candidate substance is bound to an expression vector capable of presenting a peptide on HLA-A24 molecule on a cell having antigen presenting ability described below, Antigen presentation transformed by the recombinant vector The CTL is reacted with cells having the ability. If a candidate substance-specific cytokine release or proliferation reaction is observed, it can be determined that the substance is a functional derivative.
- Examples of the functional derivative include an amino acid sequence represented by any one of SEQ ID NOs: 1 to 6 in the amino acid sequence of the peptide, in order to strengthen the binding to the HLA-A24 molecule,
- the second amino acid is substituted with an amino acid selected from Tyr, Phe, Met, and Trp characteristic of a peptide that binds to an HLA-A24 molecule, and / or the amino acid at the C-terminal is HLA.
- HLA— HLA- capable of binding to A24 molecule A complex with an A24 molecule is represented by SEQ ID NO: 1 to 6, and a peptide in which CTL recognizes a complex between a peptide described in any of the above and HLA-A24 molecule.
- a peptide represented by an amino acid sequence in which one or several amino acids of the amino acid sequence described in any one of SEQ ID NOs: 1 to 6 are substituted the similarity of each amino acid to be substituted and the side chain Among the peptides substituted with an amino acid or amino acid analog, those which have a binding ability to an HLA-A24 molecule and whose complex with the HLA-A24 molecule is recognized by the CTL of the present invention are also exemplified.
- amino acids having similar side chains include glycine (G 1) and alanine (Al a); valine (Va 1), isoleucine (I 1 e), leucine (Leu) and methionine (Me t); Asn) and glumin (G1n); aspartic acid (Asp) and gluminic acid (G1u); serine (Ser) and threonine (Thr); lysine (Lys) and arginine (Ar g); phenylalanine (Phe) and tyrosine (Tyr).
- a peptide represented by the amino acid sequence of SEQ ID NO: 24 in which the sixth amino acid (Va 1) from the N-terminus is substituted with I 1 e And a peptide represented by the amino acid sequence of SEQ ID NO: 46 in which the eighth amino acid (Gly) is substituted with Ala.
- the amino acid sequence described in SEQ ID NOS: 53 and 55 in which the amino acid (Va 1) at the sixth position from the N-terminus has been substituted with I 1 e and Met, respectively.
- the peptide represented or the fourth amino acid (Cy s) from the N-terminus is substituted with a thiol group of Cy s and 4,5-dihydroxy-2-cyclopenten-1-one (4,5-dihydroxy—2-eye) 1 open ten-1 - ⁇ e: hereinafter abbreviated as DHCP).
- the fourth amino acid from the N-terminus is the Cys thiol group and N-Ethylmaleimid, dithiothreitol (Dithi 0 threit 01), and 4-vinylpyridine (4 — It may be a product obtained by reaction with Viny l pyr idi ne).
- the functional derivative may be a substitution of an amino acid having no similar side chain, 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 complex with the molecule is SEQ ID NO: 1 to 6, and the peptide described in any of the above and the peptide recognized by CTL which recognizes the complex with the HLA-A24 molecule.
- a peptide represented by the amino acid sequence of any one of SEQ ID NOs: 39, 40, 42 to 45, and 48 examples include the peptides represented by the amino acid sequences of SEQ ID NOs: 26, 27, 30 and 32.
- the amino acid at the position that can be substituted for Ala in the above peptide may be able to be substituted with another amino acid or an amino acid analog different from the original amino acid.
- the fourth amino acid of the antigenic peptide represented by the amino acid sequence set forth in SEQ ID NO: 5 is an amino acid other than an acidic amino acid such as Asa and G1u in addition to Ala.
- it can be substituted with an amino acid analog.
- a peptide substituted with Ser or Gly represented by the amino acid sequence of SEQ ID NO: 51 or 52 can be mentioned.
- Examples of the dimer of a peptide formed by a disulfide bond or the like include a dimer of a peptide represented by the amino acid sequence of SEQ ID NO: 5 formed by a disulfide bond via a thiol group of cysteine.
- functional derivatives of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 include those recognized by CTL induced using the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1. MAGE-3 and other peptides derived from MAGE family proteins are also included.
- functional derivatives of the peptide represented by the amino acid sequence of SEQ ID NO: 2 include MAGE-recognized by the CTLs induced by the peptide represented by the amino acid sequence of SEQ ID NO: 2. 3 and other peptides derived from MAGE family proteins.
- Functional derivatives of the peptide represented by the amino acid sequence shown in SEQ ID NO: 3 include MAGE-1 which is recognized by CTL induced by the peptide represented by the amino acid sequence shown in SEQ ID NO: 3. And peptides derived from MAGE family proteins.
- Functional derivatives of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 include CEA and non-specific, which are recognized by CTLs induced using the peptide consisting of the amino acid sequence shown in SEQ ID NO: 4. Peptides derived from non-specific cross-reacting antigens (NCA) are also included.
- Functional derivatives of the peptide represented by the amino acid sequence represented by SEQ ID NO: 5 include CEA and NCA recognized by CTLs induced by the peptide represented by the amino acid sequence represented by SEQ ID NO: 5. Peptides derived therefrom are also included.
- the peptide represented by SEQ ID NO: 1 or 2 and the functional derivative thereof are referred to as MAGE-3 antigen peptide and the peptide represented by SEQ ID NO: 3 and the functional derivative thereof are referred to as MAGE-1 antigen.
- MAGE-3 antigen peptide the peptide represented by SEQ ID NO: 3 and the functional derivative thereof are referred to as MAGE-1 antigen.
- SEQ ID NO: 4 or The peptide represented by 5 and its functional derivative are collectively referred to as CEA antigen peptide
- the peptide represented by SEQ ID NO: 6 and its functional derivative are collectively referred to as HER2Zneu antigen peptide.
- the “antigen peptide” used in the present specification may be any of MAGE-3 antigen peptide, MAGE-1 antigen peptide, CEA antigen peptide and / or HER2 / neu antigen peptide unless otherwise specified. Indicates that this is the selected peptide.
- Antigen peptides and derivatives thereof can be prepared by an organic chemical method by force coupling of a liquid phase or solid phase amino acid. It can also be prepared using recombinant DNA technology using a nucleic acid encoding the amino acid sequence of the antigenic peptide. The peptide obtained by the recombinant DNA technology may be modified as necessary by using an appropriate organic chemical or biochemical technique.
- CTLs induced using MAGE-3 antigen peptide are tumor cells positive for both HLA-A24 and MAGE-3
- CTLs induced using MAGE-1 antigen peptide are HLA-A24 and Tumor cells positive for both MAGE-1 and tumor cells positive for both induced by using 2428 antigen peptide and A28-24 and CEA were used for HER2Zn eu antigen peptide.
- the induced CTL can selectively injure HLA-A24 and HER2Zneu-positive tumor cells, respectively, so that it can be used as a cell medicine for tumors and for the detection of such CTL-sensitive cells. it can.
- a second aspect of the present invention relates to an anticancer agent comprising the CTL of the first aspect as an active ingredient.
- the anticancer agent is provided in a form in which the CTL is suspended in a pharmaceutically acceptable diluent.
- the diluent mentioned here is, for example, a medium, physiological saline, or phosphate buffered saline suitable for storing the CTL.
- a medium such as RPMI or AIM-V is generally used.
- a pharmaceutically acceptable carrier may be added to the anticancer agent for the purpose of stabilization.
- the carrier mentioned here is human serum albumin or the like.
- the carcinostatic agent may be administered with a syringe for administration to a human Bok, adult human normal CTL numbers as those Rino dose, CTL 1 kind per 0 6-1 0 1 (1 and The above range is only a guide and is not limited to the above.Since the active ingredient CTL is derived from the human being administered, the toxicity of the anticancer agent is particularly recognized. Absent.
- the anticancer agent of the present invention is administered to a cancer patient having a tumor cell expressing an antigen peptide capable of recognizing CTL contained as an active ingredient.
- MAGE is initially administered to a melanoma patient. It is an antigen identified from malignant tumor cells, but it has been subsequently confirmed that it is expressed at a frequency of about 10 to 50% of cancer patients other than melanoma.
- MAGE-K MAGE-2 or MAGE-3 expressing tumor cells are derived from melanoma, lung cancer, breast cancer, cervix cancer, stomach cancer, esophagus cancer, bladder cancer, etc. B. Gougler et al., Journal of Experimental Medicine, Vol. 179, pp. 921-930 (1994)].
- the anticancer agent containing the CTL of the first aspect of the present invention as an active ingredient using the MAGE-3 antigen peptide as an active ingredient is positive for both HLA-A24 and MAGE-3. It is useful for treating patients with the above cancers consisting of tumor cells.
- anticancer agent when used, it may be used in combination with other anticancer agents, but it is not preferable to use an anticancer agent which acts as an immunosuppressant.
- a third aspect of the present invention relates to the method for inducing CTL of the first aspect.
- the CTL uses at least one antigen peptide selected from the HLA-A24 binding antigen peptide represented by the amino acid sequence of any one of SEQ ID NOs: 1 to 6 and a functional derivative thereof. Can be guided by
- the CTL When the CTL is induced in vitro, it can be induced using, for example, a sample extracted from a living body expressing HLA-A24.
- the extracorporeally extracted sample in this specification includes, in addition to blood, lymph nodes, spleen, and various other organs extracted by surgery or the like, and lymphocytes and infiltrating lymphocyte cells present in these samples are preferably used. Is done.
- antigen stimulation is repeatedly performed on lymphocytes obtained from peripheral blood mononuclear cells prepared from HLA-A24-positive human blood (Peripheral monolide 1 s; hereinafter, abbreviated as PBMC).
- PBMC peripheral blood mononuclear cells prepared from HLA-A24-positive human blood
- antigen stimulation for example, use antigen-presenting cells, etc., which present antigen-peptide on the cell surface as a complex with HLA-A24 molecule on cells having antigen-presenting ability prepared from the same blood prepared from lymphocytes. This can be implemented.
- the induced CTL can be proliferated by further stimulation with an anti-CD-3 antibody or various stimuli in the presence of an antigen peptide.
- CTL induction can be performed by mixing antigen presenting cells with lymphocytes 1 prepared from PBMC at a ratio of 0.01 to 1.
- the antigen peptide is added to PBMC in a cell culture medium, and 1 n per antigen peptide is used.
- CTL induction can be performed by adding gZm 1 to 100 g / m 1, preferably 1 Ong Zml to 1 gZm 1.
- the induced CTLs can be maintained as stable cytotoxic lymphocytes by cloning.
- the induced CTLs can be proliferated by stimulation with antigens, various cytokines, and anti-CD3 antibodies.
- the specific CTLs induced are the antigen-specific increase of CTL proliferation or the antigen-induced increase in CTLs or target cells, which can be measured by the toxicity to the target cells labeled with the radioactive substance and the antigen peptide that induced the CTLs.
- GM-CSF, IFN specifically released-Detectable by measuring the amount of cytokines such as y Can be.
- it can also be directly confirmed by using an antigen peptide-conjugate labeled with a fluorescent dye or the like.
- CTL is brought into contact with a first fluorescent marker coupled to a CTL-specific antibody, and then brought into contact with an antigen-beptide MHC complex coupled with a second fluorescent marker, and then double-labeled.
- the presence of cells can be determined by FACS (fluorescence-activated cell sorting) analysis.
- the induction of CTLs in vivo can be carried out, for example, by administering an antigen-presenting cell presented on the cell surface as a complex of an antigen peptide and an HLA-A24 molecule to a living body.
- the dose per adult is usually 10 4 to 10 9 .
- CTLs specific to the peptide can be induced by mixing the antigenic peptide with an appropriate adjuvant and administering the mixture to a living body.
- Adjuvants include: (1) Freund's complete adjuvant, (2) Freund's incomplete adjuvant, (3) inorganic gels such as aluminum hydroxide and alum, (4) surfactants such as lysolecithin and dimethyloctadecyl ammonium bromide, ⁇ ⁇ Polyanion such as dextran sulfate and poly IC; ⁇ such as muramyl peptide and tuftsin; and ⁇ monophosphoryl lipid (MPL) A manufactured by Ribi. Not limited. For the induction of CTL in a living body, a mixture of an antigen peptide and an adjuvant, or a helper T cell antigen peptide restricted to MHC class 11 may be mixed and administered.
- the antigen peptide may be covalently bonded to a higher fatty acid or a helper T cell antigen peptide via an appropriate linker for stabilization in a living body, and may be administered.
- the dose per adult is 0.1 zgZkg to 10 mgZkg per antigen peptide, preferably 1 g / kg to 1 mg / kg, more preferably 1 g / kg per antigen peptide concentration. ⁇ L 0 O ⁇ gZkg.
- antigen peptides and H A complex with the LA-A24 molecule can also be used.
- the HLA-A24 molecule does not need to be a natural HLA-A24 molecule, but may be a fragment that essentially preserves the binding to the antigenic peptide.
- These fragments can be prepared, for example, by proteolysis of natural HLA-A24 molecules or by recombinant DNA technology.
- This complex can be stabilized by coexisting yfi 2 -microglobulin and an antibody that recognizes the complex.
- a fourth aspect of the present invention relates to a method for preparing at least one antigen peptide selected from the HLA-A24-restricted antigen peptide represented by the amino acid sequence of any one of SEQ ID NOs: 1 to 6 and a functional derivative thereof. It relates to a CTL inducer as an active ingredient.
- the CTL inducer is supplied in the form of an antigenic peptide alone or as a mixture with another molecule (helper T cell antigenic peptide and amino acid or adjuvant) suspended in physiological saline or phosphate buffered saline.
- the antigen peptide may be a covalent complex with a higher fatty acid or a helper T cell antigen peptide or a complex with an HLA-A24 molecule.
- the inducer contains an antigen peptide in an amount of 0.01 to 100% by weight, preferably 0.1 to 95% by weight, per one kind of the antigen peptide.
- the CTL inducer may be used as an additive to a culture medium for growing the CTL of the present invention in vitro, or for diagnosing an immunization state using T lymphocyte proliferation activity or delayed skin reaction as an index.
- a culture medium for growing the CTL of the present invention in vitro, or for diagnosing an immunization state using T lymphocyte proliferation activity or delayed skin reaction as an index.
- the amount used is 1 ng Zm 1 to 100 zg / m 1 per antigen peptide, preferably 1 ng / m 1 to 1 as the peptide concentration in the medium. g / m1.
- the present invention relates to an anticancer drug containing a peptide as an active ingredient.
- the anticancer agent comprises 1) an antigenic peptide alone, 2) a mixture of an antigenic peptide and a pharmaceutically acceptable carrier and / or a diluent, or 3) a supplement as described in 1) or 2) above if necessary.
- the carrier used here is, for example, human serum albumin
- the diluent is, for example, a phosphate buffer, distilled water, physiological saline, or the like.
- the adjuvant includes the pharmaceutically acceptable adjuvants and the like.
- the anticancer agent When the anticancer agent is administered to humans, it may be administered with a syringe, or may be administered by percutaneous absorption through a mucous membrane by a method such as spraying.
- the anticancer agent contains 0.01 to 100% by weight, preferably 0.1 to 95% by weight, of the antigen peptide per antigen peptide.
- the dose per adult is 0.1 l ⁇ gZkg to 10 mgZkg, preferably 1 ⁇ g / kg to 1 mg / kg, more preferably 1 gZkg to 100 zgZkg per antigen peptide as the peptide concentration.
- the toxicity of the peptide is not particularly observed.
- the anticancer agent when used, it may be used in combination with other anticancer agents, but it is not preferable to use an anticancer agent which acts immunosuppressively.
- a sixth aspect of the present invention relates to an HLA-A24-restricted antigen peptide represented by the amino acid sequence of any one of SEQ ID NOs: 1 to 6 and at least one antigen peptide selected from functional derivatives thereof.
- the present invention relates to an antigen presenting cell which presents a complex with HLA-A24 molecule on its cell surface.
- antigen presenting cell in the present specification means at least selected from MAGE-3 antigen peptide, MAGE-1 antigen peptide, CEA antigen peptide and Z or HER2Zn eu antigen peptide unless otherwise specified. 1 shows an antigen-presenting cell that presented one antigen peptide.
- the antigen presenting cells can be used for inducing the CTL of the present invention.
- the antigen presenting cells can be prepared by using cells having antigen presenting ability.
- Cells that have the ability to present antigen include, for example, macrophage II B cells and dendritic cells (DC), which can present a complex of an antigen peptide and HLA-A24 molecule on the cell surface. And other white blood cells.
- DC has a large amount of antigen present per cell, and is a cell surface molecule necessary for antigen presentation (costimulatory signal (c0-stimu 1 at ory signal) molecules such as CD80 and CD86) Cells with high antigen expression ability due to high expression level It is particularly suitable as
- the cells having the antigen-presenting ability can be prepared from the in vitro extracted sample in the present specification.
- DC is 1) isolated and purified from PBMC based on several cell surface markers, 2) derived from monocytes by GM-CSF and IL-4, 3) CD- It can be prepared by any method such as induction from 34 positive cells with cytokines such as GM-CSF, TNF-H, and SCF.
- HLA-A24 There is a method of loading an antigen peptide onto a molecule.
- an antigen peptide irrelevant to the present invention is already presented on the HLA-A24 molecule like the cell prepared by the method 1) in the method for preparing a cell having the antigen presenting ability described above.
- the cells to be obtained may be subjected to an acid treatment or the like before loading with the antigen peptide to remove the peptide present on the HLA-A24 molecule that cannot induce the CTL of the present invention.
- the cells having the antigen-presenting ability may be cells loaded with a single antigen peptide, or may be loaded with several kinds of antigen peptides per cell.
- an expression vector capable of presenting the antigen peptide to the cells include commercially available plasmids such as pcDNA3, pMQMneo.pCEP4, and at least one antigen peptide to be displayed on the cell surface by recombinant DNA technology.
- the expression vector may have a gene encoding an appropriate amino acid sequence added to both ends of the antigen peptide to be displayed on the cell surface so that protease is efficiently decomposed in the cell.
- retrovirus vectors and adenovirus vectors are also preferably used.
- the antigen presenting cells are non-proliferative.
- irradiation with X-rays or the like or treatment with an agent such as mitomycin (mitomicin) may be performed.
- mitomycin mitomycin
- a seventh aspect of the present invention relates to a CTL inducer comprising the antigen-presenting cell 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, physiological saline, or phosphate-buffered saline suitable for storing the cells.
- RPMI, AIM-V, X-VI VO10 and the like are generally used.
- Antigen presenting cells to the inducing agent antigen-presenting cells one per 10 3 to 5 X 10 6 cells / ml, preferably is contained 10 4 to 10 6 ZML.
- the CTL inducer is used not only as an additive to a medium for growing the CTL of the present invention in vitro, but also for diagnosing an immunization state using T lymphocyte proliferation activity as an index. be able to.
- a medium for growing the CTL of the present invention in vitro, but also for diagnosing an immunization state using T lymphocyte proliferation activity as an index. be able to.
- An eighth aspect of the present invention is a carcinostatic agent comprising the antigen-presenting cell of the sixth aspect as an active ingredient.
- the anticancer agent is provided in a form in which the antigen-presenting cells are suspended in a pharmaceutically acceptable diluent.
- the diluent mentioned here is a medium, a phosphate buffer, a physiological saline, or the like suitable for storing cells.
- Examples of the medium generally include RPMI, AIM-V, and XVIVO10.
- a pharmaceutically acceptable carrier may be added to the anticancer agent for stabilization.
- the carrier mentioned here is human serum albumin or the like.
- the ⁇ cancer agent antigen-presenting cells, antigen presenting cells one per 10 5-10 8 Zm 1, preferably 5x l 0 5 ⁇ 5x l 0 7 amino Roh m 1 Ru is contained.
- the anticancer agent When the anticancer agent is administered to humans, it can be administered with a syringe, and the dose per adult is usually 10 4 to 10 9 per antigen presenting cell as the number of antigen presenting cells. Note that the above range is only a guide and is not limited to this.
- the antigen-presenting cells as the active ingredient are derived from the human being administered, no toxicity of the anticancer agent is observed.
- a ninth embodiment of the present invention relates to a method for detecting a CTL-sensitive cell, using a change generated when the CTL of the first embodiment is brought into contact with a test cell as an index.
- the change caused by contact of the CTL with a sensitive cell in the test cell includes, for example, target cell lysis by the CTL, release of cytodynamic force, or proliferation of the CTL.
- cytoforce release can be performed by measuring the release amount of GM-CSF, TNF, IFN- ⁇ , etc. from CTLs or test cells.
- CTL proliferation can be carried out by measuring the amount of 3 H-thymidine incorporated into cells, or by measuring the number of CTL cells by microscopic observation.
- the tumor cells present in the test cells are positive for both HLA-A24 and MAGE-3 or positive for both HLA-A24 and MAGE-3 related antigens, and positive for both HLA-A24 and MAGE-1.
- Tumor cells positive for both HLA-A24 and MAGE-1 related antigens tumor cells positive for both HLA-A24 and CEA or positive for both HLA-A24 and CEA related antigens, and / or ⁇ 11 ⁇ -8 It is possible to detect tumor cells positive for both 24 and HER2 / neu or positive for both HLA-A24 and HER2Zneu-related antigen.
- a tenth aspect of the present invention relates to an agent for detecting a cell sensitive to the CTL of the first aspect, comprising the CTL of the first aspect as an active ingredient.
- the detecting agent is supplied in a form suspended in a medium, physiological saline, or phosphate buffered saline suitable for storing the CTL.
- a medium a medium such as RPMI, octane or — ⁇ 1 ⁇ 100 is generally used. Serum albumin or the like may be added to the medium or buffer for the purpose of stabilizing CTL.
- the on the detected polishes the CTL of the first aspect CTL 1 kind per 0 4 to 1 0 to 8 Zml contained.
- the detection agent is used not only for detection of CTLs susceptible to the CTL of the present invention in test cells, but also for HLA typing for identifying whether HLA expressed in test cells is HLA-A24. Can also be used.
- a CTL capable of recognizing a complex of an antigen peptide and an HLA molecule in the presence of at least one antigen peptide selected from an HLA-restricted antigen peptide is provided.
- the present invention relates to a method for detecting an HLA molecule, which is brought into contact with a test cell.
- the HLA-A24 molecule on the surface of the test cell can be detected using, as an index, a change caused by contacting the test cell with the CTL of the first embodiment.
- the change caused by the contact of the test cell with the CTL includes, for example, lysis of the target cell by CTL, release of cytodynamic force, or proliferation of CTL.
- Target cell lysis can be detected by, for example, measuring the amount of radioisotope released from the test cells divided by the amount of fluorescent dye when the test cells are mixed with CTL after being labeled with radioisotope or a fluorescent dye.
- Detection of cytoforce in release can be carried out by measuring the release amount of GM-CSF, TNF, IFN-y, etc. from CTL or test cells.
- CTL proliferation can be carried out by measuring the amount of 3 H-thymidine incorporated into cells, or by measuring the number of CTL cells by microscopic observation or the like.
- the reaction solution is selected from the test cells and an antigen peptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 6 and a functional derivative thereof so that the final concentration is 0.01 to 50 gZm1.
- the CTL and the test cell may be contacted in the presence of at least one antigen peptide, and the CTL of the first embodiment, which recognizes a complex of the HLA-A24 molecule and the antigen peptide.
- the antigen peptide used in the present method is at least 10 HLA-A24 restricted selected from the peptide represented by the amino acid sequence described in any one of SEQ ID NOs: 1 to 6 or a functional derivative thereof.
- various HLA molecules can be detected by using various HLA-restricted antigen peptides.
- HLA-A2 molecules can be detected by using an HLA-A2-restricted antigen peptide.
- the HLA-A2-restricted antigen peptide include a peptide derived from MAGE-3 represented by SEQ ID NO: 37 and a peptide derived from an influenza matrix peptide represented by SEQ ID NO: 38.
- HLA-A2 is classified into 10 or more subtypes including A * 0201, A * 0206 and A * 0207. Since HLA-restricted antigen peptides differ between subtypes, for example, when using CTLs as anticancer agents, it is important to determine the subtype of HLA molecules.
- This method can classify subtypes, which were not possible with the detection (typing) of HLA molecules using anti-HLA antibodies. For example, when a peptide having the amino acid sequence of SEQ ID NO: 37 is used, A * 0201-restricted CTL can be induced. By using this CTL in combination with the peptide, the HLA-A Up to two subtypes can be determined. Note that this method is superior to the DNA typing method using DNA extracted from test cells in that it can detect HLA molecules actually expressed on the cell surface.
- Example 1 Preparation of MAGE-3 Antigen Peptide-Specific CTL (1)
- the CTL in this example was prepared by an induction method using Staphylococcus aureus Cowan-1 (SAC-I).
- Blood was collected by blood sampling from healthy individuals having HLA-A24, and leukocyte fractions were collected, and PBMCs were separated according to the following separation method. That is, the collected blood was diluted about 2-fold with RPMI 1640 medium, layered on a Ficoll-Paque separation solution (manufactured by Pharmacia), and centrifuged at 500 xg for 20 minutes at room temperature. The PBMCs in the middle layer were collected with a pipette, washed, and suspended in a stock solution containing 90% fetal calf serum (FCS, Intergen) and 10% dimethyl sulfoxide (Sigma). Stored in liquid nitrogen.
- FCS fetal calf serum
- Sigma dimethyl sulfoxide
- PBMC prepared in (2) was thawed, it was suspended in 5H-RPMI so that the cell concentration was 4 ⁇ 10 6 Zml.
- RPMI 1640 medium contains human AB serum (manufactured by Avain Science) at a final concentration of 5% (v / v) and non-essential amino acids (manufactured by Gibco BRL) at a final concentration of 0.1 lmM.
- the cell suspension 25ml bound anti CD 4 antibodies T-0.99 flasks (AIS MicroCELLector®, Applied I made mu capacitors Iensu Co.) After reaction for 1 hour at room temperature placed in, to recover the non-adherent cells 2 XI 0 6 pieces
- the suspension was suspended in 5H-RPMI so as to be / m1.
- This suspension was prepared using any of the peptides MA3-1, MA3-2, MA3-4, MA3-6, and MA3-7 by the method described in Example 8, (1) below.
- Individually mix equal amounts of the five antigen-presenting cells for primary stimulation in 1 treatment add IL-17 (Genzym) to a final concentration of 15 ng / ml, and add each well of a 24-well culture plate.
- IL-10 manufactured by R & D
- IL-7 was added to a final concentration of 1 OngZml.
- TISI As target cells for measuring cytotoxic activity, TISI (WSNO 9042), an EBV transform B cell expressing HLA-A24, was used.
- MA3-1, MA3-2, and MA3 prepared in (1) the day before the measurement of TISI cells.
- TISI (+) 5 types of media containing 10 g / m1 of any of the peptides of MA4, MA3-6 and MA3-7 individually
- TISI (+) 5 types of media containing 10 g / m1 of any of the peptides of MA4, MA3-6 and MA3-7 individually
- TISI cultured in the medium is referred to as TISI (+)
- TISI (-) the cells were cultured overnight in a medium containing no peptide
- Equation 1 (Maximum release value-minimum release value)] X 100 (Equation 1)
- the minimum release value is the amount of 51 Cr in a well containing only target cells and K562 cells. Shows the amount of spontaneous release of 51 Cr.
- the maximum release value is determined by adding Triton X-100 detergent to target cells and destroying the cells. Shows the amount of 51 Cr released at the time. The results are shown in Table 2.c Table 2 Specific cytotoxic activity
- EZT indicates the ratio of effector cells to target cells.
- effector cells obtained by induction with MA32 showed antigen-peptide-specific cytotoxicity to TISI (+) to which MA3-2 peptide was added.
- the CTL in this example was prepared by an induction method using KLH.
- antigen peptides five kinds of peptides, MA3-1, MA3-2, MA3-4, MA3-6, and MA3-7, prepared in (1) of Example 1 were used.
- Blood samples were collected from healthy persons carrying HLA-A24, and PBMCs were separated according to the following separation method. That is, the collected blood is suspended in the same volume of RPMI 1640 medium. The mixture was overlaid on a ficoll-one-pack separated solution, and centrifuged at 500 xg for 25 minutes at room temperature. The middle layer of PBMC was collected with a pipette, placed in a 15 ml centrifuge tube, and washed three times with RPMI 1640 medium. Next, cells were suspended in 5H-RPMI so that the final cell concentration was 4 ⁇ 10 6 1.
- the PBMC prepared in (1) was mixed with an equal amount of 5H-RPMI to which 20 Zml of the peptide was added, followed by standing in a C02 incubator at 37 ° C for 2 to 3 hours.
- a KLH (manufactured by Calbiochem) solution and an IL-7 solution were added to a final concentration of 5 g / mK and 25 ng / m1, respectively.
- the cell suspension is dispensed at 2m 1 to 24 Ueru culture play Bok 2 Ueru were cultured in C0 2 incubator within one 37 ° C.
- 5H-RPM containing 1 000 I UZm1 of rIL-2 was added at 60 ⁇ 1 (final rIL-12 concentration 30 IU / m1).
- the cells After culturing for one week, the cells are collected by centrifugation, and suspended in 5H-RPI so that the cell concentration becomes 5 ⁇ 10 5 cells / ml.
- This suspension was prepared using any one of the peptides MA3-1, MA3-2, MA3-4, MA3-6, and MA3_7 in Example 8 described later.
- TISI (-) and five types of TISI (+) prepared in the same manner as (4) in Example 1 as labeled target cells WiDr (colorectal cancer) positive for both MAGE-3 and HLA-A24 Cell line), TE11 (esophageal cancer cell line), and MRKnu1 (breast cancer cell line).
- WiDr colonal cancer
- TE11 esophageal cancer cell line
- MRKnu1 breast cancer cell line
- MAGE-3 positive, HLA-A24 negative cancer cell line KAT0-III gastric cancer cell line
- WiDr, TE11, MRKnuU and KAT0-III was 1 hour mixed with Na 2 61 C r 0 4 in solution in 37 ° C of each 5 X 1 0 6 cells to 200 / Ci, then 1 0% Rei_3 Washed with 1 ⁇ ?] 640 culture solution for labeling with 51 Cr.
- the cytotoxic activity was measured using the effector cells prepared in (2) and the target cells prepared in (3).
- TISI (1) and TISI (+) were used as target cells, measurement was performed in the same manner as in Example 1, (5).
- the reaction time between one effector cell and the target cells was 4.5 hours.
- the effector-cell suspension of (2) above was added to each cell of a 96-well culture plate at a rate of 100 1 / well (4 ⁇ 10 5 Aliquots / ml), and add a 100/1 cell suspension containing 5 x 10 3 51 Cr labeled target cells and 1.5 x 10 5 562 cells. added. After centrifugation for 1 minute at 400 X g, 4.
- EZT indicates the ratio of effector-cell to target cell
- ND indicates that the ratio was not measured.
- FIGS. 1 and 2 show the specific cytotoxicity curves of the effector cells induced by ⁇ ⁇ ⁇ 3-2 or ⁇ 3-4 against ⁇ ISI ( ⁇ ) and TISI (+) in various ⁇ .
- FIGS. 3 and 4 show the specific cytotoxicity curves of cells with various EZTs.
- FIG. 1 shows the results obtained using an effector cell induced by MA 3-1
- FIG. 2 shows the results obtained using effector cells induced by MA 3-1.
- the squares ( ⁇ ) show the results using T I S I (+) as the target cells, and the diamonds ( ⁇ ) show the results using T I S I (—).
- FIG. 3 shows the results obtained using the effector cells induced by MA 2-2
- FIG. 4 shows the results obtained using the effector cells induced by MA 3-1.
- the squares ( ⁇ ) show the results using WiDr as the target cells
- the diamonds ( ⁇ ) show the results using TE-11
- the white circles ( ⁇ ) show the results using MRKnu1.
- cytotoxic activity shown in Fig. 3 and Fig. 4 indicates that the class I antibody (W6 / 32 ) Was inhibited.
- effector cells induced by MA3-2 or MA3-4 did not show cytotoxicity to KATO-III, a MAGE-3 positive and HLA-A24 negative cancer cell line.
- CTL in this example was prepared by a CTL induction method using KLH.
- the amino acid sequence of MAGE-1 protein consisting of 309 amino acids [Molecular Immunology, Vol. 31, Vol. 14, Nos. 1423-1430 (1994)] Similar to 1), the search was carried out focusing on the sequence having the HLA-A24 binding motif structure, taking into account the types of amino acids at other positions. As a result, the five peptides shown in Table 4 were selected as candidate antigen peptides.
- One effector cell was prepared in the same manner as described above, and the cytotoxic activity against various target cells was measured.
- the target cells used were TISI (1), MA1-1, MA1-2, MA1-3, MA1-4, and MA1-5.
- FIG. 5 shows specific cytotoxicity curves of various EZTs against various target cells of one effector cell induced by MA1-1.
- a black circle ( ⁇ ) indicates NUGC-3 as a target cell
- a black square (country) indicates TE11
- an inverted triangle ( ⁇ ) indicates KATO III
- a diamond ( ⁇ ) indicates Raji
- Triangles ( ⁇ ) show the results using TISI (—).
- the cytotoxic activity against the GC-3 cell line and the TE-11 cell line was inhibited by the coexistence of a class I antibody (W6Z32) in the reaction system.
- CTL in this example was prepared by the CTL induction method using SAC-I in the same manner as in Example 1.
- the numbers in the sequence numbers indicate the amino acid sequence of each peptide.
- SEQ ID No. in the Sequence Listing is shown below.
- the number of the term at the position in the CE A protein indicates the number of amino acids from the N-terminus in the CE A protein.
- the symbol in the name column indicates the name of the peptide named by the present inventors.
- Example 8 Using any of the stored PBMC, CE-K CE-3, CE-4, and CE-5 peptides prepared in the same manner as (2) in Example 1, and using the method described in (1) in Example 8 below. Using the prepared antigen-presenting cells for initial stimulation by treatment with SAC-1 and any peptide of CE-1, CE-3, CE-4, CE-5, the following Example 8
- the cells were cultured in a medium containing any of the peptides CE-1, CE-3, CE-4, and CE-5 in the same manner as in (4) of Example 1.
- the obtained five kinds of TISI (+) and gastric cancer cell line MKN-45 (HLA-A24 and CEA positive) were used.
- Each of the above cells was labeled with 51 Cr in the same manner as in (4) of Example 1 on the day of measuring the cytotoxic activity.
- Table 6 shows the results.
- Table 6 Peptide name E / T TISI (+) MKN-45
- EZT indicates the ratio of efek yuichi cells to target cells.
- the CTL in this example was prepared by an induction method using dendritic cells (DC) for antigen presentation.
- DC dendritic cells
- Example 2 (2) After thawing the PBMC prepared in Example 1 (2), the cell concentration 2 XI 0 7 or Zm
- the suspension was suspended in PBS containing 1 human AB serum at 4 ° C (hereinafter, abbreviated as 1H-PBS) at 1 ° C.
- 2 X 1 0 7 or Zm 1 bead (Dynabeads M450, manufactured by Dynal Co.) conjugated anti-CD 8 antibody was washed with 1 H- PBS respect PBMC with 14 / z 1 was added, in 1 hour 4 ° C After the reaction, non-adherent cells were removed. After removing non-adherent cells were suspended in 1 H- PBS of 0.
- the beads for cell dissociation of 0. 1 m 1 (DETACHaBEAD, Dynal Was added. After mixing at room temperature for 1 hour, the dissociated cells were collected and washed with 1 H-PBS. The washed cells are suspended in 5H-RPMI at a concentration of 2 ⁇ 10 6 cells / ml, and prepared using the peptide of either CE-2 or CE-3 according to the method described in Example 8, (3). It was mixed in equal amounts with the DC suspension.
- the IL one 7 to a final concentration of 1 OngZml to this suspension pressurized example, 48 to each Ueru of Weru culture plates 0..5 m 1 by the dispensed 37 ° C C 0 2 Inkyube in one coater Incubate. The next day, 50 1 of 5 H RPMI containing 100 ng / ml of IL-11 was added.
- the cytotoxic activity against TISI (10) and TISI (-) was measured for each effector cell in each well.
- Effector cells that showed cytotoxic activity were grown individually using anti-CD3 antibodies or each of the peptides used for antigen stimulation.
- the proliferated cells were suspended in 5 H-RPMI, and the cytotoxic activity was measured by the following method (3).
- TISI (1) As target cells, use TISI (1), CE-2 or CE as in Example 4 (3).
- Two types of TISI (+) were prepared by culturing one to three in a supplemented medium. MKN-45 was treated with 10 OUZml of IFN-48 for 48 hours at 37 ° C.
- E / T indicates the ratio of effector cells to target cells.
- effector cells obtained by induction with either C C-2 or C ⁇ -3 peptides showed antigen-peptide-specific cytotoxicity against TISI (+) and ⁇ -45. However, it was revealed that it was a C ⁇ -specific CTL.
- Example 6 1 ⁇ 2 11 6 1 Antigen peptide specific (preparation of Ding (1)
- CTL in this example was prepared by the CTL induction method using SAC-I in the same manner as in Example 1.
- the numbers in the SEQ ID No. column indicate the SEQ ID numbers in the sequence listing showing the amino acid sequences of the respective peptides.
- the number of the term at the position in the HER2Zneu protein indicates the number of amino acids from the N-terminus in the HER2Zneu protein.
- the symbol in the name column indicates the name of the peptide named by the present inventors.
- PBMC Stored PBMC prepared in (2) of Example 1 or any of HE-1 to HE-5 Using an antigen-presenting cell for initial stimulation by SAC-1 treatment prepared by the method described in Example 8 (1) described below and a peptide of any of HE-1 to HE-5 prepared in Example 8 (1) 2) Using five types of plates containing any one of the antigen-presenting cells prepared by the method described in the above, stimulated with any of HE-1 to HE-5 peptides in the same manner as (3) in Example 1. Five types of effector cells were prepared.
- TISI (+) cultured in a medium containing any of HE-1 to HE-5 peptides in the same manner as in (4) of Example 1
- the ovarian cancer cell line SKOV3 HLA-83 and 11 £ 1 ⁇ 2 11 eu positive
- HLA-A24 transformant S KO V-A24 HLA-A24 and HERSZn eu positive
- the cells were labeled with 51 Cr in the same manner as in (1) of Example 1 on the day of measuring the cytotoxic activity.
- EZT indicates the ratio of Effecta-to-target cells.
- the CTL in this example was prepared by an induction method using dendritic cells (DC) for antigen presentation.
- DC dendritic cells
- TISI (1) and TISI (+) cultured in a culture medium supplemented with HE-1 were prepared in the same manner as in Example 6, (3).
- SKOV3 and SKOV3-A24 were treated with 10 OUZml of IFN-a for 48 hours at 37 ° C.
- the four target cell is cytotoxic activity measured day was 51 C r mark 5 Hara in the same manner as in Example 1 (4).
- EZT indicates the ratio of effector cells to target cells.
- effector cells obtained by induction with HE-1 peptide were (+)
- SKOV3-A24 showed antigen-peptide-specific cytotoxicity, indicating that the CTL was HER2 / neu-specific CTL.
- Example 2 After thawing the stored PBMC prepared in Example 1 (2), were suspended as a 5H-RPM I cell concentration becomes 2 XI 0 6 cells / m 1.
- the final concentration of SAC-I (Pans orbin cells, Calbiochem) in the cell suspension is 0.005%
- the final concentration of Immunobeads (Rabb it anti-Human IM, BioRad) is 20 g / m1
- IL- added 4 Jenzaimu Co.
- the cells were suspended in a phosphate buffer containing 1% BSA, 3 micrograms / ml of 2 microglobulins and 50 g / ml of a peptide, and reacted at 20 ° C for 4 hours. After the reaction, the cells were irradiated with X-rays (5500 Rad) and suspended in 5H-RPMI so that the cell concentration became 1 ⁇ 10 6 Zm 1 to prepare antigen-presenting cells for initial stimulation.
- X-rays 5500 Rad
- Example 2 After thawing the stored PBMC prepared in Example 2 (1), were suspended in manner 5H-RPM I cell concentration becomes 2 XI 0 6 cells / m 1, T one 25 the cell suspension 1 OML The flask was placed in a flask (Nunc). 1. After leaving at 37 ° C for 5 hours, remove the non-adherent cells, and add 100 OUZml of GM-CSF (Genzim) and 2000 U / m of IL-4 (Genzim) to the remaining adherent cells. the 5 H- RPM I containing 1, 1 OML added, and incubated in a C0 2 incubator 37 ° C to induce DC cells. After 7 days, the floating cells were collected.
- the adherent cells were collected with a cell dissociation buffer (manufactured by Gibco BRL), washed with 5H-RPMI, and combined with the floating cells.
- the recovered cells peptide 40 g / m 1, ⁇ 2 micro glow purine 3 ⁇ g / m 1 and saline plus 1% human serum albumin containing 3 X 1 0 so that six Zm l It was suspended and reacted in a thermostat at 20 ° C for 4 hours. After the reaction, the cells were irradiated with X-rays (550 ORad), and diluted with a physiological saline containing 1% human serum albumin to 1 ⁇ 10 6 Zm 1 to obtain antigen-presenting cells.
- X-rays 550 ORad
- Example 9 Preparation of CEA Antigen Beptide Functional Derivative
- CE—201 to CE—212 and CE—301 to CE shown in Tables 11 and 12 -315 peptides were produced using a peptide synthesizer.
- the CE-3 16 in Table 12 was obtained by adding 100 (mg / ml) of a DHCP (Takara Shuzo) aqueous solution to CE-3 (2.85 nmo 1) prepared in (1) of Example 5. 1 was added, reacted at 37 ° C. for 19 hours, and purified by HPLC.
- CE-3 17 is a CE in which two molecules of CE-3 are linked by a disulfide bond via a thiol residue of the fourth cysteine from the N-terminal in the amino acid sequence of CE-3 represented by SEQ ID NO: 5. — It is a dimer of 3. This peptide was prepared by reacting CE- 3 in a 0.32 mM aqueous solution of 0.02M NH 4 HC ⁇ 3 at 37 for 24 hours and then purifying by HPLC.
- Example 4 which recognizes the complex of ⁇ 5-2 or 0 £ _3 and ⁇ : 1 ⁇ 8-824 molecules was the peptide or the peptide shown in Tables 11 and 12. It was confirmed whether it recognized a complex between the peptide analog and the HLA-A24 molecule.
- CTs recognizing CE-2 prepared in the same manner as in (2) of Example 4 were obtained from 51 Cr-labeled TISI (1) cells and CE-201 to CE-212 peptide. Using either one of them, it was confirmed whether or not it shows toxicity to 12 kinds of 61 Cr-labeled TISI (ten) cells prepared in the same manner as (3) of Example 4.
- the CTLs that recognize CE-2 are CE-201, CE-203, CE-204, CE-206, CE-207, CE-208, CE-209, CE-210, CE - showed clear cell injury property to 2 1 51 C r obtained by cultivation of 2 in medium supplemented with labeled TISI (+).
- the CTL did not show cytotoxicity against 51 (: 1 "-labeled 13 I (one) cells, CE-201, CE-203, CE-204, CE-206, CE-207. , CE-208, CE-209, CE-210, CE-212 were revealed to be functional derivatives of CE-2.
- PBMC blood cells
- CE- 3 recognizes the starting material four different CTL is, 51 Cr-labeled TISI (—) Cells and 17 kinds of 51 Cr-labeled TISI (+) cells prepared in the same manner as (3) in Example 4 using one of the 17 kinds of peptides shown in Table 12 It was confirmed whether or not it showed any injuries.
- the CTLs that recognize CE-3 are CE-301, CE-302, CE-305, CE-307, CE-310, CE-311, CE-312, CE-314, CE_316, It showed clear cytotoxicity against 10 kinds of 51 Cr-labeled TISI (+) obtained by culturing in a medium to which any of CE-317 peptides were individually added. Note The CTL did not show cytotoxicity against 51 C r-labeled TISI (I) cells, CE- 30 1, CE- 302, CE - 305, CE - 307, CE- 31 0, CE - 31 1 , CE-312, CE-314, CE-316, CE-317 were found to be functional derivatives of CE-3.
- HLA-A24 molecule can be detected by the CTL of the present invention was examined using human-derived PBMC as test cells.
- PBMCs were prepared from eight individuals with known HLA types in the same manner as in Example 1, (2), and used as PBMC8 specimens for test cells. Each sample was divided into two, and one was suspended in 5H-RPMI in which CE-3 was dissolved so as to have a concentration of 10 g / ml, and this was used as a CE-3 added sample group. The other was suspended in 5H-RPI, and this was used as a sample group without CE-3. After leaving each group at 37 for 2 hours, the cells were collected by centrifugation, and the supernatant was removed. This operation removed excess CE-3 from the CE-3 added group.
- HLA-A2 (A * 0201) was synthesized in the same manner as in Example 1 (3) using FLWGPRALV, which is a MAGE3-derived HLA-A2-restricted peptide represented by the amino acid sequence of SEQ ID NO: 37.
- FLWGPRALV which is a MAGE3-derived HLA-A2-restricted peptide represented by the amino acid sequence of SEQ ID NO: 37.
- One effector cell was prepared from PBMCs derived from a healthy person.
- .221 (A2.1) cells which are HLA-A2 cells as target cells, were cultured on the day before the cytotoxicity measurement in a medium containing the above peptide 10 / gZm1 (.221 (A2.1) (+)) or A culture cultured in a medium (.221 (A2. 1) (1)) not containing was used. Measurements day, 51 C r-labeled .221 (.
- A2 1) (+ ) or .221 (. A2 1) (- ) cells and effector cells were mixed, 51 C r amount liberated into the culture medium after 5 hours was measured.
- the prepared effector It was revealed that the vesicles were CTL showing peptide-specific cytotoxicity.
- .221 (A2.1) (+) or .221 (A2.1) (-) cells were mixed with CTL, and IFN-7 in the supernatant one day later was measured. It was revealed that one cell, CTL, released IFN-a in a peptide-specific manner. After increasing this CTL, the presence or absence of a peptide-specific IFN-migrating effect on various HLA-A2 cells was examined.
- the fourth amino acid in the amino acid sequence of SEQ ID NO: 33 is 2-hydroxy-4 (R, S) -1L-cysteine-S-yl-1-2-cyclopentene-11one.
- the CTL of the present invention can be used to express 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. It can be selectively injured and used as a cell medicine for the treatment of cancer.
- HLA-A24 and MAGE-3 positive tumor cells, HLA-A24 and MA in extracorporeal samples Detection of the presence of tumor cells positive for both GE-1 and HLA-A24 and CEA, or HLA-A24 and HER2Z neu, and the HLA of cells in the extracorporeal sample when used together with the antigen peptide. Useful for typing, etc.
- the antigen-presenting cell of the present invention is useful for preparing the CTL or as an anticancer agent.
- the pharmaceutical composition containing the antigen peptide of the present invention as an active ingredient is useful as a CTL inducer and further as a carcinostatic agent.
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Abstract
Cytotoxic T lymphocytes (CTL) capable of recognizing cells presenting on the surface thereof complexes of at least one antigen peptide selected from among human major histocompatibility antigen (HLA)-A24-restrained antigen peptides represented by any of the amino acid sequences of SEQ ID NOS:1 to 6 and functional derivatives thereof with HLA-A24 molecules; carcinostatics containing the above CTL; a method for inducing the above CTL; inducers for the above CTL; carcinostatics containing at least one antigen peptide selected from among the above HLA-A24-restrained antigen peptides and functional derivatives thereof; antigen presenting cells which present on the surface thereof complexes of at least one antigen peptide selected from among the above HLA-A24-restrained antigen peptides and functional derivatives thereof with HLA-A24 molecules; CTL inducers containing the above antigen presenting cells; carcinostatics containing the above antigen presenting cells; methods for detecting cells sensitive to the above CTL; agents for detecting cells sensitive to the above CTL which contain the above CTL; and methods for detecting HLA molecules.
Description
明細書 細胞傷害性 Tリンパ球 Description Cytotoxic T lymphocytes
技術分野 Technical field
本発明は、 がんの治療及び診断に有用な細胞傷害性 Τリンパ球 （細胞障害性 Τ リンパ球と記載される場合もある） （cytotoxic T lymphocytes ；以下、 CTL と略す） 及び該 CTLを誘導する際等に有用な CTL誘導剤に関する。 The present invention relates to cytotoxic T lymphocytes (sometimes referred to as cytotoxic T lymphocytes) useful for the treatment and diagnosis of cancer (cytotoxic T lymphocytes; hereinafter abbreviated as CTL) and to induce the CTL The present invention relates to a CTL inducer that is useful in such cases.
背景技術 Background art
CTLには、 抗原ペプチドと主要組織適合性抗原遺伝子複合体 （major histocompatibility gene complex；以下、 MHCと略す） にコードされる主要組織適 合性抗原 MHCクラス I (ヒトの場合 human leukocyte antigen クラス Iと呼 ばれ、 以下、 HLAクラス Iと略す） 分子との結合物である複合体を特異的な T 細胞レセプター （T cell receptor ；以下、 TCRと略す） によって認識し、 そ の複合体を細胞表面に提示してレ、る細胞を傷害することのできるものがある。 こ のような CTLは自身と同じ MHCクラス I分子を有する標的細胞のみを認識し 傷害することから、 MHCクラス I分子拘束性 CTLと呼ばれる。 したがって該 細胞傷害反応が成立するためには、 1)特異的 TCRを持った CTLが存在する こと、 2) HLAクラス I分子に提示され CTLに認識される抗原ペプチドとな るために、 MHCクラス I抗原分子との結合だけでなく、 TCRによる認識を受 ける複合体を形成できる抗原べプチドが存在すること、 が必要である。 CTLs contain major histocompatibility gene complex (MHC), a major histocompatibility antigen complex (MHC), which is encoded by an antigenic peptide and a major histocompatibility gene complex (MHC). (Hereinafter referred to as HLA class I) A complex that binds to a molecule is recognized by a specific T cell receptor (TCR), and the complex is recognized on the cell surface. Some can present and damage cells. Such CTLs are called MHC class I molecule-restricted CTLs because they recognize and damage only target cells having the same MHC class I molecule as themselves. Therefore, in order for the cytotoxic reaction to be established, 1) the presence of CTL having a specific TCR, and 2) the MHC class which is presented on HLA class I molecules and becomes an antigen peptide recognized by CTL It is necessary that there be an antigen peptide that can form a complex that can be recognized by the TCR as well as binding to the I antigen molecule.
すなわち本明細書における 「抗原ペプチド」 とは、 MHCクラス I分子に結合 し MHCクラス I分子との複合体形成能を有すると共に該複合体が特異的な CT Lの TCRによって認識される抗原性を有するぺプチドを意味する。 また本明細 書における 「HL A拘束性抗原ペプチド」 とは MHCクラス I分子が HLA分子
である抗原ペプチドであることを意味する。 なお本明細書における 「ペプチド」 とは、 アミノ酸のみからなるホモメリックペプチドに特に限定されず、 非ァミノ 酸成分を含むヘテロメリックぺプチドをも包含する。 ァミノ酸も天然型に特に限 定されず、 化学修飾されたアミノ酸であってもよい。 また本明細書におけるぺプ チドは単量体に限定されず、 多量体であってもよい。 That is, the “antigen peptide” in the present specification refers to an antigenicity that binds to an MHC class I molecule and forms a complex with the MHC class I molecule, and that the complex is recognized by a specific CCR TCR. Means a peptide having The term “HLA-restricted antigen peptide” used herein refers to an MLA class I molecule as an HLA molecule. Is an antigenic peptide. The “peptide” in the present specification is not particularly limited to a homomeric peptide consisting of only amino acids, but also includes a heteromeric peptide containing a non-amino acid component. Amino acids are not particularly limited to natural forms, and may be chemically modified amino acids. Further, the peptide in the present specification is not limited to a monomer, and may be a multimer.
このような抗原べプチドは、 例えば哺乳類細胞の細胞内で合成された抗原等が 小胞体でプロセスされ、 小さいェピトープぺプチドに分解されることにより生じ Such antigen peptides are produced, for example, by the processing of antigens and the like synthesized in mammalian cells in the endoplasmic reticulum and decomposing them into small epitope peptides.
、 更に MHCクラス I分子と会合し、 細胞表面に提示される。 すなわち、 多くの サブユニッ トよりなるプロテオソ一ム複合体の中で、 タンパク質は 8〜1 5アミ ノ酸よりなるぺプチドに分解され、 そのうちのいくつかが TAPトランスポー夕 一により細胞質から小胞体に運ばれる。 これらのぺプチドは小胞体でクラス IZ β2 ミクログロブリン （microglobulin ) のへテロダイマーに結合できれば、 3 分子複合体として安定化され、 ゴルジ装置を通って、 細胞表面に輸送される。 腫 瘍関連抗原又は腫瘍特異的抗原タンパク質を発現している腫瘍細胞は、 腫瘍細胞 表面に Tリンパ球に認識される、 MHCクラス I分子拘束性抗原べプチドを提示 できるはずである。 In addition, it associates with MHC class I molecules and is displayed on the cell surface. That is, in a proteosome complex composed of many subunits, proteins are decomposed into peptides composed of 8- to 15-amino acids, some of which are converted from the cytoplasm to the endoplasmic reticulum by the TAP transport enzyme. Carried. If these peptides can bind to class IZ β2 microglobulin heterodimers in the endoplasmic reticulum, they will be stabilized as trimolecular complexes and transported to the cell surface through the Golgi apparatus. Tumor cells expressing tumor-associated antigens or tumor-specific antigen proteins should be able to present the MHC class I molecule-restricted antigen peptides recognized by T lymphocytes on the tumor cell surface.
Tリンパ球に認識される最初の腫瘍特異抗原として、 T. ブーン （T. B o o n) らによりメラノ一マ （me l a n oma) 抗原 E (以下、 MAGEと略す） と呼ばれる遺伝子がクローニング、 同定された 〔P. ファン デル ブルガン （ P. van der Bruggen) ら、 サイエンス （Science ) 、 第 2 5 4巻、 第 1 6 4 3〜 1 6 4 7頁 （ 1 9 9 1 ) 〕 。 彼らは、 メラノーマ患者由来の CTLクローン、 及 びその C T Lに認識される同じ患者由来の一連のメラノ一マ細胞株、 そしてそれ らのうちいくつかはその CTLに耐性であるように免疫選択された株を用いて、 発現クローニング法により MAGEを単離した。 すなわち CTLに認識されたメ ラノーマ細胞株より調製したある 5 kbの DN A断片を元々認識されなかった細 胞株に形質導入すると、 上記 C T Lクローンによって認識されるようになること
が示された。 この DNA断片は推定分子量 26, 000の夕ンパクをコードしていた。 このタンパクをコードした遺伝子は MAGE— 1と呼ばれているが mRNA分析 によって、 転移性だけでなく初期段階のメラノーマ由来の 4 0%の細胞株が MA GE- 1を発現していることが示された。 該夕ンパク由来の抗原は HLA— A 1 拘束性であり、 MAGE— 1特異的 CTLクローンは、 HLAクラス I分子の一 種である H L A— A 1分子に結合した、 配列番号 7に記載した 9ァミノ酸配列か らなるペプチド配列 （EADPTGHSY ) を認識した 〔C. トラヴァーサリ （C. Trave rsari ) ら、 ジャーナル ォブ イクスペリメンタル ォブ メデイシン （Jour nal of Experimental of Medicine ) 、 第 1 76巻、 第 1 4 5 3〜 1 4 57頁 （ 1 9 92 ) 3 As the first tumor-specific antigen recognized by T lymphocytes, a gene called melanoma antigen E (hereinafter abbreviated as MAGE) was cloned and identified by T. Boon et al. [P. van der Bruggen, et al., Science, Vol. 254, pp. 1643-16447 (1991)]. They are CTL clones from melanoma patients, and a series of melanoma cell lines from the same patient that are recognized by the CTL, and some of which are immunoselected to be resistant to the CTL Was used to isolate MAGE by the expression cloning method. That is, when a 5 kb DNA fragment prepared from a melanoma cell line recognized by CTL is transduced into an originally unrecognized cell line, it will be recognized by the CTL clone. It has been shown. This DNA fragment encoded a protein with an estimated molecular weight of 26,000. The gene encoding this protein is called MAGE-1, but mRNA analysis indicates that 40% of early-stage melanoma-derived cell lines express MAGE-1 as well as metastatic. Was done. The antigen derived from the protein is HLA-A1-restricted, and the MAGE-1-specific CTL clone binds to HLA-A1 molecule, which is a kind of HLA class I molecule, and is represented by SEQ ID NO: 7. Recognized a peptide sequence consisting of an amino acid sequence (EADPTGHSY) [C. Trave rsari et al., Journal of Experimental of Medicine, Vol. 176, No. 1 4 5 3 to 1 4 57 pages (1992) 3
腫瘍抗原として知られているものは多くのものがある力 現在 C D— 8陽性の CTLに認識されるものとしては、 上記の MAGE— 1を始めとして 1 0種以上 存在することが知られている MAGEファミ リー、 gp 100、 チロシナ一ゼ （tyro sinase) 、 またがん胎児性抗原 （carcinoembryonic antigen；以下、 CEAと略 す） 、 HER2/neu等がよく知られている。 C T Lに認識されるものは、 腫瘍抗原夕 ンパク質由来のペプチドであり、 HLA クラス I分子との複合体として提示される There are many known tumor antigens. Currently, it is known that there are more than 10 types of CD-8-positive CTL, including MAGE-1, described above. The MAGE family, gp100, tyrosine synthase, carcinoembryonic antigen (CEA), HER2 / neu, and the like are well known. Recognized by CTL is a peptide derived from the tumor antigen protein and presented as a complex with HLA class I molecules
HLA クラス I分子は主として HLA-A、 -B、 -Cがあり、 これらに結合して提示さ れる抗原ペプチドは、 9〜 0個のアミノ酸よりなり、 更に各々の HLA分子に よって異なる一定の構造上の特徴があることが知られている。 例えば、 世界的に 最も頻度の高い HLA-A2.1分子に結合するべプチドとしては N末端より 2番目に Le u 、 且つ C末端に Leu 又は Val を有する 9〜1 0個のアミノ酸よりなるペプチド が最も良く知られているものである。 また、 日本人を始めとするアジアの人種に 多い HLA- A24 分子に結合するペプチドは N末端より 2番目に Tyr 、 Phe 、 Met 、 Trp のいずれか、 且つ C末端に Leu、 He、 Trp 、 Phe のいずれかを有する 9〜 1 0個のアミノ酸よりなるものであるペプチドが最もよく知られている 〔A. コ
ンド一 （A. Kondo ) ら、 ジャーナル ォブ ィムノロジ一 （Journal of Immun ology ) 、 第 1 55巻、 第 4 307〜 43 1 2頁 （ 1 9 9 5 ) 〕 。 HLA class I molecules are mainly HLA-A, -B, and -C, and the antigenic peptide presented by binding to them consists of 9 to 0 amino acids, and has a certain structure that differs depending on each HLA molecule. It is known to have the above characteristics. For example, the world's most frequent peptide that binds to the HLA-A2.1 molecule is a peptide consisting of 9 to 10 amino acids having Leu at the N-terminus and Leu or Val at the C-terminus. Are the best known. In addition, peptides that bind to HLA-A24 molecules, which are common in Asian peoples including Japanese, are Tyr, Phe, Met, or Trp at the second position from the N-terminal, and Leu, He, Trp, or C-terminal at the C-terminal. Peptides consisting of 9 to 10 amino acids with any of the Phe are best known [A. A. Kondo et al., Journal of Immunology, Vol. 155, No. 4, 307-4312 (1995)].
現在までに抗原べプチドが同定されている腫瘍抗原としては、 HLA-A1に対する MAGE-1, MAGE - 3、 HLA-A2.1に対する MAGE_3、 MARTI 、 チロシナーゼ、 gplOO、 HE R2/neu、 CEA等、 HLA-Cwl に対する MAGE-3、 HLA-B44 に対する MAGE- 3等、 HLA-A2 4 に対してはチロシナ一ゼ、 8—力テニン （c a t e n i n) 等がある。 CEA 由来の抗原べプチドに関しては、 配列番号 34に示した 9個のァミノ酸配列から なるペプチドが HL A— A 2. 1拘束性抗原ペプチドとして同定されている [ジ ヤーナル ォブ ナショナル キャンサー インスティテユート （ J 0 u r n a 1 o f Na t i on a l C an c e r I n s t i t u t e ) 、 第 8 7巻 、 第 982〜 990頁 （ 1 995) ] 。 同様に、 HER 2Zn e u由来の抗原べ プチドに関しては、 配列番号 35又は 36に示した 9個のアミノ酸配列からなる ペプチドが HLA— A2. 1拘束性抗原ペプチドとして同定されている [ジャー ナル ォブ ェクスペリメンタル メデイシン、 第 1 8 1巻、 第 2 1 0 9〜2 1 1 7頁 （ 1 995 ) 、 キャンサー リサーチ （Can c e r Re s e a r c h ：) 、 第 54巻、 第 1 07 1〜 1 076頁 （ 1 994) ] 。 これらの中の多くは、 まず腫瘍細胞を認識するクラス I拘束性の C T Lを株化し、 この C T Lの認識す る腫瘍抗原を同定し、 続レ、て遺伝子工学的方法により腫瘍抗原タンパク質中最小 単位を見出し、 更に HL Aクラス I分子への結合モチーフに関する情報を基に最 小単位中のペプチドが見出されている 〔Y. 力ワカミ （Y. Kawakami ) ら、 プロ シ一ディングズ ォブ ザ ナショナル アカデミー ォブ ザ サイエンシー ズ ォブ ザ USA (Proceedings of the National Academy of the Science s of the United States of America ) 、 第 9 1巻、 第 35 1 5〜 35 1 9頁 （ 1 9 94 ) 〕 。 また、 まず前記の HLAクラス I分子結合ペプチドに共通したモ チーフ構造を基に、 腫瘍抗原タンパク質中の HL Aクラス I分子結合べプチドを 見出し、 続レ、て抗原提示細胞を利用して C T Lを誘導可能なものを選択した後、
最終的に腫瘍細胞に対して傷害性を有する CTLが誘導できているかどうかによ り抗原ペプチドが決定されている 〔E. セリス （E. Celis) ら、 プロシーディン グズ ォブ ザ ナショナル アカデミー ォブ サイエンシーズ ォブ ザ US A、 第 9 1巻、 第 2 1 0 5〜2 1 0 9頁 （ 1 9 9 4 ) 、 B. ゴーグラ一 （B.To date, tumor antigens whose antigenic peptides have been identified include MAGE-1, MAGE-3 for HLA-A1, MAGE_3 for HLA-A2.1, MARTI, tyrosinase, gplOO, HE R2 / neu, CEA, etc. There are MAGE-3 for HLA-Cwl, MAGE-3 for HLA-B44, and tyrosinase, 8-cathenin for HLA-A24. Regarding the CEA-derived antigenic peptide, a peptide consisting of the nine amino acid sequences shown in SEQ ID NO: 34 has been identified as an HLA-A2.1-restricted antigenic peptide [Journal of National Cancer Institute] Ute (Journa 1 of Nation al Cancer Institute), Vol. 87, pp. 982-990 (1995)]. Similarly, as for the antigen peptide derived from HER2Zneu, a peptide consisting of the nine amino acid sequences shown in SEQ ID NO: 35 or 36 has been identified as an HLA-A2.1-restricted antigen peptide [Journal of Experimental Medicine, Volume 181, Volume 209-211 (1995), Cancer Research (Cancer Research), Volume 54, Volume 1071-1076 (1 994)]. Many of these cells first establish a class I-restricted CTL that recognizes tumor cells, identify tumor antigens that are recognized by these CTLs, and then use genetic engineering methods to construct the smallest unit of tumor antigen protein. And the peptide in the smallest unit has been found based on information on the binding motif to HLA class I molecules [Y. Kawakami et al., Processings of the National. Academy of the Sciences of the USA (Proceedings of the National Academy of the Sciences of the United States of America), Vol. 91, pp. 3515-3519 (1994)]. First, based on the motif structure common to the HLA class I molecule-binding peptides described above, we found the HLA class I molecule-binding peptide in the tumor antigen protein, and then used the antigen-presenting cells to convert the CTL. After choosing what is navigable, The ultimate determination of antigenic peptides depends on whether CTLs that are toxic to tumor cells can be induced [E. Celis et al., Proceedings of the National Academy of Sciences. Science of the USA, Vol. 91, Vol. 210-210, p. 9 (1994), B. Gogra (B.
Gaugler) ら、 ジャーナル ォブ イクスペリメンタル ォブ メデイシン、 第Gaugler) et al., Journal of Experimental Medicine, Med.
1 7 9巻、 第 9 2 1〜 9 3 0頁 （ 1 9 94) 〕 。 Vol. 179, pp. 921-930 (1994)].
一方、 HLAクラス I分子はいくつかのサブタイプに分類されるが、 その保有 サブタイプの種類は人種間で大きく異なり、 世界的には HLA-A2が最も多く、 白色 人種の 4 5 %を占めている。 そして、 この HLA-A2拘束性の抗原ペプチドの同定が 最も進んでいる。 日本人では HLA- A2は 4 0%を占めるが、 そのサブタイプを見る と白色人種と同じ HLA-A * 0201は 2 0 で、 残りの多くは A * 0206である。 これ らのサブタイプへの結合べプチドは異なり、 主として研究されている HLA-A2は HL A-A * 0201である。 一方、 日本人では HLA-A24が 6 0%以上を占めており、 この HLA-A24 はアジア人種では他の人種に比べて比率が高い。 し力、し、 現在までにこ の HLA— A24拘束性の抗原べプチドが発見されている腫瘍抗原として上記の チロシナーゼ、 S—力テニンがあるが、 その解析は非常に遅れている。 したがつ てァジァ人種、 特に日本人において腫瘍細胞特異的に作用する C T Lの解析も遅 れており、 腫瘍治療に有用な CTLの提供が不可能であった。 本発明の目的は該 CTLを提供することにある。 発明の開示 On the other hand, HLA class I molecules are classified into several subtypes, but the types of possessed subtypes vary greatly among races, with HLA-A2 being the most common worldwide and 45% of white races Occupy. The identification of this HLA-A2-restricted antigen peptide is the most advanced. HLA-A2 accounts for 40% of the Japanese population, but its subtype is HLA-A * 0201, which is the same as that of Caucasians, and the rest is A * 0206. The binding peptides to these subtypes are different, and the HLA-A2 studied primarily is HLA-A * 0201. On the other hand, HLA-A24 accounts for more than 60% of the Japanese, and this HLA-A24 is higher in Asian races than in other races. The above tyrosinase and S-force tenin have been identified as tumor antigens for which the HLA-A24-restricted antigenic peptide has been discovered to date, but its analysis has been very slow. Therefore, the analysis of CTL that acts specifically on tumor cells in Azia races, especially in Japanese, has been delayed, and it has been impossible to provide CTLs useful for tumor treatment. An object of the present invention is to provide the CTL. Disclosure of the invention
本発明の第 1の態様は、 配列番号 1〜 6のいずれかに記載のァミノ酸配列で表 される HLA— A24拘束性抗原べプチド及びその機能的誘導体から選択される 少なくとも 1つの抗原ペプチドと HL A— A 24分子との複合体を細胞表面に提 示する細胞を認識する CTLに関する。 本発明の第 2の態様は、 第 1の態様の C TLを有効成分とする制がん剤に関する。 本発明の第 3の態様は、 第 1の態様の
CTLの誘導方法に関する。 本発明の第 4の態様は、 配列番号 1〜6のいずれか に記載のァミノ酸配列で表される HL A— A 24拘束性抗原べプチド及びその機 能的誘導体から選択される少なくとも一つの抗原べプチドを有効成分とする C T Lの誘導剤に関する。 本発明の第 5の態様は、 配列番号 1〜6のいずれかに記載 のァミノ酸配列で表される HLA— A24拘束性抗原べプチド及びその機能的誘 導体から選択される少なくとも一つの抗原べプチドを有効成分とする制がん剤に 関する。 本発明の第 6の態様は、 配列番号 1〜6のいずれかに記載のアミノ酸配 列で表される H L A— A 24拘束性抗原べプチド及びその機能的誘導体から選択 される少なくとも一つの抗原ペプチドと HLA— A 24分子との複合体を細胞表 面に提示する抗原提示細胞に関する。 なお本明細書では HL A— A 24拘束性抗 原べプチドと HL A— A 24分子との複合体を細胞表面に提示する細胞として、 「標的細胞」 なる語と 「抗原提示細胞」 なる語を使用する。 本明細書において 「 標的細胞」 とは該細胞を特異的に認識する CTLにより傷害を受ける細胞とする 。 一方、 本明細書において 「抗原提示細胞」 とは MHC分子と共に抗原を提示し Tリンパ球の活性化を惹起する機能を有する細胞を意味し、 特に断りのない限り MHC分子が HLA— A24分子である細胞とする。 また本明細書における Γ抗 原提示能を有する細胞」 とは該細胞に存在する MHC分子と抗原べプチドが複合 体を形成することにより抗原提示細胞となることができる細胞を意味し、 特に断 りのない限り MHC分子が HLA— A24分子である細胞とする。 本発明の第 7 の態様は、 第 6の態様の抗原提示細胞を有効成分とする C T Lの誘導剤に関する 。 本発明の第 8の態様は、 第 6の態様の抗原提示細胞を有効成分とする制がん剤 に関する。 本発明の第 9の態様は、 第 1の態様の CTLを被検細胞と接触させた 際に生じる変化を指標として該 C T Lに感受性の細胞の検出方法に関する。 なお 本明細書における 「被検細胞」 とは、 末梢血リ ンパ球、 組織等の体外摘出試料由 来のヒト細胞又は株化細胞である。 また本明細書における 「感受性細胞」 とは、 CTLにより認識され細胞溶解あるいはサイトカイン遊離を引き起こされる腫瘍
細胞をはじめとする、 異常細胞を意味する。 本発明の第 1 0の態様は、 第 1の態 様の C T Lを有効成分として含有することを特徴とする該 C T Lに感受性の細胞 の検出剤に関する。 更に、 本発明の第 1 1の態様は、 HL A拘束性抗原ペプチド 及びその機能的誘導体から選択される少なくとも一つの抗原ペプチド存在下、 該 抗原べプチドと H L A分子との複合体を認識可能な C T Lを被検細胞と接触させ ることを特徴とする HL A分子の検出方法に関する。 A first aspect of the present invention relates to at least one antigen peptide selected from an HLA-A24-restricted antigen peptide represented by the amino acid sequence set forth in any one of SEQ ID NOS: 1 to 6 and a functional derivative thereof. HL A—Relates to a CTL that recognizes cells that present a complex with the A24 molecule on the cell surface. A second aspect of the present invention relates to an anticancer agent comprising the CTL of the first aspect as an active ingredient. A third embodiment of the present invention is directed to the first embodiment, The present invention relates to a method for inducing CTL. A fourth embodiment of the present invention provides at least one selected from HLA-A24-restricted antigenic peptide represented by the amino acid sequence of any one of SEQ ID NOs: 1 to 6 and a functional derivative thereof. The present invention relates to a CTL inducer comprising an antigen peptide as an active ingredient. A fifth aspect of the present invention relates to an HLA-A24-restricted antigenic peptide represented by the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 6 and at least one antigenic receptor selected from functional derivatives thereof. It relates to an anticancer drug containing peptide as an active ingredient. A sixth aspect of the present invention relates to at least one antigen peptide selected from an HLA-A24-restricted antigenic peptide represented by the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 6 and a functional derivative thereof. And an antigen presenting cell that presents a complex of HLA-A24 and HLA-A24 on the cell surface. In this specification, the term "target cell" and the term "antigen-presenting cell" are used as cells that present a complex of an HLA-A24-restricted antigen peptide and an HLA-A24 molecule on the cell surface. Use As used herein, the term “target cell” refers to a cell that is damaged by CTL that specifically recognizes the cell. On the other hand, the term “antigen-presenting cell” as used herein refers to a cell having a function of presenting an antigen together with an MHC molecule and inducing the activation of T lymphocytes. Unless otherwise specified, the MHC molecule is an HLA-A24 molecule. Some cells. The term “cells having antigen-presenting ability” as used herein means cells that can become antigen-presenting cells by forming a complex between an MHC molecule and an antigen peptide present in the cells, and in particular, may be used in particular. Unless otherwise indicated, cells whose MHC molecules are HLA-A24 molecules are used. A 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 an anticancer agent 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 a cell that is sensitive to the CTL of the first aspect, using a change generated when the CTL is brought into contact with the test cell as an index. The “test cell” in the present specification is a human cell or a cell line derived from an extracorporeal sample such as peripheral blood lymphocytes and tissues. The term “sensitive cell” as used herein refers to a tumor that is recognized by CTL and causes cell lysis or cytokine release. Abnormal cells, including cells. A tenth aspect of the present invention relates to an agent for detecting a cell sensitive to CTL, comprising the CTL of the first aspect as an active ingredient. Furthermore, the first embodiment of the present invention is capable of recognizing a complex between the antigen peptide and an HLA molecule in the presence of at least one antigen peptide selected from an HLA-restricted antigen peptide and a functional derivative thereof. The present invention relates to a method for detecting an HLA molecule, which comprises contacting a CTL with a test cell.
本発明者らは、 腫瘍抗原である MAGE— 3タンパク質中の HLA— A24結 合モチーフを有するぺプチドを中心として多くの候補べプチドの中から抗原ぺプ チドを探索した結果、 配列番号 1又は 2で表されるァミノ酸配列からなるぺプチ ドを用いると、 HLA— A24を発現している健常人の末梢血単核球より該腫瘍 抗原発現細胞を選択的に傷害する C T Lを誘導できることを明らかにした。 同様 に、 腫瘍抗原である各種タンパク質中の HL A— A 24結合モチーフを有するぺ プチドを中心とした候補べプチドの中から HL A— A 24拘束性抗原べプチドを 探索した結果、 MAGE— 1タンパク質ァミノ酸配列中に存在する配列番号 3で 表されるアミノ酸配列からなるぺプチド、 CE Aタンパク質アミノ酸配列中に存 在する配列番号 4又は 5で表されるァミノ酸配列からなるペプチド、 更に HER 2/n e uタンパク質ァミノ酸配列中に存在する配列番号 6で表されるァミノ酸 配列からなるぺプチドを用いると、 HLA— A24を発現している健常人の末稍 血単核球より該腫瘍抗原発現細胞を選択的に傷害する C T Lを誘導できることを 明らかにし、 本発明を完成させた。 図面の簡単な説明 The present inventors searched for an antigen peptide from many candidate peptides centering on the peptide having the HLA-A24 binding motif in the MAGE-3 protein, which is a tumor antigen, and found that SEQ ID NO: 1 or The use of a peptide consisting of the amino acid sequence represented by 2 can induce CTLs that selectively damage the tumor antigen-expressing cells from peripheral blood mononuclear cells of healthy humans expressing HLA-A24. Revealed. Similarly, as a result of searching for HLA-A24-restricted antigen peptides from candidate peptides centered on peptides having an HLA-A24 binding motif in various tumor antigen proteins, MAGE-1 A peptide consisting of the amino acid sequence represented by SEQ ID NO: 3 present in the protein amino acid sequence; a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 or 5 present in the amino acid sequence of CEA protein; and HER When a peptide consisting of the amino acid sequence represented by SEQ ID NO: 6 present in the 2 / neu protein amino acid sequence is used, the tumor antigen is obtained from peripheral blood mononuclear cells of a healthy human expressing HLA-A24. The present inventors have clarified that it is possible to induce CTLs that selectively damage expression cells, and have completed the present invention. BRIEF DESCRIPTION OF THE FIGURES
第 1図は、 抗原べプチド MA 3一 2の刺激により誘導したエフェクタ一細胞の 、 標的細胞に対する特異的細胞傷害活性を示す図である。 FIG. 1 is a diagram showing the specific cytotoxic activity of effector cells on target cells induced by stimulation of antigen peptide MA321.
第 2図は、 抗原べプチド MA 3一 4の刺激により誘導したェフユクタ一細胞の 、 標的細胞に対する特異的細胞傷害活性を示す図である。
第 3図は、 抗原べプチド MA 3— 2の刺激により誘導したエフヱクタ一細胞の 、 各種がん細胞に対する特異的細胞傷害活性を示す図である。 FIG. 2 is a graph showing specific cytotoxic activity of target cells on efjucuta cells induced by stimulation of antigen peptide MA314. FIG. 3 is a view showing the specific cytotoxic activity of effector cells induced by stimulation of the antigen peptide MA3-2 against various cancer cells.
第 4図は、 抗原べプチド MA 3 - 4の刺激により誘導したエフヱク夕一細胞の 、 各種がん細胞に対する特異的細胞傷害活性を示す図である。 FIG. 4 is a view showing specific cytotoxic activity of the effect cells induced by the stimulation of the antigen peptide MA3-4 on various cancer cells.
第 5図は、 抗原べプチド MA 1 - 1の刺激により誘導したエフヱクタ一細胞の 、 各種標的細胞に対する特異的細胞傷害活性を示す図である。 発明を実施するための最良の形態 FIG. 5 is a graph showing specific cytotoxic activity of effector cells induced by stimulation of antigen peptide MA1-1 against various target cells. BEST MODE FOR CARRYING OUT THE INVENTION
本発明の第 1の態様は、 配列番号 1〜 6いずれかに記載のァミノ酸配列で表さ れる H L A— A 24拘束性抗原べプチド及びその機能的誘導体から選択される少 なくとも一つの抗原ペプチドと HLA— A24分子との複合体を細胞表面に提示 する細胞を認識する CTLであり、 該 CTLは標的細胞に対して特異的に細胞溶 解又はサイト力イン遊離反応等を起こす。 なお、 本明細書における CTLは HL A-A24分子上に提示された抗原べプチドの抗原性を認識し傷害活性を有する ものを指し、 その他の抗原性認識などにより限定されるものではない。 A first aspect of the present invention relates to at least one antigen selected from an HLA-A24-restricted antigen peptide represented by the amino acid sequence shown in any one of SEQ ID NOs: 1 to 6 and a functional derivative thereof. A CTL that recognizes a cell that presents a complex of a peptide and an HLA-A24 molecule on the cell surface, and the CTL specifically causes cell lysis or cytoforce release reaction on a target cell. In the present specification, CTL refers to one that recognizes the antigenicity of the antigenic peptide presented on the HLA-A24 molecule and has a damaging activity, and is not limited by other antigenicity recognition or the like.
本明細書において、 HLA— A24拘束性抗原ペプチドの機能的誘導体とは、 HLA— A24分子との複合体形成能を有すると共に、 形成された複合体が配列 番号 1〜 6レ、ずれか記載のァミノ酸配列で表される抗原べプチドと H L A— A 2 4分子との複合体を認識する CTLに認識されるものを意味する。 例えば、 配列 番号 1〜 6いずれかに記載のァミノ酸配列で表されるぺプチドのァミノ酸配列に おいて、 1又は数個のアミノ酸が、 欠失、 他のアミノ酸若しくはアミノ酸アナ口 グに置換、 及び/又は 1又は数個のァミノ酸若しくはアミノ酸アナログの付加、 又はそれらの組み合わせによってァミノ酸配列は異なるが、 HL A— A 24分子 との複合体形成能を有すると共に、 形成された複合体が本発明の CTLに認識さ れるペプチドであり、 ジスルフィ ド結合等によるペプチドの 2量体も含まれる。 機能的誘導体のアミノ酸配列長は、 9〜1 0が好ましいが、 これに特に限定され
ない。 なお、 本明細書におけるアミノ酸アナログとは、 種々のアミノ酸の N—ァ シル化物、 0—ァシル化物、 エステル化物、 酸アミ ド化物、 アルキル化物等を意 味する。 In the present specification, the functional derivative of the HLA-A24-restricted antigen peptide has the ability to form a complex with an HLA-A24 molecule, and the formed complex has a sequence number of 1 to 6 or It means the one recognized by CTL which recognizes the complex of the antigen peptide represented by the amino acid sequence and the HLA-A24 molecule. For example, in the amino acid sequence of the peptide represented by any one of SEQ ID NOs: 1 to 6, one or several amino acids are deleted or replaced with another amino acid or an amino acid analog. , And / or the addition of one or several amino acids or amino acid analogs, or a combination thereof, wherein the amino acid sequence differs, but has the ability to form a complex with HLA-A24 molecule, and the complex formed Is a peptide recognized by the CTL of the present invention, and also includes a dimer of the peptide due to a disulfide bond or the like. The amino acid sequence length of the functional derivative is preferably 9 to 10, but is not particularly limited thereto. Absent. The term “amino acid analog” as used herein means N-acylated, 0-acylated, esterified, acid amide, alkylated, and the like of various amino acids.
また、 HL A— A 24分子との複合体が CTLに認識されれば、 抗原ペプチド の N末端や遊離のアミノ基は、 ホルミル基、 ァセチル基、 t一ブトキシカルボ二 ル （t一 Bo c)基等が結合していてもよい。 また、 HLA— A24分子との複 合体が CTLに認識されれば、 抗原べプチドの C末端や遊離のカルボキシル基は 、 メチル基、 ェチル基、 t—ブチル基、 ベンジル基等が結合していてもよい。 同 様に HLA— A24分子との複合体が CTLに認識されれば、 抗原ペプチドに含 まれるシスティンのチオール基はァセトアミ ドメチル基、 メトキシベンジル基等 が結合していてもよい。 Also, if the complex with HL A-A 24 molecule is recognized by CTL, the N-terminus and free amino group of the antigen peptide will be changed to formyl group, acetyl group, t-butoxycarbon (t-Boc) A group or the like may be bonded. Also, if the complex with HLA-A24 molecule is recognized by CTL, the C-terminus of the antigen peptide and the free carboxyl group are bound by methyl group, ethyl group, t-butyl group, benzyl group, etc. Is also good. Similarly, if a complex with an HLA-A24 molecule is recognized by CTL, the thiol group of cysteine contained in the antigen peptide may be bound to an acetamidomethyl group, a methoxybenzyl group, or the like.
機能的誘導体は、 配列番号 1〜 6いずれか記載のァミノ酸配列で表される抗原 ペプチドと HL A— A 24分子との複合体を認識する CTLを用いることにより 同定することが出来、 例えば、 下記方法が挙げられる。 A functional derivative can be identified by using a CTL that recognizes a complex of the antigenic peptide represented by any one of SEQ ID NOs: 1 to 6 and the HLA-A24 molecule. The following method is mentioned.
第 1の方法は、 機能的誘導体としての候補物質と HLA— A 24発現細胞を混 合し、 HLA— A 24分子に未結合の候補物質を洗浄後、 CTLと反応させる。 候補物質特異的な、 細胞傷害性、 サイトカイン遊離、 又は増殖反応が認められれ ば機能的誘導体であると判断出来る。 In the first method, a candidate substance as a functional derivative is mixed with HLA-A24-expressing cells, the candidate substance not bound to the HLA-A24 molecule is washed, and then reacted with CTL. If a candidate substance-specific cytotoxicity, cytokine release, or proliferative response is observed, it can be determined that the compound is a functional derivative.
第 2の方法としては、 候補物質を抗原提示能を有する細胞に添加し、 適当な時 間、 例えば、 抗原が細胞内に取り込まれプロセッシングを受け、 抗原ペプチドと HLA分子複合体が細胞表面に提示されるために要する時間、 反応させた後、 C TLと反応させる。 候補物質特異的なサイトカイン遊離又は増殖反応が認められ れば、 該物質は機能的誘導体であると判断出来る。 As a second method, a candidate substance is added to cells capable of presenting antigen, and the antigen is incorporated into the cells and processed for an appropriate period of time, and the antigen peptide and HLA molecule complex are displayed on the cell surface. After reacting for the required time, react with CTL. If a candidate substance-specific cytokine release or proliferation reaction is observed, it can be determined that the substance is a functional derivative.
第 3の方法としては、 後述の抗原提示能を有する細胞上の HLA— A 24分子 上にぺプチドを提示させることが可能な発現ベクターに候補物質のァミノ酸配列 をコードする核酸を結合させ、 該組換えべクタ一により形質転換された抗原提示
能を有する細胞と CTLを反応させる。 候補物質特異的なサイトカイン遊離又は 増殖反応が認められれば、 該物質は機能的誘導体であると判断出来る。 As a third method, a nucleic acid encoding an amino acid sequence of a candidate substance is bound to an expression vector capable of presenting a peptide on HLA-A24 molecule on a cell having antigen presenting ability described below, Antigen presentation transformed by the recombinant vector The CTL is reacted with cells having the ability. If a candidate substance-specific cytokine release or proliferation reaction is observed, it can be determined that the substance is a functional derivative.
上記機能的誘導体としては、 例えば、 配列番号 1〜6いずれかに記載のァミノ 酸配列で表されるぺプチドのァミノ酸配列において、 H L A— A 24分子への結 合を強めるために、 N末端より 2番目のアミノ酸が HLA— A24分子に結合す るべプチドに特徴的な Ty r、 Ph e、 Me t、 Tr pより選択されるァミノ酸 に置換、 及び 又は C末端のァミノ酸が HL A— A 24分子に結合するべプチド に特徴的な Leu、 I 1 e、 Trp、 P h eより選択されるアミノ酸に置換され たペプチドのうち、 HLA— A24分子との結合能を有し、 HLA— A24分子 との複合体が配列番号 1〜 6レ、ずれか記載のぺプチドと HLA— A24分子との 複合体を CTLに認識されるべプチドが挙げられる。 Examples of the functional derivative include an amino acid sequence represented by any one of SEQ ID NOs: 1 to 6 in the amino acid sequence of the peptide, in order to strengthen the binding to the HLA-A24 molecule, The second amino acid is substituted with an amino acid selected from Tyr, Phe, Met, and Trp characteristic of a peptide that binds to an HLA-A24 molecule, and / or the amino acid at the C-terminal is HLA. — Among the peptides substituted with amino acids selected from Leu, I1e, Trp, and Phe, which are characteristic of peptides that bind to A24 molecule, HLA— HLA- capable of binding to A24 molecule, A complex with an A24 molecule is represented by SEQ ID NO: 1 to 6, and a peptide in which CTL recognizes a complex between a peptide described in any of the above and HLA-A24 molecule.
また、 配列番号 1〜 6いずれかに記載のァミノ酸配列の 1又は数個のァミノ酸 が置換されたァミノ酸配列で表されるぺプチドにおいて、 置換されるそれぞれの ァミノ酸と側鎖の類似したァミノ酸若しくはアミノ酸アナログに置換したぺプチ ドのうち、 HLA— A24分子と結合能を有し、 HLA— A24分子との複合体 が本発明の CTLに認識されるべプチドも挙げられる。 側鎖の類似したアミノ酸 としては、 例えば、 グリシン （G 1 ) とァラニン （Al a) ；バリン （Va 1 ) 、 イソロイシン （I 1 e)、 ロイシン （Leu) とメチォニン （Me t) ；ァ スパラギン （As n) とグル夕ミン （G 1 n) ；ァスパラギン酸 （As p) とグ ル夕ミン酸 （G 1 u) ；セリン （S e r) とスレオニン （Th r) ； リジン （L y s) とアルギニン （Ar g) ； フエ二ルァラニン （Ph e) とチロシン （Ty r)が挙げられる。 In addition, in a peptide represented by an amino acid sequence in which one or several amino acids of the amino acid sequence described in any one of SEQ ID NOs: 1 to 6 are substituted, the similarity of each amino acid to be substituted and the side chain Among the peptides substituted with an amino acid or amino acid analog, those which have a binding ability to an HLA-A24 molecule and whose complex with the HLA-A24 molecule is recognized by the CTL of the present invention are also exemplified. Examples of amino acids having similar side chains include glycine (G 1) and alanine (Al a); valine (Va 1), isoleucine (I 1 e), leucine (Leu) and methionine (Me t); Asn) and glumin (G1n); aspartic acid (Asp) and gluminic acid (G1u); serine (Ser) and threonine (Thr); lysine (Lys) and arginine (Ar g); phenylalanine (Phe) and tyrosine (Tyr).
例えば、 配列番号 4に示す抗原ペプチドの機能的誘導体として、 N末端より 6 番目のアミノ酸 （Va 1)を I 1 eに置換した配列番号 24に記載のアミノ酸配 列で表されるペプチド、 N末端より 8番目のアミノ酸 （G l y) を Al aに置換 した配列番号 46に記載のアミノ酸配列で表されるペプチドが挙げられる。 一方
、 配列番号 5に示す抗原ペプチドの機能的誘導体として、 N末端より 6番目のァ ミノ酸 （Va 1) を I 1 e、 Me tにそれぞれ置換した配列番号 53、 55に記 載のアミノ酸配列で表されるペプチド、 又は N末端より 4番目のアミノ酸 （Cy s ) を Cy sのチオール基と 4, 5—ジハイドロキシー 2—シクロペンテン一 1 —オン (4， 5— d i hydr oxy— 2— eye 1 open t en- 1 -οη e ：以下 DHCPと略す） との反応により得られる生成物に置換した配列番号 3 3に記載したペプチドが挙げられる。 なお、 N末端より 4番目のアミノ酸は、 C y sのチオール基と N—ェチルマレイミ ド （N— E thy lma l e imi de ) 、 ジチオスレィトール （D i t h i 0 t h r e i t 01 )、 4一ビニルピリジ ン （4— Vi ny l pyr i d i ne) との反応により得られる生成物であって もよい。 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 SEQ ID NO: 24 in which the sixth amino acid (Va 1) from the N-terminus is substituted with I 1 e, And a peptide represented by the amino acid sequence of SEQ ID NO: 46 in which the eighth amino acid (Gly) is substituted with Ala. on the other hand As a functional derivative of the antigenic peptide shown in SEQ ID NO: 5, the amino acid sequence described in SEQ ID NOS: 53 and 55 in which the amino acid (Va 1) at the sixth position from the N-terminus has been substituted with I 1 e and Met, respectively. The peptide represented or the fourth amino acid (Cy s) from the N-terminus is substituted with a thiol group of Cy s and 4,5-dihydroxy-2-cyclopenten-1-one (4,5-dihydroxy—2-eye) 1 open ten-1 -οη e: hereinafter abbreviated as DHCP). The fourth amino acid from the N-terminus is the Cys thiol group and N-Ethylmaleimid, dithiothreitol (Dithi 0 threit 01), and 4-vinylpyridine (4 — It may be a product obtained by reaction with Viny l pyr idi ne).
また、 上記機能的誘導体としては、 側鎖の類似していないアミノ酸の置換であ つてもよく、 例えば、 配列番号 1〜6いずれかに記載のアミノ酸配列で表される ぺプチドのァミノ酸配列において、 1又は数個のァミノ酸が他のァミノ酸又はァ ミノ酸アナログ、 例えば、 Al aに置換したアミノ酸配列で表されるペプチドの うち、 HLA— A24分子と結合能を有し、 HLA— A24分子との複合体が配 列番号 1〜 6レ、ずれか記載のぺプチドと HLA— A24分子との複合体を認識す る CTLに認識されるべプチドが挙げられる。 The functional derivative may be a substitution of an amino acid having no similar side chain, 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. One or several amino acids, other amino acids or amino acid analogs, for example, peptides represented by the amino acid sequence substituted with Ala, having the ability to bind to the HLA-A24 molecule; The complex with the molecule is SEQ ID NO: 1 to 6, and the peptide described in any of the above and the peptide recognized by CTL which recognizes the complex with the HLA-A24 molecule.
例えば、 配列番号 4に示す抗原ペプチドの機能的誘導体として、 配列番号 39 、 40、 42〜45、 48いずれかに記載のアミノ酸配列で表されるペプチド、 配列番号 5に示す抗原ペプチドの機能的誘導体として、 配列番号 26、 27、 3 0、 32に記載のアミノ酸配列で表されるペプチドが挙げられる。 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, and 48, and a functional derivative of the antigenic peptide shown in SEQ ID NO: 5 Examples include the peptides represented by the amino acid sequences of SEQ ID NOs: 26, 27, 30 and 32.
なお、 上記ペプチドのうち Al aに置換可能である位置のアミノ酸は、 本来の ァミノ酸と異なる更に他のァミノ酸、 又はァミノ酸アナログに置換できる可能性 がある。 例えば、 配列番号 5記載のアミノ酸配列で表される抗原ペプチドの 4番 目のアミノ酸は、 Al aの他、 Asp、 G 1 u等の酸性アミノ酸以外のアミノ酸
若しくはアミノ酸アナログに置換することができる。 例えば、 配列番号 5 1又は 52記載のァミノ酸配列で表される、 S e r又は G 1 yで置換されたべプチドが 挙げられる。 In addition, the amino acid at the position that can be substituted for Ala in the above peptide may be able to be substituted with another amino acid or an amino acid analog different from the original amino acid. For example, the fourth amino acid of the antigenic peptide represented by the amino acid sequence set forth in SEQ ID NO: 5 is an amino acid other than an acidic amino acid such as Asa and G1u in addition to Ala. Alternatively, it can be substituted with an amino acid analog. For example, a peptide substituted with Ser or Gly represented by the amino acid sequence of SEQ ID NO: 51 or 52 can be mentioned.
また、 ジスルフイ ド結合等によるペプチドの 2量体としては、 システィンのチ オール基を介したジスルフィ ド結合による配列番号 5記載のァミノ酸配列で表さ れるぺプチドの 2量体が例示される。 Examples of the dimer of a peptide formed by a disulfide bond or the like include a dimer of a peptide represented by the amino acid sequence of SEQ ID NO: 5 formed by a disulfide bond via a thiol group of cysteine.
上記の他、 例えば配列番号 1に記載されたァミノ酸配列からなるぺプチドの機 能的誘導体には、 配列番号 1記載のァミノ酸配列からなるぺプチドを用いて誘導 した CTLに認識される、 MAGE— 3を始めとする MAGEファミリ一タンパ ク質由来のぺプチドも含まれる。 同様に配列番号 2に記載されたァミノ酸配列で 表されるぺプチドの機能的誘導体には、 配列番号 2記載のァミノ酸配列で表され るべプチドで誘導した CTLに認識される、 MAGE— 3を始めとする MAGE ファミリータンパク質由来のペプチドも含まれる。 配列番号 3に記載されたアミ ノ酸配列で表されるぺプチドの機能的誘導体には、 配列番号 3記載のァミノ酸配 列で表されるペプチドで誘導した CTLに認識される、 MAGE— 1を始めとす る MAGEファミリータンパク質由来のペプチドも含まれる。 In addition to the above, for example, functional derivatives of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 include those recognized by CTL induced using the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1. MAGE-3 and other peptides derived from MAGE family proteins are also included. Similarly, functional derivatives of the peptide represented by the amino acid sequence of SEQ ID NO: 2 include MAGE-recognized by the CTLs induced by the peptide represented by the amino acid sequence of SEQ ID NO: 2. 3 and other peptides derived from MAGE family proteins. Functional derivatives of the peptide represented by the amino acid sequence shown in SEQ ID NO: 3 include MAGE-1 which is recognized by CTL induced by the peptide represented by the amino acid sequence shown in SEQ ID NO: 3. And peptides derived from MAGE family proteins.
配列番号 4に記載されたァミノ酸配列からなるぺプチドの機能的誘導体には、 配列番号 4記載のァミノ酸配列からなるべプチドを用いて誘導した C T Lに認識 される、 CE A及び非特異的交差反応性抗原 （non-sp e c i f i c c r o s s— r e a c t i ng an t i gen : NC A) 由来のぺプチドも挙げら れる。 配列番号 5に記載されたァミノ酸配列で表されるぺプチドの機能的誘導体 には、 配列番号 5記載のァミノ酸配列で表されるぺプチドで誘導した C T Lに認 識される、 CEA及び NCA由来のペプチドも挙げられる。 Functional derivatives of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 include CEA and non-specific, which are recognized by CTLs induced using the peptide consisting of the amino acid sequence shown in SEQ ID NO: 4. Peptides derived from non-specific cross-reacting antigens (NCA) are also included. Functional derivatives of the peptide represented by the amino acid sequence represented by SEQ ID NO: 5 include CEA and NCA recognized by CTLs induced by the peptide represented by the amino acid sequence represented by SEQ ID NO: 5. Peptides derived therefrom are also included.
以下、 本明細書において配列番号 1又は 2で表されるぺプチド及びその機能的 誘導体を MAGE— 3抗原べプチド並びに配列番号 3で表されるぺプチド及びそ の機能的誘導体を MAGE— 1抗原ペプチドと総称する。 同様に配列番号 4又は
5で表されるぺプチド及びその機能的誘導体を CE A抗原べプチド、 配列番号 6 で表されるぺプチド及びその機能的誘導体を HER 2Zn e u抗原べプチドと総 称する。 以下本明細書において使用する 「抗原ペプチド」 とは、 特に断りの無い 限り MAGE - 3抗原ペプチド、 MAGE— 1抗原ペプチド、 CE A抗原べプチ ド及び/又は HER 2/n e u抗原べプチドから任意に選択されたべプチドであ ることを示す。 Hereinafter, the peptide represented by SEQ ID NO: 1 or 2 and the functional derivative thereof are referred to as MAGE-3 antigen peptide and the peptide represented by SEQ ID NO: 3 and the functional derivative thereof are referred to as MAGE-1 antigen. Collectively referred to as peptides. Similarly, SEQ ID NO: 4 or The peptide represented by 5 and its functional derivative are collectively referred to as CEA antigen peptide, and the peptide represented by SEQ ID NO: 6 and its functional derivative are collectively referred to as HER2Zneu antigen peptide. Hereinafter, the “antigen peptide” used in the present specification may be any of MAGE-3 antigen peptide, MAGE-1 antigen peptide, CEA antigen peptide and / or HER2 / neu antigen peptide unless otherwise specified. Indicates that this is the selected peptide.
抗原べプチド及ぴその誘導体は、 液相又は固相のァミノ酸の力ップリングによ る有機化学的方法により調製することができる。 また、 抗原ペプチドのアミノ酸 配列をコードした核酸を用いた組換え DN A技術を利用しても調製することがで きる。 組換え DNA技術によって得られたぺプチドは必要に応じ適当な有機化学 的又は生化学的手法等を用いることにより、 修飾してもよい。 Antigen peptides and derivatives thereof can be prepared by an organic chemical method by force coupling of a liquid phase or solid phase amino acid. It can also be prepared using recombinant DNA technology using a nucleic acid encoding the amino acid sequence of the antigenic peptide. The peptide obtained by the recombinant DNA technology may be modified as necessary by using an appropriate organic chemical or biochemical technique.
本発明の CTLにおいて、 MAGE— 3抗原べプチドを用い誘導された CTL は HLA— A24及び MAGE— 3共に陽性の腫瘍細胞を、 MAGE - 1抗原べ プチドを用い誘導された CTLは HLA— A24及び MAGE— 1共に陽性の腫 瘍細胞を、 〇£八抗原ぺプチドを用ぃ誘導された〇丁しは1 1^ ー八24及び C E A共に陽性の腫瘍細胞を、 HER 2Zn e u抗原べプチドを用い誘導された C TLは HLA - A 24及び HER 2 Zn e u共に陽性の腫瘍細胞を、 それぞれ選 択的に傷害できることから腫瘍に対する細胞医薬として、 また該 C T L感受性紬 胞の検出等に使用することができる。 In the CTL of the present invention, CTLs induced using MAGE-3 antigen peptide are tumor cells positive for both HLA-A24 and MAGE-3, and CTLs induced using MAGE-1 antigen peptide are HLA-A24 and Tumor cells positive for both MAGE-1 and tumor cells positive for both induced by using 24²8 antigen peptide and A²8-24 and CEA were used for HER2Zn eu antigen peptide. The induced CTL can selectively injure HLA-A24 and HER2Zneu-positive tumor cells, respectively, so that it can be used as a cell medicine for tumors and for the detection of such CTL-sensitive cells. it can.
本発明の第 2の態様は第 1の態様の C T Lを有効成分とする制がん剤に関する 。 該制がん剤は、 該 CTLを医薬的に許容される希釈剤に懸濁した形で提供され る。 なお、 ここで言う希釈剤とは例えば該 CTLの保存に適した培地、 生理食塩 水、 又はリン酸緩衝生理食塩水である。 培地としては RPMI、 AIM— Vなど の培地が一般的に挙げられる。 また該制がん剤には医薬的に許容される担体が安 定化の目的で添加されていてもよい。 なおここで言う担体とはヒト血清アルブミ ン等である。 該制がん剤には第 1の態様の CTLを、 CTL 1種類当り 1 04〜
1 08 個 Zm 1、 好ましくは 5 x 1 05 〜5 x 1 07 個 1含有させる。 A second aspect of the present invention relates to an anticancer agent comprising the CTL of the first aspect as an active ingredient. The anticancer agent is provided in a form in which the CTL is suspended in a pharmaceutically acceptable diluent. The diluent mentioned here is, for example, a medium, physiological saline, or phosphate buffered saline suitable for storing the CTL. As the medium, a medium such as RPMI or AIM-V is generally used. Further, a pharmaceutically acceptable carrier may be added to the anticancer agent for the purpose of stabilization. The carrier mentioned here is human serum albumin or the like. The CTL of the first aspect to該制cancer agent, CTL 1 kind per 0 4 - 10 8 Zm 1, preferably 5 × 10 5 to 5 × 10 7
上記制がん剤をヒ卜に投与する場合注射器で投与することができ、 成人 1人当 りの投与量としては通常 CTL数が、 CTL 1種類当り 1 06 〜1 01(1個となる ようにする。 なお上記範囲は目安でありこれに限定されるものではない。 また有 効成分である CTLは投与するヒト由来のものであるため、 該制がん剤の毒性は 特に認められない。 The carcinostatic agent may be administered with a syringe for administration to a human Bok, adult human normal CTL numbers as those Rino dose, CTL 1 kind per 0 6-1 0 1 (1 and The above range is only a guide and is not limited to the above.Since the active ingredient CTL is derived from the human being administered, the toxicity of the anticancer agent is particularly recognized. Absent.
本発明の制がん剤は、 有効成分として含有する C T Lが認識可能な抗原べプチ ドを発現している腫瘍細胞を有するがん患者に投与されるカ 例えば、 MAGE は当初メラノ一マ患者の悪性腫瘍細胞から同定された抗原であるが、 その後メラ ノーマ以外のがん患者の約 1 0〜50%の頻度で発現していることが確認されて いる。 MAGE— K MAGE— 2又は MAGE— 3の発現している腫瘍細胞と してはメラノ一マのほか、 例えば肺がん、 乳がん、 頭けい部がん、 胃がん、 食道 がん、 膀胱がん等に由来する腫瘍細胞が挙げられる 〔B. ゴーグラー （B.Gaugle r ) ら、 ジャーナル ォブ イクスペリメンタル ォブ メディスン、 第 1 79 巻、 第 921〜930頁 （1 994 ) 〕 。 したがって、 例えば、 MAGE— 3抗 原べプチドを用レ、誘導された本発明の第 1の態様の C T Lを有効成分として含有 する制がん剤は H L A— A 24及び MA GE- 3共に陽性の腫瘍細胞よりなる上 記がんを有する患者の治療に有用である。 The anticancer agent of the present invention is administered to a cancer patient having a tumor cell expressing an antigen peptide capable of recognizing CTL contained as an active ingredient.For example, MAGE is initially administered to a melanoma patient. It is an antigen identified from malignant tumor cells, but it has been subsequently confirmed that it is expressed at a frequency of about 10 to 50% of cancer patients other than melanoma. MAGE-K MAGE-2 or MAGE-3 expressing tumor cells are derived from melanoma, lung cancer, breast cancer, cervix cancer, stomach cancer, esophagus cancer, bladder cancer, etc. B. Gougler et al., Journal of Experimental Medicine, Vol. 179, pp. 921-930 (1994)]. Therefore, for example, the anticancer agent containing the CTL of the first aspect of the present invention as an active ingredient using the MAGE-3 antigen peptide as an active ingredient is positive for both HLA-A24 and MAGE-3. It is useful for treating patients with the above cancers consisting of tumor cells.
なお、 上記制がん剤を用いる場合他の制がん剤と併用してもよいが、 免疫抑制 的に働く制がん剤の併用は好ましくない。 When the above anticancer agent is used, it may be used in combination with other anticancer agents, but it is not preferable to use an anticancer agent which acts as an immunosuppressant.
本発明の第 3の態様は、 第 1の態様の CTLの誘導方法に関する。 該 CTLは 、 配列番号 1〜 6のいずれかに記載のァミノ酸配列で表される H L A— A 24拘 束性抗原べプチド及びその機能的誘導体から選択される少なくとも一つの抗原べ プチドを使用することにより誘導することができる。 A third aspect of the present invention relates to the method for inducing CTL of the first aspect. The CTL uses at least one antigen peptide selected from the HLA-A24 binding antigen peptide represented by the amino acid sequence of any one of SEQ ID NOs: 1 to 6 and a functional derivative thereof. Can be guided by
イン ビトロ （i n v i t r o) で該 CTLを誘導する場合、 例えば HLA 一 A 24を発現している生体よりの体外摘出試料を用い誘導することができる。
本明細書における体外摘出試料とは、 血液のほか、 手術などにより摘出したリン パ節、 脾臓、 その他各種臓器が包含され、 これらの試料に存在するリンパ球や浸 潤リンパ球細胞が好適に使用される。 When the CTL is induced in vitro, it can be induced using, for example, a sample extracted from a living body expressing HLA-A24. The extracorporeally extracted sample in this specification includes, in addition to blood, lymph nodes, spleen, and various other organs extracted by surgery or the like, and lymphocytes and infiltrating lymphocyte cells present in these samples are preferably used. Is done.
例えば血液を用いる場合、 HLA— A24陽性のヒト血液より調製した末梢血 単核球 (pe r i phe r a l b l ood mononuc l ear e e l 1 s ；以下、 P BMCと略す） より得られるリンパ球に繰り返し抗原刺激を与え ることにより CTLを誘導することができる。 抗原刺激は、 例えばリンパ球を調 製した同一血液より調製した抗原提示能を有する細胞に抗原べプチドを H LA- A 24分子との複合体として細胞表面に提示した抗原提示細胞等を用いることに より実施できる。 誘導された CTLは、 更に抗 CD— 3抗体等による刺激や抗原 ぺプチド存在下各種刺激を加え増殖させることができる。 例えば I L一 7存在下 、 PBMCより調製したリンパ球 1に対し抗原提示細胞を 0. 01〜1の割合で 混合することにより CTL誘導を実施することができる。 別の態様として、 I L 一 7及びキーホール リムペット へモシァニン （K e y h 01 e L impe t Hemocyan i n ; KLH)存在下、 PBMCに抗原べプチドを細胞培 養液中、 抗原べプチド 1種類当り 1 n gZm 1〜 100 g/m 1、 好ましくは 1 OngZml〜l gZm 1添加することにより C T L誘導を実施することが できる。 For example, in the case of using blood, antigen stimulation is repeatedly performed on lymphocytes obtained from peripheral blood mononuclear cells prepared from HLA-A24-positive human blood (Peripheral monolide 1 s; hereinafter, abbreviated as PBMC). Can induce CTL. For antigen stimulation, for example, use antigen-presenting cells, etc., which present antigen-peptide on the cell surface as a complex with HLA-A24 molecule on cells having antigen-presenting ability prepared from the same blood prepared from lymphocytes. This can be implemented. The induced CTL can be proliferated by further stimulation with an anti-CD-3 antibody or various stimuli in the presence of an antigen peptide. For example, in the presence of IL-17, CTL induction can be performed by mixing antigen presenting cells with lymphocytes 1 prepared from PBMC at a ratio of 0.01 to 1. In another embodiment, in the presence of IL-17 and keyhole rimpet hemocyanin (Keyh01eLimpet Hemocyanin; KLH), the antigen peptide is added to PBMC in a cell culture medium, and 1 n per antigen peptide is used. CTL induction can be performed by adding gZm 1 to 100 g / m 1, preferably 1 Ong Zml to 1 gZm 1.
誘導された C T Lは、 クローン化することにより安定した細胞傷害性を有する リンパ球として維持することもできる。 例えば、 誘導された CTLに抗原、 各種 サイトカイン、 抗 CD 3抗体刺激を与えることにより増殖させることができる。 誘導された特異的 C T Lは、 C T Lを誘導した抗原べプチドと放射性物質等で 標識した標的細胞に対する傷害性、 放射能の取り込みによって測定できる C T L 増殖の抗原特異的な増加若しくは CTLや標的細胞より抗原特異的に遊離される GM-CSF, I FN—《y等のサイトカイン量を測定することにより検出するこ
とができる。 その他蛍光色素等によって標識された抗原べプチドゃ複合体の使用 によって直接確認することもできる。 この場合、 例えば CTLを CTL特異的抗 体とカツプリングさせた第 1蛍光マーカーと接触させてから第 2蛍光マ一カーと カップリングさせた抗原べプチドー MH C複合体を接触させ、 そして二重標識細 胞の存在を F ACS (fluorescence-activated cell sorting)分析によって行 ことができる。 The induced CTLs can be maintained as stable cytotoxic lymphocytes by cloning. For example, the induced CTLs can be proliferated by stimulation with antigens, various cytokines, and anti-CD3 antibodies. The specific CTLs induced are the antigen-specific increase of CTL proliferation or the antigen-induced increase in CTLs or target cells, which can be measured by the toxicity to the target cells labeled with the radioactive substance and the antigen peptide that induced the CTLs. GM-CSF, IFN specifically released-Detectable by measuring the amount of cytokines such as y Can be. In addition, it can also be directly confirmed by using an antigen peptide-conjugate labeled with a fluorescent dye or the like. In this case, for example, CTL is brought into contact with a first fluorescent marker coupled to a CTL-specific antibody, and then brought into contact with an antigen-beptide MHC complex coupled with a second fluorescent marker, and then double-labeled. The presence of cells can be determined by FACS (fluorescence-activated cell sorting) analysis.
—方、 イン ビボ （i n v i vo) における CTLの誘導は、 例えば抗原べ プチドと HLA— A24分子との複合体として細胞表面に提示した抗原提示細胞 を生体に投与することにより実施することができる。 ヒトに投与する場合、 成人 1人当りの投与量は通常 104 〜109 個である。 別法として、 抗原ペプチドを 適当なアジュバントと混合し生体に投与することにより、 該ぺプチドに特異的な CTLを誘導することもできる。 アジュバントとしては、 ①フロイント （Fr e und)完全アジュバント、 ②フロイント不完全アジュバント、 ③水酸化アルミ 二ゥム、 みょうばん等の無機物ゲル、 ④リゾレシチン、 ジメチルォクタデシルァ ンモニゥムブロミ ド等の界面活性剤、 ⑤硫酸デキストラン、 ポリ I C等のポリア 二オン、 ⑥ムラミルぺプチド、 タフトシン等のぺプチド、 ⑦リビ （R i b i ) 社 製のモノホスホリルリピド （monophosphoryl lipid; MP L) A等があるが、 特 に限定されない。 生体における CTLの誘導には抗原べプチドとアジュバントの 混合物のほか、 MH Cクラス 11拘束性のヘルパー T細胞抗原べプチドを混合し投 与してもよい。 なお、 該抗原ペプチドは生体内における安定化のため適当なリン カーを介して高級脂肪酸やヘルパー T細胞抗原べプチドと共有結合させ投与して もよい。 ヒトに投与する場合、 成人 1人当りの投与量は抗原ペプチド濃度として 、 抗原ペプチド 1種類当り 0. 1 zgZkg〜l 0mgZkg、 好ましくは 1 g/kg~lmg/kg, 更に好ましくは 1 g/kg〜l 0 O^gZkgであ る。 On the other hand, the induction of CTLs in vivo can be carried out, for example, by administering an antigen-presenting cell presented on the cell surface as a complex of an antigen peptide and an HLA-A24 molecule to a living body. When administered to humans, the dose per adult is usually 10 4 to 10 9 . Alternatively, CTLs specific to the peptide can be induced by mixing the antigenic peptide with an appropriate adjuvant and administering the mixture to a living body. Adjuvants include: (1) Freund's complete adjuvant, (2) Freund's incomplete adjuvant, (3) inorganic gels such as aluminum hydroxide and alum, (4) surfactants such as lysolecithin and dimethyloctadecyl ammonium bromide,ポ リ Polyanion such as dextran sulfate and poly IC; ぺ such as muramyl peptide and tuftsin; and 、 monophosphoryl lipid (MPL) A manufactured by Ribi. Not limited. For the induction of CTL in a living body, a mixture of an antigen peptide and an adjuvant, or a helper T cell antigen peptide restricted to MHC class 11 may be mixed and administered. The antigen peptide may be covalently bonded to a higher fatty acid or a helper T cell antigen peptide via an appropriate linker for stabilization in a living body, and may be administered. When administered to humans, the dose per adult is 0.1 zgZkg to 10 mgZkg per antigen peptide, preferably 1 g / kg to 1 mg / kg, more preferably 1 g / kg per antigen peptide concentration. ~ L 0 O ^ gZkg.
CTLを誘導するための抗原べプチドによる刺激のために、 抗原べプチドと H
LA-A24分子との複合体を用いることもできる。 この場合 HLA— A24分 子は天然の HLA— A24分子である必要はなく、 抗原ペプチドとの結合性を本 質的に保存している断片であってもよい。 これらの断片は、 例えば、 天然の HL A— A 24分子のタンパク分解又は組換え D N A技術により調製することができ る。 この複合体は yfi 2 —ミクログロブリン、 更に複合体を認識する抗体を共存さ せて安定化させることができる。 For stimulation with antigen peptides to induce CTLs, antigen peptides and H A complex with the LA-A24 molecule can also be used. In this case, the HLA-A24 molecule does not need to be a natural HLA-A24 molecule, but may be a fragment that essentially preserves the binding to the antigenic peptide. These fragments can be prepared, for example, by proteolysis of natural HLA-A24 molecules or by recombinant DNA technology. This complex can be stabilized by coexisting yfi 2 -microglobulin and an antibody that recognizes the complex.
本発明の第 4の態様は、 配列番号 1〜 6のいずれかに記載のァミノ酸配列で表 される HLA— A24拘束性抗原べプチド及びその機能的誘導体から選択される 少なくとも一つの抗原ペプチドを有効成分とした CTL誘導剤に関する。 CTL 誘導剤は、 抗原ペプチドを単独又は他の分子 （ヘルパー T細胞抗原ペプチド及び ノ又はアジュバント） との混合物として生理食塩水又はリン酸緩衝生理食塩水に 懸濁した形で供給される。 該抗原ペプチドは、 高級脂肪酸やヘルパー T細胞抗原 ぺプチドとの共有結合体あるいは HLA— A 24分子との複合体としてもよい。 該誘導剤には抗原べプチドを、 抗原べプチド 1種類当り 0. 0 1〜 1 00重量％ 、 好ましくは 0. 1〜95重量％含有させる。 A fourth aspect of the present invention relates to a method for preparing at least one antigen peptide selected from the HLA-A24-restricted antigen peptide represented by the amino acid sequence of any one of SEQ ID NOs: 1 to 6 and a functional derivative thereof. It relates to a CTL inducer as an active ingredient. The CTL inducer is supplied in the form of an antigenic peptide alone or as a mixture with another molecule (helper T cell antigenic peptide and amino acid or adjuvant) suspended in physiological saline or phosphate buffered saline. The antigen peptide may be a covalent complex with a higher fatty acid or a helper T cell antigen peptide or a complex with an HLA-A24 molecule. The inducer contains an antigen peptide in an amount of 0.01 to 100% by weight, preferably 0.1 to 95% by weight, per one kind of the antigen peptide.
該 CTL誘導剤は、 イン ビトロで本発明の CTLを増殖させるための培地へ の添加物、 Tリンパ球増殖活性、 遅延型皮膚反応を指標とした免疫感作状態の診 断などに利用することができる。 例えば培地への添加物として使用する場合、 使 用量は培地中のぺプチド濃度として、 抗原べプチド 1種類当り 1 n gZm 1〜 1 00 z g/m 1、 好ましくは 1 O n g/m 1〜 1 g/m 1である。 The CTL inducer may be used as an additive to a culture medium for growing the CTL of the present invention in vitro, or for diagnosing an immunization state using T lymphocyte proliferation activity or delayed skin reaction as an index. Can be. For example, when used as an additive to a culture medium, the amount used is 1 ng Zm 1 to 100 zg / m 1 per antigen peptide, preferably 1 ng / m 1 to 1 as the peptide concentration in the medium. g / m1.
本発明の第 5の態様は、 配列番号 1〜 6のいずれかに記載のァミノ酸配列で表 される HL A— A 24拘束性抗原べプチド及びその機能的誘導体から選択される 少なくとも一つの抗原べプチドを有効成分とした制がん剤に関する。 該制がん剤 は、 1) 抗原ペプチドを単独、 2) 抗原ペプチドと医薬的に許容される担体及び 又は希釈剤との混合物、 又は 3) 更に必要ならば上記 1 ) 若しくは 2) に補助 剤を加えた形で提供される。 なおここで言う担体とは例えば、 ヒト血清アルブミ
ンであり、 また希釈剤とは例えばリン酸緩衝液、 蒸留水、 生理食塩水等である。 また補助剤とは薬学的に許容される上記アジュバント等が挙げられる。 該制がん 剤をヒトに投与する場合、 注射器で投与することもできるし、 噴霧等の方法で粘 膜からの経皮吸収等で投与してもよい。 該制がん剤には抗原ペプチドを、 抗原べ プチド 1種類当り 0. 01〜100重量％、 好ましくは 0. 1〜95重量％含有 させる。 成人 1人当りの投与量はペプチド濃度として、 抗原ペプチド 1種類当り 0. l〃gZkg〜10mgZkg、 好ましくは 1〃g/kg〜lmg/ kg、 更に好ましくは 1 gZkg〜l 00 zgZkgである。 なおヒトに投与した場 合、 該ペプチドの毒性は特に認められない。 According to a fifth aspect of the present invention, there is provided at least one antigen selected from the HLA-A24-restricted antigen peptide represented by the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 6 and a functional derivative thereof. The present invention relates to an anticancer drug containing a peptide as an active ingredient. The anticancer agent comprises 1) an antigenic peptide alone, 2) a mixture of an antigenic peptide and a pharmaceutically acceptable carrier and / or a diluent, or 3) a supplement as described in 1) or 2) above if necessary. Is provided with The carrier used here is, for example, human serum albumin The diluent is, for example, a phosphate buffer, distilled water, physiological saline, or the like. The adjuvant includes the pharmaceutically acceptable adjuvants and the like. When the anticancer agent is administered to humans, it may be administered with a syringe, or may be administered by percutaneous absorption through a mucous membrane by a method such as spraying. The anticancer agent contains 0.01 to 100% by weight, preferably 0.1 to 95% by weight, of the antigen peptide per antigen peptide. The dose per adult is 0.1 l〃gZkg to 10 mgZkg, preferably 1〃g / kg to 1 mg / kg, more preferably 1 gZkg to 100 zgZkg per antigen peptide as the peptide concentration. When administered to humans, the toxicity of the peptide is not particularly observed.
該制がん剤を使用する場合、 他の制がん剤と併用してもよいが免疫抑制的に働 く制がん剤の併用は好ましくない。 When the anticancer agent is used, it may be used in combination with other anticancer agents, but it is not preferable to use an anticancer agent which acts immunosuppressively.
本発明の第 6の態様は、 配列番号 1〜 6のいずれかに記載のァミノ酸配列で表 される HLA— A24拘束性抗原べプチド及びその機能的誘導体から選択される 少なくとも一つの抗原ペプチドと HLA— A 24分子との複合体をその細胞表面 に提示する抗原提示細胞に関する。 以下、 本明細書における 「抗原提示細胞」 と は、 特に断りの無い限り MAGE— 3抗原ペプチド、 MAGE— 1抗原ペプチド 、 CE A抗原べプチド及び Z又は HER 2Zn e u抗原べプチドから選択される 少なくとも 1つの抗原べプチドを提示した抗原提示細胞を示す。 該抗原提示細胞 は、 本発明の CTLの誘導に用いることができる。 A sixth aspect of the present invention relates to an HLA-A24-restricted antigen peptide represented by the amino acid sequence of any one of SEQ ID NOs: 1 to 6 and at least one antigen peptide selected from functional derivatives thereof. The present invention relates to an antigen presenting cell which presents a complex with HLA-A24 molecule on its cell surface. Hereinafter, “antigen presenting cell” in the present specification means at least selected from MAGE-3 antigen peptide, MAGE-1 antigen peptide, CEA antigen peptide and Z or HER2Zn eu antigen peptide unless otherwise specified. 1 shows an antigen-presenting cell that presented one antigen peptide. The antigen presenting cells can be used for inducing the CTL of the present invention.
該抗原提示細胞は、 抗原提示能を有する細胞を用いることにより調製すること ができる。 抗原提示能を有する細胞には、 例えば抗原ペプチドと HL A— A 24 分子との複合体をその細胞表面に提示することが可能な、 マクロファージゃ B紬 胞、 樹状細胞 （DC： dendritic cells )を始めとする白血球細胞が挙げられる 。 DCは、 細胞当り抗原提示量が多く、 また抗原提示に必要な細胞表面分子 〔C D80、 CD 86等のコスティミュラトリー ·シグナル （c 0— s t imu 1 a t ory s i gna l)分子等〕 の発現量も高いため抗原提示能を有する細胞
として特に好適である。 上記抗原提示能を有する細胞は、 本明細書における体外 摘出試料より調製することができる。 例えば DCは、 1) PBMCよりいくつか の細胞表面マーカ一等を基に単離精製してくる、 2)単球より GM-CSFと IL-4によ り誘導してくる、 3) CD— 34陽性細胞より GM-CSF、 TNF-ひ、 SCF等のサイト カインで誘導してくる等いずれかの方法により調製することができる。 The antigen presenting cells can be prepared by using cells having antigen presenting ability. Cells that have the ability to present antigen include, for example, macrophage II B cells and dendritic cells (DC), which can present a complex of an antigen peptide and HLA-A24 molecule on the cell surface. And other white blood cells. DC has a large amount of antigen present per cell, and is a cell surface molecule necessary for antigen presentation (costimulatory signal (c0-stimu 1 at ory signal) molecules such as CD80 and CD86) Cells with high antigen expression ability due to high expression level It is particularly suitable as The cells having the antigen-presenting ability can be prepared from the in vitro extracted sample in the present specification. For example, DC is 1) isolated and purified from PBMC based on several cell surface markers, 2) derived from monocytes by GM-CSF and IL-4, 3) CD- It can be prepared by any method such as induction from 34 positive cells with cytokines such as GM-CSF, TNF-H, and SCF.
抗原提示能を有する細胞表面に抗原べプチドを提示させるには、 例えば上記抗 原提示能を有する細胞と抗原ペプチドの少なくとも一つを混合後必要に応じて過 剰量を洗浄し、 HLA— A24分子上に抗原ペプチドを負荷させる方法が挙げら れる。 なお、 上記抗原提示能を有する細胞の調製方法における 1) の方法で調製 した細胞のように、 既に H L A— A 24分子上に本発明とは無関係の抗原べプチ ドを提示していると考えられる細胞は、 抗原べプチドを負荷する前に酸処理など を行うことにより、 HL A— A 24分子上に存在する本発明の CTLを誘導出来 ないぺプチドを除去してもよい。 該抗原提示能を有する細胞は単一の抗原べプチ ドが負荷された細胞のほか、 細胞 1個当り数種類の抗原べプチドが負荷されてい てもよい。 また、 別法として、 B細胞や CD 34陽性細胞に対して上記抗原ぺプ チドを提示させることのできる発現べクタ一により該細胞を形質転換する方法が 挙げられる。 このようなベクターとしては、 p cDNA3、 pMQMn e o. p CEP 4等の市販のプラスミ ドに、 組換え DN A技術によって細胞表面に提示さ せたい抗原べプチドの少なくとも 1つをコ一ドする遺伝子を組込んだ、 発現用プ ラスミ ドベクタ一が挙げられる。 更に該発現べクタ一は、 細胞表面に提示させた い抗原べプチドの両端に細胞内で効率良くプロテアーゼの分解を受けるように適 当なアミノ酸配列をコードする遺伝子が付加されていてもよい。 更に、 レトロゥ ィルスベクターやアデノウイルスベクターも好適に利用される。 In order to present the antigen peptide on the cell surface having antigen presenting ability, for example, after mixing at least one of the cells having antigen presenting ability and the antigen peptide, an excess amount is washed as necessary, and HLA-A24 There is a method of loading an antigen peptide onto a molecule. In addition, it is considered that an antigen peptide irrelevant to the present invention is already presented on the HLA-A24 molecule like the cell prepared by the method 1) in the method for preparing a cell having the antigen presenting ability described above. The cells to be obtained may be subjected to an acid treatment or the like before loading with the antigen peptide to remove the peptide present on the HLA-A24 molecule that cannot induce the CTL of the present invention. The cells having the antigen-presenting ability may be cells loaded with a single antigen peptide, or may be loaded with several kinds of antigen peptides per cell. As another method, there is a method of transforming B cells or CD34-positive cells with an expression vector capable of presenting the antigen peptide to the cells. Such vectors include commercially available plasmids such as pcDNA3, pMQMneo.pCEP4, and at least one antigen peptide to be displayed on the cell surface by recombinant DNA technology. An expression plasmid vector in which a gene has been incorporated. Further, the expression vector may have a gene encoding an appropriate amino acid sequence added to both ends of the antigen peptide to be displayed on the cell surface so that protease is efficiently decomposed in the cell. Further, retrovirus vectors and adenovirus vectors are also preferably used.
抗原提示細胞は、 非増殖性とすることが好ましい。 細胞を非増殖性とするため には、 X線等の放射線照射又はマイトマイシン （m i t omy c i n)等の薬剤 による処理を行えばよい。
本発明の第 7の態様は、 第 6の態様の抗原提示細胞を有効成分として含有する CTLの誘導剤に関する。 該誘導剤は、 該抗原提示細胞を、 細胞の保存に適した 培地、 生理食塩水、 又はリン酸緩衝生理食塩水に懸濁した形で供給される。 培地 としては RPMI、 A IM— V、 X— V I VO 10などの培地が一般的である。 また該培地又は緩衝液には血清ァルブミン等を安定化の目的で添加してもよレ、。 該誘導剤には抗原提示細胞を、 抗原提示細胞 1種類当り 103 〜5 X 106 個/ ml、 好ましくは 104 〜106 個 Zml含有させる。 Preferably, the antigen presenting cells are non-proliferative. In order to make the cells non-proliferating, irradiation with X-rays or the like or treatment with an agent such as mitomycin (mitomicin) may be performed. A seventh aspect of the present invention relates to a CTL inducer comprising the antigen-presenting cell 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, physiological saline, or phosphate-buffered saline suitable for storing the cells. As the medium, RPMI, AIM-V, X-VI VO10 and the like are generally used. Further, serum albumin or the like may be added to the medium or the buffer for the purpose of stabilization. Antigen presenting cells to the inducing agent, antigen-presenting cells one per 10 3 to 5 X 10 6 cells / ml, preferably is contained 10 4 to 10 6 ZML.
該 CTLの誘導剤は、 イン ビトロで本発明の CTLを増殖させるための培地 への添加物としての利用のほか、 Tリンパ球増殖活性を指標とした免疫感作状態 の診断などにも利用することができる。 例えば培地への添加物として使用する場 合、 CTLへ誘導するリンパ球 1に対し通常 0. 01〜1の割合で用いる。 本発明の第 8の態様は、 第 6の態様の抗原提示細胞を有効成分として含有する 制がん剤である。 該制がん剤は、 該抗原提示細胞を医薬的に許容される希釈剤に 懸濁した形で提供される。 ここで言う希釈剤とは、 細胞の保存に適した培地、 リ ン酸緩衝液、 生理食塩水等である。 培地としては RPMI、 A IM— V、 X-V I VO l 0などの培地が一般的に挙げられる。 また、 該制がん剤には医薬的に許 容される担体を安定化のために添加してもよい。 ここで言う担体とは、 ヒト血清 アルブミン等である。 該制がん剤には抗原提示細胞を、 抗原提示細胞 1種類当り 105〜 108 個 Zm 1、 好ましくは 5x l 05 〜5x l 07 個ノ m 1含有させ る。 該制がん剤をヒトに投与する場合注射器で投与することができ、 成人 1人当 りの投与量は抗原提示細胞数として、 抗原提示細胞 1種類当り通常 104 〜10 9 個である。 なお、 上記範囲はあくまで目安であり、 これに限定されるものでは ない。 また、 有効成分である抗原提示細胞は投与するヒト由来のものであるため 、 該制がん剤の毒性は特に認められない。 The CTL inducer is used not only as an additive to a medium for growing the CTL of the present invention in vitro, but also for diagnosing an immunization state using T lymphocyte proliferation activity as an index. be able to. For example, when used as an additive to a medium, it is usually used in a ratio of 0.01 to 1 per lymphocyte 1 induced to CTL. An eighth aspect of the present invention is a carcinostatic agent comprising the antigen-presenting cell of the sixth aspect as an active ingredient. The anticancer agent is provided in a form in which the antigen-presenting cells are suspended in a pharmaceutically acceptable diluent. The diluent mentioned here is a medium, a phosphate buffer, a physiological saline, or the like suitable for storing cells. Examples of the medium generally include RPMI, AIM-V, and XVIVO10. In addition, a pharmaceutically acceptable carrier may be added to the anticancer agent for stabilization. The carrier mentioned here is human serum albumin or the like. The該制cancer agent antigen-presenting cells, antigen presenting cells one per 10 5-10 8 Zm 1, preferably 5x l 0 5 ~5x l 0 7 amino Roh m 1 Ru is contained. When the anticancer agent is administered to humans, it can be administered with a syringe, and the dose per adult is usually 10 4 to 10 9 per antigen presenting cell as the number of antigen presenting cells. Note that the above range is only a guide and is not limited to this. In addition, since the antigen-presenting cells as the active ingredient are derived from the human being administered, no toxicity of the anticancer agent is observed.
該制がん剤を使用する場合、 他の制がん剤と併用してもよいが免疫抑制的に働 く制がん剤の併用は好ましくない。
本発明の第 9の態様は、 第 1の態様の CTLを被検細胞と接触させた際に生じ る変化を指標とした該 C T Lの感受性細胞の検出方法に関する。 C T Lと被検細 胞中の感受性細胞との接触により生じる変化としては、 例えば C T Lによる標的 細胞溶解、 サイト力イン遊離、 又は CTLの増殖が挙げられる。 標的細胞溶解の 検出は、 例えばラジオアイソト一プゃ蛍光色素により被検細胞を標識後 C T Lと 混合した際に、 被検細胞より遊離されるラジオァイソトープ量ゃ蛍光色素量を測 定することにより実施できる。 サイト力イン遊離の検出は、 CTL又は被検細胞 からの GM-CSF、 TNF、 IFN-ァ等の遊離量を測定することにより実施することがで きる。 また CTLの増殖は、 3 H—チミジンの細胞への取り込み量の測定や、 顕 微鏡観察などによる C T L細胞数の測定などにより実施することが出来る。 この 変化により、 被検細胞中に存在する HL A— A 24及び MAGE— 3共に陽性若 しくは HLA— A24及び M A G E— 3関連抗原共に陽性の腫瘍細胞、 H L A— A24及び MAGE— 1共に陽性若しくは HLA— A 24及び MAGE— 1関連 抗原共に陽性の腫瘍細胞、 HLA— A24及び CE A共に陽性若しくは HL A— A 24及び CE A関連抗原共に陽性の腫瘍細胞、 及び//又は^11^ ー八24及び HER 2/n e u共に陽性若しくは HLA— A 24及び HER 2 Zn e u関連抗 原共に陽性の腫瘍細胞を検出することが可能である。 When the anticancer agent is used, it may be used in combination with other anticancer agents, but it is not preferable to use an anticancer agent which acts immunosuppressively. A ninth embodiment of the present invention relates to a method for detecting a CTL-sensitive cell, using a change generated when the CTL of the first embodiment is brought into contact with a test cell as an index. The change caused by contact of the CTL with a sensitive cell in the test cell includes, for example, target cell lysis by the CTL, release of cytodynamic force, or proliferation of the CTL. To detect target cell lysis, for example, when the test cells are labeled with radioisotope fluorescent dye and mixed with CTL, the amount of radioisotope released from the test cells and the amount of fluorescent dye are measured. Can be implemented. Detection of cytoforce release can be performed by measuring the release amount of GM-CSF, TNF, IFN-α, etc. from CTLs or test cells. In addition, CTL proliferation can be carried out by measuring the amount of 3 H-thymidine incorporated into cells, or by measuring the number of CTL cells by microscopic observation. Due to this change, the tumor cells present in the test cells are positive for both HLA-A24 and MAGE-3 or positive for both HLA-A24 and MAGE-3 related antigens, and positive for both HLA-A24 and MAGE-1. Tumor cells positive for both HLA-A24 and MAGE-1 related antigens, tumor cells positive for both HLA-A24 and CEA or positive for both HLA-A24 and CEA related antigens, and / or ^ 11 ^ -8 It is possible to detect tumor cells positive for both 24 and HER2 / neu or positive for both HLA-A24 and HER2Zneu-related antigen.
本発明の第 1 0の態様は、 第 1の態様の CTLを有効成分として含有する、 第 1の態様の CTLに感受性の細胞の検出剤に関する。 該検出剤は、 該 CTLの保 存に適した培地、 生理食塩水、 又はリン酸緩衝生理食塩水に懸濁した形で供給さ れる。 培地としては RPMI、 八 11^ー 又は —¥ 1¥01 0などの培地が一 般的である。 また該培地又は緩衝液には血清アルブミン等を CTL安定化の目的 で添加してもよい。 該検出剤には第 1の態様の CTLを、 CTL 1種類当り 1 0 4〜1 08 個 Zml含有させる。 A tenth aspect of the present invention relates to an agent for detecting a cell sensitive to the CTL of the first aspect, comprising the CTL of the first aspect as an active ingredient. The detecting agent is supplied in a form suspended in a medium, physiological saline, or phosphate buffered saline suitable for storing the CTL. As a medium, a medium such as RPMI, octane or — ¥ 1 ¥ 100 is generally used. Serum albumin or the like may be added to the medium or buffer for the purpose of stabilizing CTL. The on the detected polishes the CTL of the first aspect, CTL 1 kind per 0 4 to 1 0 to 8 Zml contained.
該検出剤は、 被検細胞中における本発明の CTLに感受性細胞の検出の他、 被 検細胞に発現する HLAが HLA— A 24であるか同定する HLAタイビングに
も用いることが出来る。 The detection agent is used not only for detection of CTLs susceptible to the CTL of the present invention in test cells, but also for HLA typing for identifying whether HLA expressed in test cells is HLA-A24. Can also be used.
本発明の第 1 1の態様は、 HL A拘束性抗原ペプチドから選択される少なくと も一つの抗原べプチド存在下、 該抗原べプチドと HL A分子との複合体を認識可 能な C T Lを被検細胞と接触させることを特徴とする H L A分子の検出方法に関 する。 In a first embodiment of the present invention, a CTL capable of recognizing a complex of an antigen peptide and an HLA molecule in the presence of at least one antigen peptide selected from an HLA-restricted antigen peptide is provided. The present invention relates to a method for detecting an HLA molecule, which is brought into contact with a test cell.
例えば、 被検細胞表面上の HL A - A 24分子は、 第 1の態様の CTLと被検 細胞を接触させることによって生じる変化を指標に検出することが出来る。 CT Lと被検細胞の接触により生じる変化としては、 例えば C T Lによる標的細胞溶 解、 サイト力イン遊離、 又は CTLの増殖が挙げられる。 標的細胞溶解の検出は 、 例えばラジオァイソト一プや蛍光色素により被検細胞を標識後 CTLと混合し た際に、 被検細胞より遊離されるラジオァイソトープ量ゃ蛍光色素量を測定する ことにより実施できる。 サイト力イン遊離の検出は、 CTL又は被検細胞からの GM-CSF, TNF、 IFN-y等の遊離量を測定することにより実施することができる。 また CTLの増殖は、 3 H—チミジンの細胞への取り込み量の測定や、 顕微鏡観 察などによる C T L細胞数の測定などにより実施することが出来る。 For example, the HLA-A24 molecule on the surface of the test cell can be detected using, as an index, a change caused by contacting the test cell with the CTL of the first embodiment. The change caused by the contact of the test cell with the CTL includes, for example, lysis of the target cell by CTL, release of cytodynamic force, or proliferation of CTL. Target cell lysis can be detected by, for example, measuring the amount of radioisotope released from the test cells divided by the amount of fluorescent dye when the test cells are mixed with CTL after being labeled with radioisotope or a fluorescent dye. Can be implemented. Detection of cytoforce in release can be carried out by measuring the release amount of GM-CSF, TNF, IFN-y, etc. from CTL or test cells. Further, CTL proliferation can be carried out by measuring the amount of 3 H-thymidine incorporated into cells, or by measuring the number of CTL cells by microscopic observation or the like.
上記測定値により HL A— A 24分子の発現の有無の検出のみならず、 被検細 胞上の HLA— A24分子の発現量を測定することも可能である。 なお被検細胞 に MAGE— 3、 MAGE— 1、 C E A又は HER 2/n e uが発現しているか 不明の場合には以下の方法により H L A— A 24分子を検出することが出来る。 本方法においては、 反応液中に被検細胞及び最終 0. 01〜 50 gZm 1とな るよう配列番号 1〜6いずれか記載のァミノ酸配列からなる抗原べプチド及びそ の機能的誘導体から選択される少なくとも 1つの抗原ペプチド、 更に該抗原ぺプ チドと HL A— A 24分子との複合体を認識する第 1の態様の CTLを共存させ 、 CTLと被検細胞を接触させればよい。 Based on the above measured values, it is possible not only to detect the presence or absence of the expression of the HLA-A24 molecule, but also to measure the expression level of the HLA-A24 molecule on the test cell. If it is not known whether MAGE-3, MAGE-1, CEA or HER2 / neu is expressed in the test cells, 24 HLA-A molecules can be detected by the following method. In this method, the reaction solution is selected from the test cells and an antigen peptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 6 and a functional derivative thereof so that the final concentration is 0.01 to 50 gZm1. The CTL and the test cell may be contacted in the presence of at least one antigen peptide, and the CTL of the first embodiment, which recognizes a complex of the HLA-A24 molecule and the antigen peptide.
本発明により、 補体活性化工程が必須であつた従来の抗 H L A抗体を用いた H L A分子の検出方法では検出不可能であつた腫瘍細胞の H L A - A 24分子の検
出が可能となる。 According to the present invention, detection of HLA-A24 molecules in tumor cells, which could not be detected by the conventional method for detecting HLA molecules using an anti-HLA antibody, which required a complement activation step, was not possible. It is possible to go out.
なお、 本方法において使用する抗原べプチドは、 配列番号 1〜 6いずれかに記 載のァミノ酸配列で表されるぺプチド又はその機能的誘導体から選択される少な くとも 1 0HLA— A24拘束性抗原ペプチドに特に限定されなく、 種々の H L A拘束性抗原べプチドを用いることにより、 種々の HL A分子の検出を行なう ことが出来る。 例えば、 HLA— A2拘束性抗原ペプチドを用いることにより H LA-A2分子の検出を行なうことも出来る。 HLA— A2拘束性抗原べプチド としては、 例えば、 配列番号 37で表される MAGE— 3由来のペプチド、 配列 番号 38で表されるインフルエンザマトリックスぺプチド由来のぺプチドが挙げ られる。 The antigen peptide used in the present method is at least 10 HLA-A24 restricted selected from the peptide represented by the amino acid sequence described in any one of SEQ ID NOs: 1 to 6 or a functional derivative thereof. There is no particular limitation on the antigenic peptide, and various HLA molecules can be detected by using various HLA-restricted antigen peptides. For example, HLA-A2 molecules can be detected by using an HLA-A2-restricted antigen peptide. Examples of the HLA-A2-restricted antigen peptide include a peptide derived from MAGE-3 represented by SEQ ID NO: 37 and a peptide derived from an influenza matrix peptide represented by SEQ ID NO: 38.
なお、 HL Aの各タイプには更にサブタイプが存在する。 例えば、 HLA— A 2は、 A* 020 1、 A* 0206 , A* 0207を始めとする 1 0種類以上の サブタイプに分類される。 HL A拘束性抗原ペプチドは、 各サブタイプ間でも異 なるため、 例えば、 制がん剤などとして CTLを利用する時には HL A分子のサ ブタイプまで決定することが重要である。 抗 HL A抗体を用いた HL A分子の検 出方法 （タイピング） では不可能であったサブタイプの分類が本方法では可能で ある。 例えば、 配列番号 37記載のアミノ酸配列を有するペプチドを用いると、 A* 020 1拘束性の CTLを誘導することが出来、 この CTLと該ペプチドを 組み合わせて用いることにより、 被検細胞の HLA— A 2分子のサブタイプまで 決定することが出来る。 なお、 本方法は、 被検細胞より抽出した DNAを用いる D N Aタイピング法より実際の細胞表面上で発現している HLA分子を検出出来 る点で優れている。 実施例 Each type of HLA has a sub-type. For example, HLA-A2 is classified into 10 or more subtypes including A * 0201, A * 0206 and A * 0207. Since HLA-restricted antigen peptides differ between subtypes, for example, when using CTLs as anticancer agents, it is important to determine the subtype of HLA molecules. This method can classify subtypes, which were not possible with the detection (typing) of HLA molecules using anti-HLA antibodies. For example, when a peptide having the amino acid sequence of SEQ ID NO: 37 is used, A * 0201-restricted CTL can be induced. By using this CTL in combination with the peptide, the HLA-A Up to two subtypes can be determined. Note that this method is superior to the DNA typing method using DNA extracted from test cells in that it can detect HLA molecules actually expressed on the cell surface. Example
以下、 実施例により本発明を更に具体的に説明するが、 本発明はこれら実施例 に限定されるものではない。
実施例 1 MAGE - 3抗原ペプチド特異的 CTLの調製 （ 1 ) 本実施例における CTLは、 スタフイロコッカス ァゥレウス コワン— I ( Staphylococcus aureus Cowan- 1 ； SAC- I) を用いた誘導法により調製した ο Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. Example 1 Preparation of MAGE-3 Antigen Peptide-Specific CTL (1) The CTL in this example was prepared by an induction method using Staphylococcus aureus Cowan-1 (SAC-I).
( 1 ) CTL誘導用ペプチドの選択及び合成 (1) Selection and synthesis of CTL-inducing peptide
3 1 4アミノ酸よりなる MAGE— 3タンパク質のアミノ酸配列について、 H L A— A 24結合性モチーフ構造を有する配列 （N末端より 2番目が Tyr、 Phe 、 Trp、 Met のいずれかのアミノ酸であり、 C末端が Leu、 lie、 Phe、 Trp の いずれかのアミノ酸であるペプチド） を中心に、 その他の位置のアミノ酸の種類 も考慮して検索した。 その結果、 MAGE— 3抗原ペプチドの候補ペプチドとし て表 1に示した 7個のぺプチドが存在することが明らかとなつた。 表 1 配列番号 MAGE - 3中の位置 名称 Regarding the amino acid sequence of MAGE-3 protein consisting of 3 14 amino acids, a sequence having an HLA-A24 binding motif structure (Tyr, Phe, Trp, Met is the second amino acid from the N-terminal, and C-terminal However, the search was carried out taking into account the types of amino acids at other positions, with the focus being on peptides that are any of the amino acids Leu, lie, Phe, and Trp. As a result, it became clear that the seven peptides shown in Table 1 exist as candidate peptides of the MAGE-3 antigen peptide. Table 1 Sequence number Position in MAGE-3 Name
8 76 - 84 MA3 - 6 8 76-84 MA3-6
2 1 1 3 - 1 2 1 MA3 - 4 2 1 1 3-1 2 1 MA3-4
9 1 42- 1 50 MA3 - 1 9 1 42- 1 50 MA3-1
1 0 1 42- 1 5 1 MA3 - 5 1 0 1 42- 1 5 1 MA3-5
1 1 1 50 - 1 58 MA3 - 7 1 1 1 50-1 58 MA3-7
1 1 95 - 203 MA3 - 2 1 1 95-203 MA3-2
1 2 1 95 - 204 MA3- 3
なお、 表 1において配列番号の項の番号は、 各ペプチドのアミノ酸配列を示し た配列表の配列番号を示す。 また MAGE— 3中の位置の項の番号は、 MAGE —3タンパクの N末端からのアミノ酸数を示す。 また名称の項の記号は、 本発明 者らが命名したべプチドの名称を示す。 1 2 1 95-204 MA3-3 In Table 1, the numbers in the SEQ ID No. column indicate the SEQ ID Nos. In the sequence listing showing the amino acid sequence of each peptide. The number at the position in MAGE-3 indicates the number of amino acids from the N-terminus of MAGE-3 protein. The symbol in the name section indicates the name of the peptide named by the present inventors.
表 1に示した M A 3 - 1〜！ VI A 3— 7の 7種のぺプチドをぺプチド合成機 （ P SSM- 8 ：島津製作所製） を用い、 固相 Fmo c法にて作製した。 M A 3-1 shown in Table 1! Seven peptides of VI A3-7 were prepared by solid phase Fmoc method using a peptide synthesizer (PSSM-8: manufactured by Shimadzu Corporation).
(2) PBMCの調製 (2) Preparation of PBMC
HLA-A24を保有する健常人から成分採血により採血を行い、 白血球画分 を集め更に以下の分離方法に従って PBMCを分離した。 すなわち、 採血液を R PM I 1 640培地で約 2倍希釈後、 フイコール—バック （Ficoll-Paque)分離 液 （フアルマシア社製） 上に重層し、 500 xgで 20分間室温で遠心した。 中 間層の PBMCをピぺットで回収、 洗浄して 90%牛胎児血清 （FCS、 インター ゲン社製） と 1 0%ジメチルスルホキシド （シグマ社製） からなる保存液に懸濁 した状態で液体窒素中に保存した。 Blood was collected by blood sampling from healthy individuals having HLA-A24, and leukocyte fractions were collected, and PBMCs were separated according to the following separation method. That is, the collected blood was diluted about 2-fold with RPMI 1640 medium, layered on a Ficoll-Paque separation solution (manufactured by Pharmacia), and centrifuged at 500 xg for 20 minutes at room temperature. The PBMCs in the middle layer were collected with a pipette, washed, and suspended in a stock solution containing 90% fetal calf serum (FCS, Intergen) and 10% dimethyl sulfoxide (Sigma). Stored in liquid nitrogen.
(3) エフェクター細胞 （e f f e c t o r c e l l) の調製 (3) Preparation of effector cells (effecctorcell)
下記の方法で MA 3— 1、 MA3 - 2、 MA3— 4、 MA3 - 6、 MA 3 - 7 いずれかのぺプチドで個別に刺激された 5種類のエフェクター細胞を調製した。 Five types of effector cells individually stimulated with any of MA3-1, MA3-2, MA3-4, MA3-6, and MA3-7 peptides were prepared in the following manner.
(2) で調製した保存 PBMCを融解後、 細胞濃度が 4 X 1 06 個 Zmlとな るように 5H— RPMIに懸濁した。 なお 5H— RPMIは、 RPMI 1 640 培地にヒト AB型血清 （ァ一バインサイエンス社製） を終濃度 5% (v/v)、 非必須アミノ酸 （ギブコ BR L社製） を終濃度 0. lmM、 ピルビン酸ナトリウ ム （ギブコ BRL社製） を終濃度 lmM、 L-グルタミン （ギブコ BRL社製） を 終濃度 4mM、 硫酸ゲンタマイシン （ァーバインサイエンス社製） を終濃度 1 0
//g/m 1 となるように加えた培地である。 細胞懸濁液 25mlを抗 CD 4抗体 を結合した T— 150フラスコ （AIS MicroCELLector、 アプライドィミューンサ ィエンス社製） に入れ 1時間室温にて反応後、 非接着細胞を回収し 2 X I 06 個 /m 1となるように 5H— RPMIに懸濁した。 この懸濁液を、 MA 3— 1、 M A3— 2、 MA3— 4、 MA3— 6、 MA 3— 7いずれかのペプチドを用い後記 実施例 8の （ 1 ) の記載の方法により調製した SAC— 1処理による 5種類の初 回刺激用抗原提示細胞と個別に等量混合し、 I L一 7 (ジェンザィム社製） を最 終濃度 15ng/mlとなるように加え、 24ゥエル培養プレートの各ゥエルに 2mlずつ分注し、 37°Cの C02 インキュベーター内で培養した。 翌日、 終濃 度 1 OngZmlとなるように I L— 10 (R&D社製） を加えた。 6日後に培 養上清を半量除き、 20 ng/m 1の I L— 7を含む 5 H - RPM Iを等量加え た。 After the stored PBMC prepared in (2) was thawed, it was suspended in 5H-RPMI so that the cell concentration was 4 × 10 6 Zml. For 5H-RPI, RPMI 1640 medium contains human AB serum (manufactured by Avain Science) at a final concentration of 5% (v / v) and non-essential amino acids (manufactured by Gibco BRL) at a final concentration of 0.1 lmM. Final concentration of sodium pyruvate (manufactured by Gibco BRL) at a final concentration of lmM, L-glutamine (manufactured by Gibco BRL) at a final concentration of 4 mM, and gentamicin sulfate (manufactured by Erbain Science) at a final concentration of 10 It is a medium added to // g / m 1. The cell suspension 25ml bound anti CD 4 antibodies T-0.99 flasks (AIS MicroCELLector®, Applied I made mu capacitors Iensu Co.) After reaction for 1 hour at room temperature placed in, to recover the non-adherent cells 2 XI 0 6 pieces The suspension was suspended in 5H-RPMI so as to be / m1. This suspension was prepared using any of the peptides MA3-1, MA3-2, MA3-4, MA3-6, and MA3-7 by the method described in Example 8, (1) below. — Individually mix equal amounts of the five antigen-presenting cells for primary stimulation in 1 treatment, add IL-17 (Genzym) to a final concentration of 15 ng / ml, and add each well of a 24-well culture plate. dispensed into each 2ml min, and incubated in a C0 2 incubator 37 ° C. The next day, IL-10 (manufactured by R & D) was added to a final concentration of 1 OngZml. Six days later, half of the culture supernatant was removed, and an equal amount of 5 H-RPM containing 20 ng / ml of IL-7 was added.
更に 3日間培養を行った後細胞を遠心により回収し、 細胞濃度が 2 X 1 06 個 /m 1となるように 5H— RPMIに懸濁した。 この懸濁液を、 MA 3— 1、 M A3_2、 MA 3— 4、 MA 3— 6、 MA 3— 7いずれかのペプチドを用い後記 実施例 8の （2)のように調製した抗原提示細胞を含むプレートに lmlZゥェ ルずつ加え同一ペプチドによる再刺激を施した。 翌日、 IL-10 を終濃度 1 Ong ノ mlとなるように添加し、 更に 2日置きに、 培養上清を半量除き、 20 IUZ m 1の r I L— 2 (塩野義製薬社製） を含む 5 H— R P M Iを等量加え、 1週間 C02 インキュベーター内で培養する。 同様の再刺激を更に 3回行った後細胞を 回収し、 エフェクター細胞とした。 After further culturing for 3 days, the cells were collected by centrifugation and suspended in 5H-RPMI so that the cell concentration was 2 × 10 6 cells / m 1. An antigen-presenting cell prepared by using this suspension as a peptide as described in Example 8 (2) below, using any of the peptides MA3-1, MA3_2, MA3-4, MA3-6, and MA3-7. Was added to the plate containing, and restimulation was performed with the same peptide. The next day, add IL-10 to a final concentration of 1 ng / ml, and remove the culture supernatant by half every 2 days, and add 20 IUZ m1 of rIL-2 (manufactured by Shionogi & Co., Ltd.) 5 H- RPMI was added an equal volume, cultured in 1 week C0 2 incubator. After performing the same restimulation three more times, the cells were collected and used as effector cells.
(4) CTL標的細胞の調製 (4) Preparation of CTL target cells
細胞傷害活性測定のための標的細胞として、 HLA— A24を発現している E BVトランスフォーム B細胞である T I S I (WSNO 9042 )を用いた。 ま ず T I S I細胞を測定前日に （ 1 ) で調製した MA 3— 1、 MA3— 2、 MA 3
一 4、 MA 3— 6、 MA 3 - 7いずれかのぺプチドを個別に 10 g/m 1を含 む 5種類の培地 〔以下、 該培地で培養した T I S Iを T I S I ( + ) と表記〕 、 又はべプチドを含まない培地 〔以下、 該培地で培養した T I S Iを T I S I (- ) と表記〕 中で一晩培養した。 測定当日、 各 5 X 106 個の、 5種類の T I S I (+ ) 及び T I S I (-) を各々 200 zC iの Na2 51Cr04 溶液中に 37 °Cで 1時間混和し、 その後 10% じ3含有1^?1^ 1 1 640培養液で洗浄し51 C r標識した標識標的細胞を調製した。 As target cells for measuring cytotoxic activity, TISI (WSNO 9042), an EBV transform B cell expressing HLA-A24, was used. First, MA3-1, MA3-2, and MA3 prepared in (1) the day before the measurement of TISI cells. (I) 5 types of media containing 10 g / m1 of any of the peptides of MA4, MA3-6 and MA3-7 individually (hereinafter, TISI cultured in the medium is referred to as TISI (+)), Alternatively, the cells were cultured overnight in a medium containing no peptide [hereinafter, TISI cultured in the medium is referred to as TISI (-)]. Measurements day, each 5 X 10 6 cells, 5 kinds of TISI (+) and TISI (-) and 1 hour mixing at each 200 zC i Na 2 51 Cr0 4 solution 37 ° C during the Ji then 10% The cells were washed with a 1 ^? 1 ^ 11640 culture solution containing 3 to prepare 51 Cr-labeled labeled target cells.
(5) CTLによる細胞傷害活性の測定 (5) Measurement of cytotoxic activity by CTL
(3)のエフェクター細胞を 5 X 105 〜1 X 106 個 Zmlとなるように 5 H-RPM Iで希釈後、 96ゥヱル培養プレートの各ゥヱルに 100〃1 /ゥェ ルずつ分注しておき、 これに （4)で調製した 1 X 104 個の51 Cr標識標的細 胞及び 3 X 105 個の K 562細胞を含む 10 の細胞懸濁液を加えた。 な お K 562細胞は混入する NK細胞による非特異的傷害活性を除くために用いた 上記細胞懸濁液を 400 xgで 1分間遠心後、 37eCの C02 インキュベータ 一中に 5時間放置した。 その後各ゥヱルの培養液上清 100 1を採取しガンマ カウンターを用いて、 遊離された51 Cr量を測定した。 After diluting the effector cells of (3) with 5H-RPM I to 5 x 10 5 to 1 x 10 6 Zml, dispense 100/1 / well into each well of a 96-well culture plate. To this, 10 cell suspensions containing 1 × 10 4 51 Cr-labeled target cells and 3 × 10 5 K562 cells prepared in (4) were added. Contact K 562 cells 1 minute after centrifuging the cell suspension was used to eliminate nonspecific cytotoxic activity by NK cells mixed with 400 xg such, was allowed to stand for 5 hours to C0 2 incubator in one of 37 e C . Thereafter, 100 1 of the culture supernatant of each cell was collected, and the amount of released 51 Cr was measured using a gamma counter.
特異的細胞傷害活性は以下の計算式 （数 1) に従って算出した。 特異的細胞傷害活性 （％) = C (各ゥニルの測定値-最小放出値） The specific cytotoxic activity was calculated according to the following formula (Equation 1). Specific cytotoxic activity (%) = C (measured value of each penil-minimum release value)
/ (最大放出値一最小放出値） 〕 X 100 (数 1 ) 上式 （数 1) において、 最小放出値は標的細胞及び K 562細胞のみ入ってい るゥエルの51 Cr量であり、 標的細胞からの51 Crの自然遊離量を示す。 また、 最大放出値は、 標的細胞に界面活性剤トリ トン X - 100を加えて細胞を破壊し
た際の51 C r遊離量を示している。 結果を表 2に示す c 表 2 特異的細胞傷害活性 / (Maximum release value-minimum release value)] X 100 (Equation 1) In the above equation (Equation 1), the minimum release value is the amount of 51 Cr in a well containing only target cells and K562 cells. Shows the amount of spontaneous release of 51 Cr. The maximum release value is determined by adding Triton X-100 detergent to target cells and destroying the cells. Shows the amount of 51 Cr released at the time. The results are shown in Table 2.c Table 2 Specific cytotoxic activity
ぺプチド名 E/T TISK-) TISK+) (Peptide name E / T TISK-) TISK +)
MA 3 - 1 10 1.6 0.8 MA 3-1 10 1.6 0.8
MA 3 - 2 10 6.6 65.6 MA 3-2 10 6.6 65.6
MA 3 - 4 5 3.1 3.4 MA 3-4 5 3.1 3.4
MA 3 - 6 5.8 0.8 0.0 MA 3-6 5.8 0.8 0.0
MA 3 - 7 10 17.0 13.7 MA 3-7 10 17.0 13.7
表 2において EZTは標的細胞に対するエフェクター細胞の比を示す。 In Table 2, EZT indicates the ratio of effector cells to target cells.
その結果、 MA 3一 2で誘導して得られたエフヱクタ一細胞が MA 3— 2ぺプ チド添加した T I S I ( + ) に対して抗原べプチド特異的な細胞傷害性を示した As a result, effector cells obtained by induction with MA32 showed antigen-peptide-specific cytotoxicity to TISI (+) to which MA3-2 peptide was added.
実施例 2 MAGE - 3抗原ペプチド特異的 CTLの調製 （2) Example 2 Preparation of MAGE-3 antigen peptide-specific CTL (2)
本実施例における CTLは、 KLHを用いた誘導法により調製した。 なお抗原 ぺプチドは、 実施例 1の （ 1 ) で調製した MA 3— 1、 MA 3— 2、 MA 3 - 4 、 M A 3— 6、 MA 3— 7の 5種のぺプチドを使用した。 The CTL in this example was prepared by an induction method using KLH. As antigen peptides, five kinds of peptides, MA3-1, MA3-2, MA3-4, MA3-6, and MA3-7, prepared in (1) of Example 1 were used.
(1) PBMCの調製 (1) Preparation of PBMC
HLA-A24を保有している健常人から採血を行い、 以下の分離方法に従つ て PBMCを分離した。 すなわち、 採血液を同量の RPMI 1 640培地に懸濁
させ、 フイコール一パック分離液上に重層し、 500 xgで 25分間室温で遠心 した。 中間層の PBMCをピペッ トで回収し、 1 5ml遠心管に入れ、 RPMI 1 640培地で 3回洗浄した。 次に、 細胞最終濃度が 4 X 1 06 個 1となる ように 5H— RPMIに懸濁した。 Blood samples were collected from healthy persons carrying HLA-A24, and PBMCs were separated according to the following separation method. That is, the collected blood is suspended in the same volume of RPMI 1640 medium. The mixture was overlaid on a ficoll-one-pack separated solution, and centrifuged at 500 xg for 25 minutes at room temperature. The middle layer of PBMC was collected with a pipette, placed in a 15 ml centrifuge tube, and washed three times with RPMI 1640 medium. Next, cells were suspended in 5H-RPMI so that the final cell concentration was 4 × 10 6 1.
(2) エフェクター細胞の調製 (2) Preparation of effector cells
下記の方法により MA 3— 1、 MA3— 2、 MA3— 4、 MA3— 6、 MA 3 - 7いずれかのぺプチドで個別に刺激された 5種類のェフ ク夕一細胞を調製し た。 Five types of cells were separately stimulated with any of the peptides MA3-1, MA3-2, MA3-4, MA3-6, and MA3-7 by the following method.
(1) で調製した PBMCにペプチド 20 Zmlを加えた 5H— RPMIを 等量混合した後、 37°Cの C02 インキュベーター内で 2〜3時間放置した。 こ れに KLH (カルビオケム社製） 溶液及び I L— 7溶液をそれぞれ最終濃度 5 g/m K 25 n g/m 1となるように添加した。 該細胞懸濁液を 24ゥエル培 養プレー卜の 2ゥエルに 2m 1ずつ分注し、 37°Cの C02 インキュベータ一内 で培養した。 3日後、 1 000 I UZm 1の r I L— 2を含む 5H— RPM I を 60〃 1 (最終 r I L一 2濃度 30 I U/m 1 ) 加えた。 The PBMC prepared in (1) was mixed with an equal amount of 5H-RPMI to which 20 Zml of the peptide was added, followed by standing in a C02 incubator at 37 ° C for 2 to 3 hours. To this, a KLH (manufactured by Calbiochem) solution and an IL-7 solution were added to a final concentration of 5 g / mK and 25 ng / m1, respectively. The cell suspension is dispensed at 2m 1 to 24 Ueru culture play Bok 2 Ueru were cultured in C0 2 incubator within one 37 ° C. Three days later, 5H-RPM containing 1 000 I UZm1 of rIL-2 was added at 60〃1 (final rIL-12 concentration 30 IU / m1).
1週間培養後、 細胞を遠心により回収し、 細胞濃度が 5 X 1 05 個/ m 1とな るように 5 H— RPM Iに懸濁する。 この懸濁液を、 MA3— 1、 MA3— 2、 MA3— 4、 MA3— 6、 MA 3 _ 7いずれかのペプチドを用い後記実施例 8のAfter culturing for one week, the cells are collected by centrifugation, and suspended in 5H-RPI so that the cell concentration becomes 5 × 10 5 cells / ml. This suspension was prepared using any one of the peptides MA3-1, MA3-2, MA3-4, MA3-6, and MA3_7 in Example 8 described later.
( 4 ) 記載の方法により調製した抗原提示細胞のいずれか 1種を含む 5種類のプ レートに lml/ゥヱルずつ加え、 再刺激した。 翌日 r I L_2溶液 （1 000(4) lml / μl was added to 5 types of plates containing any one of the antigen-presenting cells prepared by the method described above, and restimulated. Next day r I L_2 solution (1 000
I U/m 1 ) を 60〃1加え、 1週間 C02 インキュベータ一内で培養した。 同 様の再刺激を更に 4回行った後、 細胞を回収し、 エフェクター細胞とした。 上記の方法で得られた 5種のエフヱクタ一細胞を 4 X 1 06 個/ m 1となるよ うに 5H— RPMIに懸濁した。
(3) CTL標的細胞の調製 IU / m 1) a 60〃1 added and incubated in the 1 week C0 2 incubator scratch. After the same restimulation was performed four more times, the cells were collected and used as effector cells. The five types of effector cells obtained by the above method were suspended in 5H-RPMI at a concentration of 4 × 10 6 cells / ml. (3) Preparation of CTL target cells
標識標的細胞として実施例 1の （4) と同様に調製した T I S I (—） 、 5種 類の T I S I ( + ) のほかに MAGE— 3及び HLA— A24共に陽性である W i Dr (大腸がん細胞株） 、 TE 1 1 (食道がん細胞株） 、 MRKnu 1 (乳が ん細胞株） を使用した。 更に、 MAGE— 3陽性、 HL A - A 24陰性のがん細 胞株である KAT0-III (胃がん細胞株） も使用した。 WiDr、 TE11、 MRKnuU 及び KAT0-IIIは各 5 X 1 06 個の細胞を 200 /Ciの Na2 61C r 04 の溶液中に 37 °Cで 1時間混和し、 その後 1 0% 〇3含有1¾?]^ 1 1 640培養液で洗浄する ことにより51 C r標識した。 In addition to TISI (-) and five types of TISI (+) prepared in the same manner as (4) in Example 1 as labeled target cells, WiDr (colorectal cancer) positive for both MAGE-3 and HLA-A24 Cell line), TE11 (esophageal cancer cell line), and MRKnu1 (breast cancer cell line). In addition, MAGE-3 positive, HLA-A24 negative cancer cell line KAT0-III (gastric cancer cell line) was also used. WiDr, TE11, MRKnuU and KAT0-III was 1 hour mixed with Na 2 61 C r 0 4 in solution in 37 ° C of each 5 X 1 0 6 cells to 200 / Ci, then 1 0% Rei_3 Washed with 1 含有?] 640 culture solution for labeling with 51 Cr.
(4) CTLによる細胞傷害活性の測定 (4) Measurement of cytotoxic activity by CTL
(2) で調製したエフェクター細胞及び （3) で調製した標的細胞を用い細胞 傷害活性を測定した。 標的細胞として T I S I (一） 及び T I S I ( + ) を用い た場合は実施例 1の （5) と同様の方法で測定した。 なおエフ クタ一細胞と標 的細胞の反応時間は 4. 5時間とした。 他の 4種類の標識がん細胞株を標的細胞 として用いた場合は、 96ゥヱル培養プレートの各ゥヱルに前記 （2) のェフエ クタ一細胞懸濁液を 1 00 1 /ゥヱル （4 X 105 個/ゥヱル） 分注しておき 、 これに 5 X 1 03 個の51 C r標識標的細胞及び 1. 5 X 1 05 個の 562細 胞を含む 1 00 /1 の細胞懸濁液を加えた。 400 X gで 1分間遠心後、 37°C の C02 インキュベータ一中に 4. 5時間放置した。 その後各ゥヱルの培養液上 清 1 00〃1 を採取しガンマカウンターを用いて、 遊離された51 C r量を測定し た。 特異的細胞傷害活性は実施例 1の （5) と同様の計算により求めた。 結果を 表 3に示す。
表 3 特異的細胞傷害活性 The cytotoxic activity was measured using the effector cells prepared in (2) and the target cells prepared in (3). When TISI (1) and TISI (+) were used as target cells, measurement was performed in the same manner as in Example 1, (5). The reaction time between one effector cell and the target cells was 4.5 hours. When the other four types of labeled cancer cell lines were used as target cells, the effector-cell suspension of (2) above was added to each cell of a 96-well culture plate at a rate of 100 1 / well (4 × 10 5 Aliquots / ml), and add a 100/1 cell suspension containing 5 x 10 3 51 Cr labeled target cells and 1.5 x 10 5 562 cells. added. After centrifugation for 1 minute at 400 X g, 4. left for 5 hours in C0 2 incubator in one of 37 ° C. Thereafter, 100 μl of the supernatant of each culture was collected, and the amount of released 51 Cr was measured using a gamma counter. The specific cytotoxic activity was determined by the same calculation as in Example 1, (5). Table 3 shows the results. Table 3 Specific cytotoxic activity
ぺプチド名 E/T TISI(-) TISK十） WiDr TE11 MRKnu 1 Peptide name E / T TISI (-) TISK10) WiDr TE11 MRKnu 1
MA 3 - 1 80 27.6 51.7 ND ND ND MA 3-1 80 27.6 51.7 ND ND ND
MA3-2 80 7.4 60.5 51.5 39.0 20.9 MA3-2 80 7.4 60.5 51.5 39.0 20.9
MA3-4 80 5.4 39.4 48.7 29.1 27.6 MA3-4 80 5.4 39.4 48.7 29.1 27.6
MA 3 - 6 80 8.7 14.8 ND ND ND MA 3-6 80 8.7 14.8 ND ND ND
MA 3 - 7 80 21.8 39.4 ND ND ND MA 3-7 80 21.8 39.4 ND ND ND
なお表 3において E ZTは標的細胞に対するエフヱクタ一細胞の比を、 N Dは 未測定であることを示す。 In Table 3, EZT indicates the ratio of effector-cell to target cell, and ND indicates that the ratio was not measured.
更に、 ΜΑ 3— 2あるいは ΜΑ 3— 4により誘導したエフェクター細胞の Τ I S I (―) 及び T I S I ( + ) に対する各種 ΕΖΤでの特異的細胞傷害性曲線を 図 1及び図 2に、 WiDr、 TE1K MRKnul細胞に対する各種 EZTでの特異的細胞傷 害性曲線を図 3及び図 4に示す。 Furthermore, the specific cytotoxicity curves of the effector cells induced by に よ り 3-2 or ΜΑ3-4 against ΤISI (−) and TISI (+) in various ΕΖΤ are shown in FIGS. 1 and 2, respectively, for WiDr and TE1K MRKnul. FIGS. 3 and 4 show the specific cytotoxicity curves of cells with various EZTs.
図 1は M A 3— 2で誘導したエフヱクタ一細胞を、 図 2は M A 3— 4で誘導し たエフェクター細胞を用いた結果を示す。 また四角印 （□) は標的細胞として T I S I ( + ) を、 ひし形印 （◊) は T I S I (—） を用いた結果を示す。 FIG. 1 shows the results obtained using an effector cell induced by MA 3-1 and FIG. 2 shows the results obtained using effector cells induced by MA 3-1. The squares (□) show the results using T I S I (+) as the target cells, and the diamonds (◊) show the results using T I S I (—).
図 3は M A 3— 2で誘導したエフヱクタ一細胞を、 図 4は M A 3— 4で誘導し たエフェクター細胞を用いた結果を示す。 また四角印 （□) は標的細胞として W iDrを、 ひし形印 （◊) は TE— 1 1を、 白丸印 （〇） は MRKnu 1を用い た結果を示す。 FIG. 3 shows the results obtained using the effector cells induced by MA 2-2, and FIG. 4 shows the results obtained using the effector cells induced by MA 3-1. The squares (□) show the results using WiDr as the target cells, the diamonds (は) show the results using TE-11, and the white circles (〇) show the results using MRKnu1.
図 3及び図 4に示された細胞傷害活性は、 反応系にクラス I抗体 （W6/32
) を共存させることによって阻害された。 また、 MA 3— 2あるいは MA3— 4 により誘導されたエフェクター細胞は MAGE— 3陽性及び HLA— A24陰性 のがん細胞株である KATO— I I Iには細胞傷害性を示さなかった。 The cytotoxic activity shown in Fig. 3 and Fig. 4 indicates that the class I antibody (W6 / 32 ) Was inhibited. In addition, effector cells induced by MA3-2 or MA3-4 did not show cytotoxicity to KATO-III, a MAGE-3 positive and HLA-A24 negative cancer cell line.
以上の結果より、 MA3— 2、 MA3— 4ペプチドにより誘導したエフヱクタ 一細胞は抗原ペプチド特異的な細胞傷害性を示すと共に、 HLA— A24及び M AGE- 3共に陽性のがん細胞株に対して細胞傷害性を示す C T Lであつた。 実施例 3 MAGE- 1抗原ペプチド特異的 CTLの調製 From the above results, the effector cells induced by MA3-2 and MA3-4 peptides show antigenic peptide-specific cytotoxicity, and are effective against cancer cell lines positive for both HLA-A24 and MAGE-3. The CTL showed cytotoxicity. Example 3 Preparation of MAGE-1 antigen peptide-specific CTL
本実施例における C T Lは K L Hを用いた C T L誘導法により調製した。 ( 1 ) CTL誘導用ペプチドの選択及び合成 CTL in this example was prepared by a CTL induction method using KLH. (1) Selection and synthesis of CTL-inducing peptide
30 9アミノ酸よりなる MAGE— 1タンパク質のァミノ酸配列 〔モレキュ ラー ィムノロジ一 （Molecular Immunology) 、 第 3 1巻、 第 1 423〜 1 4 3 0頁 （ 1 9 94 ) 〕 について、 実施例 1の （ 1 ) と同様に HLA— A24結合性 モチーフ構造を有する配列を中心に、 その他の位置のアミノ酸の種類も考慮して 検索した。 その結果、 表 4に示した 5個のペプチドを候補抗原ペプチドとして選 択した。 The amino acid sequence of MAGE-1 protein consisting of 309 amino acids [Molecular Immunology, Vol. 31, Vol. 14, Nos. 1423-1430 (1994)] Similar to 1), the search was carried out focusing on the sequence having the HLA-A24 binding motif structure, taking into account the types of amino acids at other positions. As a result, the five peptides shown in Table 4 were selected as candidate antigen peptides.
表 4 配列番号 MAGE - 1中の位置 名称 Table 4 Sequence number Position in MAGE-1 Name
3 1 35 - 1 4 3 MA 1一 13 1 35-1 4 3 MA 1 1 1
1 4 1 35 - 1 44 MA 1 - 3 1 4 1 35-1 44 MA 1-3
1 3 1 8 8 - 1 9 6 MA 1一 2 1 3 1 8 8-1 9 6 MA 1 1 2
1 5 1 8 8 - 1 97 MA 1一 4 1 5 1 8 8-1 97 MA 1 1 4
1 6 275 - 284 MA 1一 5
なお、 表 4において配列番号の項の番号は、 各ペプチドのアミノ酸配列を示し た配列表の配列番号を示す。 また MAGE— 1中の位置の項の番号は、 MAGE — 1タンパク質の N末端からのアミノ酸数を示す。 また名称の項の記号は、 本発 明者らが命名したべプチドの名称を示す。 1 6 275-284 MA 1 1 5 In Table 4, the numbers in the SEQ ID No. column indicate the SEQ ID Nos. In the sequence listing showing the amino acid sequence of each peptide. The number at the position in MAGE-1 indicates the number of amino acids from the N-terminus of MAGE-1 protein. The symbol in the name section indicates the name of the peptide named by the present inventors.
表 4に示した M A 1— 1〜M A 1— 5の 5種のぺプチドをぺプチド合成機を用 いて作製した。 Five kinds of peptides, MA1-1-1 to MA1-5, shown in Table 4 were prepared using a peptide synthesizer.
(2) KLHを用いた誘導法による MAGE— 1抗原ペプチド特異的 C T Lの調 製 (2) Preparation of MAGE-1 antigen peptide-specific CTL by induction method using KLH
実施例 3の （1) で調製した MA1— 1、 MA 1— 2、 MA 1— 3、 MA 1— 4、 MA 1— 5のいずれかのぺプチド、 及び MA 1— 1、 MA 1— 2、 MA 1— 3、 MA 1— 4、 MA 1— 5のいずれかのぺプチドを用い後記実施例 8の （4) 記載の方法により調製した抗原提示細胞を用いて実施例 2の （2) と同様の方法 でエフェクタ一細胞を調製し、 各種標的細胞に対する細胞傷害活性を測定した。 使用した標的細胞は T I S I (一） 、 MA 1— 1、 MA 1— 2、 MA 1— 3、 M A 1— 4、 MA 1— 5のいずれかのぺプチド添加培地で培養した 5種類の T I S I ( + ) の他に MAGE— 1及び HLA— A24共に陽性である NUGC— 3 ( 胃がん細胞株） 、 TE— 1 1、 MAGE- 1陽性及び H LA— A 24陰性である KATO-III 、 MAGE- 1及び H LA— A 24共に陰性である R a j i (リ ンフォーマ） である。 MA 1— 1により誘導したエフヱクタ一細胞の各種標的細 胞に対する各種 EZTでの特異的細胞傷害性曲線を図 5に示す。 Any peptide of MA1-1, MA1-2, MA1-3, MA1-4, MA1-5 prepared in (1) of Example 3, and MA1-1, MA1-2 , MA1-3, MA1-4, and MA1-5, using the antigen-presenting cells prepared by the method described in (4) of Example 8 below, using the peptide of any one of MA1-5 and MA1-5. One effector cell was prepared in the same manner as described above, and the cytotoxic activity against various target cells was measured. The target cells used were TISI (1), MA1-1, MA1-2, MA1-3, MA1-4, and MA1-5. NUGC-3 (gastric cancer cell line), TE-11, MAGE-1 positive and HLA-A24 negative KATO-III, MAGE-1 positive for both MAGE-1 and HLA-A24 And HLA-A24 are both negative Raji (reformer). FIG. 5 shows specific cytotoxicity curves of various EZTs against various target cells of one effector cell induced by MA1-1.
図 5において黒丸印 （鲁） は標的細胞として NUGC— 3を、 黒四角 （國） 印 は TE 1 1を、 逆三角印 （▽) は KATO I I Iを、 ひし形印 （◊) は Ra j i を、 三角印 （△) は T I S I (—） を用いた結果を示す。 In Fig. 5, a black circle (鲁) indicates NUGC-3 as a target cell, a black square (country) indicates TE11, an inverted triangle (▽) indicates KATO III, a diamond (◊) indicates Raji, Triangles (△) show the results using TISI (—).
更に MA 1一 1添加培地を用い得られた T I S I ( + )、 図 5に示された NU
GC— 3細胞株及び TE— 1 1細胞株に対する細胞傷害活性は、 反応系にクラス I抗体 （W6Z32) を共存させることによって阻害された。 In addition, TISI (+) obtained using MA-11 supplemented medium, NU shown in FIG. The cytotoxic activity against the GC-3 cell line and the TE-11 cell line was inhibited by the coexistence of a class I antibody (W6Z32) in the reaction system.
したがって MA 1 - 1で誘導したエフェクター細胞は HLA— A24及び MA GE- 1共に陽性のがん細胞株に対する C T Lであることが明らかとなつた。 実施例 4 C E A抗原べプチド特異的 C T Lの調製 （ 1 ) Therefore, it was revealed that the effector cells induced by MA1-1 were CTL against cancer cell lines positive for both HLA-A24 and MAGE-1. Example 4 Preparation of CEA antigen peptide specific CTL (1)
本実施例における CTLは、 実施例 1 と同様に SAC— Iを用いる CTL誘導 法により調製した。 CTL in this example was prepared by the CTL induction method using SAC-I in the same manner as in Example 1.
( 1) CTL誘導用ペプチドの選択及び合成 (1) Selection and synthesis of CTL-inducing peptide
4 64アミノ酸よりなる CEAタンパク質のアミノ酸配列について、 HLA— A 24結合性モチーフ構造を有する配列を中心に、 その他の位置のアミノ酸の種 類も考慮して検索した。 その結果、 表 5に示した 5個のペプチドが存在すること が明らかとなった。 表 5 配列番号 CE Aタンパク質中の位置 名称 The amino acid sequence of the CEA protein consisting of 4 64 amino acids was searched mainly for the sequence having the HLA-A24 binding motif structure, taking into account the types of amino acids at other positions. As a result, it became clear that the five peptides shown in Table 5 existed. Table 5 SEQ ID NO: Position in CE A protein
1 7 1 0 1 - 1 0 9 CE- 1 1 7 1 0 1-1 0 9 CE- 1
1 8 234 - 242 CE- 5 1 8 234-242 CE- 5
4 268 - 277 CE- 2 4 268-277 CE-2
1 9 3 1 8 - 326 CE- 4 1 9 3 1 8-326 CE-4
5 652- 6 60 CE- 3 5 652- 6 60 CE- 3
なお、 表 5において配列番号の項の番号は、 各ペプチドのアミノ酸配列を示し
た配列表の配列番号を示す。 また CE Aタンパク質中の位置の項の番号は、 CE Aタンパク質中の N末端からのアミノ酸数を示す。 また名称の項の記号は、 本発 明者等が命名したべプチドの名称を示す。 In Table 5, the numbers in the sequence numbers indicate the amino acid sequence of each peptide. SEQ ID No. in the Sequence Listing is shown below. The number of the term at the position in the CE A protein indicates the number of amino acids from the N-terminus in the CE A protein. The symbol in the name column indicates the name of the peptide named by the present inventors.
表 5に示した 5種のぺプチドをぺプチド合成機を用いて作製した。 Five kinds of peptides shown in Table 5 were prepared using a peptide synthesizer.
(2) エフヱクタ一細胞の調製 (2) Preparation of effector cells
実施例 1の （2) と同様の方法で調製した保存 PBMC、 CE- K CE- 3 、 CE- 4, CE— 5のいずれかのペプチドを用い後記実施例 8の （ 1 )記載の 方法により調製した SAC - 1処理による初回刺激用抗原提示細胞、 及び CE— 1、 CE— 3、 CE— 4、 CE— 5のいずれかのペプチドを用い後記実施例 8の Using any of the stored PBMC, CE-K CE-3, CE-4, and CE-5 peptides prepared in the same manner as (2) in Example 1, and using the method described in (1) in Example 8 below. Using the prepared antigen-presenting cells for initial stimulation by treatment with SAC-1 and any peptide of CE-1, CE-3, CE-4, CE-5, the following Example 8
( 2 ) 記載の方法により調製した抗原提示細胞レ、ずれか 1種を含む 4種のプレー トを用い、 実施例 1の （3) と同様の方法で CE— 1、 CE_3、 CE— 4、 C E— 5のいずれかのぺプチドで個別に刺激された 4種類のェフエク夕一細胞を調 製した。 (2) Using four types of plates, including one of the antigen-presenting cells prepared by the method described in the above, and using the same method as in (3) of Example 1, CE-1, CE_3, CE-4, Four types of Fefek Yuichi cells, individually stimulated with any of the peptides of CE-5, were prepared.
(3) CTL標的細胞の調製 (3) Preparation of CTL target cells
細胞傷害活性測定のための標的細胞として、 実施例 1の （4) と同様の方法で CE— 1、 CE— 3、 CE— 4、 C E— 5のいずれかのペプチドを添加した培地 で培養し得られた 5種の T I S I ( + ) 及び胃がん細胞株 MKN— 45 (HLA 一 A24及び CEA陽性） を用いた。 なお上記各細胞は、 細胞傷害活性測定当日 実施例 1の （4) と同様の方法で51 Cr標識した。 As target cells for measuring cytotoxic activity, the cells were cultured in a medium containing any of the peptides CE-1, CE-3, CE-4, and CE-5 in the same manner as in (4) of Example 1. The obtained five kinds of TISI (+) and gastric cancer cell line MKN-45 (HLA-A24 and CEA positive) were used. Each of the above cells was labeled with 51 Cr in the same manner as in (4) of Example 1 on the day of measuring the cytotoxic activity.
(4) CTLによる細胞傷害活性の測定 (4) Measurement of cytotoxic activity by CTL
(2) のエフェクタ一細胞及び （3) の標的細胞を用い、 実施例 1の （5) と 同様の方法で特異的細胞傷害活性 （％) を算出した。 Using one effector cell of (2) and the target cell of (3), the specific cytotoxic activity (%) was calculated in the same manner as in (5) of Example 1.
結果を表 6に示す。
表 6 ぺプチド名 E/T TISI ( + ) MKN-45 Table 6 shows the results. Table 6 Peptide name E / T TISI (+) MKN-45
10 24.8 86.7 0.0 10 24.8 86.7 0.0
CE- 3 10 E- 1.9 92.5 9.2 CE- 3 10 E- 1.9 92.5 9.2
O O
CE - 4 10 7.5 5.9 0.2 CE-4 10 7.5 5.9 0.2
CE- 5 10 4.8 4.6 0.0 CE-5 10 4.8 4.6 0.0
表 6において EZTは標的細胞に対するェフエク夕一細胞の比を示す。 In Table 6, EZT indicates the ratio of efek yuichi cells to target cells.
その結果、 CE— 3で誘導して得られたエフヱクタ一細胞が CE— 3ぺプチド を添加培地で培養した T I S I ( + ) に対して抗原べプチド特異的な細胞傷害性 を示す共に、 MKN— 4 5に対しても細胞傷害活性を示した。 実施例 5 C E A抗原べプチド特異的 C T Lの調製 （ 2 ) As a result, effector cells obtained by induction with CE-3 showed antigen-peptide-specific cytotoxicity against TISI (+) cultured in a medium supplemented with CE-3 peptide, and MKN- It also exhibited cytotoxic activity against 45. Example 5 Preparation of CEA antigen peptide specific CTL (2)
本実施例における CTLは抗原提示用樹状細胞 （DC) を用いた誘導法により 調製した。 The CTL in this example was prepared by an induction method using dendritic cells (DC) for antigen presentation.
実施例 4の （ 1) で調製した CE— 2及び CE— 3の 2種のペプチドについて 、 健常人の P BMCより各々のべプチドを認識するエフェク夕一細胞を誘導し細 胞傷害活性を測定した。 With respect to the two peptides, CE-2 and CE-3, prepared in Example 4, (1), cell effect was measured by inducing effector cells recognizing each of the peptides from PBMC of a healthy subject. did.
( 1) エフェクター細胞の調製 (1) Preparation of effector cells
下記の方法により CE— 2又は CE— 3いずれかのぺプチドで個別に刺激され た 2種類のェフエクタ一細胞を調製した。 Two types of efecta cells individually stimulated with either CE-2 or CE-3 peptide were prepared by the following method.
実施例 1の （2) で調製した PBMCを融解後、 細胞濃度が 2 X I 07 個 Zm
1となるように 4 °Cの 1 ヒト A B血清を含む PBS (以下、 1 H— PBSと略 す） に懸濁した。 2 X 1 07 個 Zm 1の PBMCに対して 1 H— PBSで洗浄し た抗 CD 8抗体を結合したビーズ （Dynabeads M450、 ダイナル社製） を 14 /z 1 加え、 1時間 4 °Cで反応後非接着細胞を除いた。 非接着細胞を除いた後、 最初に 用いた細胞数 1 X 1 08 個に対して 0. 9m 1の 1 H— PBSに懸濁し、 0. 1 m 1の細胞解離用ビーズ （DETACHaBEAD、 ダイナル社製） を加えた。 1時間室温 で混和後解離した細胞を回収し 1 H— PBSで洗浄した。 洗浄した細胞を 2 X 1 06 個/ mlとなるように 5H— RPMIに懸濁し、 CE— 2又は CE—3いず れかのペプチドを用い実施例 8の （3)記載の方法で調製した DC懸濁液と等量 ずつ混和した。 この懸濁液に最終濃度 1 OngZmlとなるように I L一 7を加 え、 48ゥェル培養プレートの各ゥエルに 0..5 m 1ずつ分注し 37 °Cの C 02 ィンキュベ一ター内で培養する。 翌日 1 00 n g/m 1の I L一 1 0を含む 5 H 一 RPM Iを 50 1加えた。 After thawing the PBMC prepared in Example 1 (2), the cell concentration 2 XI 0 7 or Zm The suspension was suspended in PBS containing 1 human AB serum at 4 ° C (hereinafter, abbreviated as 1H-PBS) at 1 ° C. 2 X 1 0 7 or Zm 1 bead (Dynabeads M450, manufactured by Dynal Co.) conjugated anti-CD 8 antibody was washed with 1 H- PBS respect PBMC with 14 / z 1 was added, in 1 hour 4 ° C After the reaction, non-adherent cells were removed. After removing non-adherent cells were suspended in 1 H- PBS of 0. 9m 1 to cell number 1 X 1 0 8 or used in the first, the beads for cell dissociation of 0. 1 m 1 (DETACHaBEAD, Dynal Was added. After mixing at room temperature for 1 hour, the dissociated cells were collected and washed with 1 H-PBS. The washed cells are suspended in 5H-RPMI at a concentration of 2 × 10 6 cells / ml, and prepared using the peptide of either CE-2 or CE-3 according to the method described in Example 8, (3). It was mixed in equal amounts with the DC suspension. The IL one 7 to a final concentration of 1 OngZml to this suspension pressurized example, 48 to each Ueru of Weru culture plates 0..5 m 1 by the dispensed 37 ° C C 0 2 Inkyube in one coater Incubate. The next day, 50 1 of 5 H RPMI containing 100 ng / ml of IL-11 was added.
1週間後に各ゥエルの培養上清を除き細胞を 0.5ml の 5H-RPMI に懸濁した。 こ の懸濁液を CE— 2又は CE— 3いずれかのペプチドを用い実施例 8の （2)記 載の方法で調製した抗原提示細胞いずれか 1種を含むプレートに加え、 再刺激し た。 翌日、 IL- 10を終濃度 10 ng/mlとなるように添加し、 更に 2日置きに、 培養 上清を半量除き、 20 IU/ml rIL-2 を含む 5H-RPMI を等量加え、 1週間 C02 ィ ンキュベータ一内で培養した。 同様の再刺激を更に 3回行った後、 各ゥエルにお けるエフヱクタ一細胞について T I S I (十） 、 T I S I (―) に対する細胞傷 害活性を測定した。 細胞傷害活性を示したエフェクター細胞は個別に抗 CD 3抗 体又は抗原刺激に用いた各べプチドを用いて増殖させた。 増殖した細胞を 5 H— RPMIに懸濁し下記 （3) の方法で細胞傷害活性を測定した。 One week later, the culture supernatant of each well was removed, and the cells were suspended in 0.5 ml of 5H-RPMI. The suspension was added to a plate containing any one of the antigen-presenting cells prepared by the method described in Example 8, (2) using either CE-2 or CE-3 peptide, and restimulated. . On the next day, add IL-10 to a final concentration of 10 ng / ml.Every 2 days, remove half of the culture supernatant and add an equal volume of 5H-RPMI containing 20 IU / ml rIL-2. They were cultured in the weekly C0 2 I Nkyubeta within one. After performing the same restimulation three more times, the cytotoxic activity against TISI (10) and TISI (-) was measured for each effector cell in each well. Effector cells that showed cytotoxic activity were grown individually using anti-CD3 antibodies or each of the peptides used for antigen stimulation. The proliferated cells were suspended in 5 H-RPMI, and the cytotoxic activity was measured by the following method (3).
(2) CTL標的細胞の調製 (2) Preparation of CTL target cells
標的細胞として実施例 4の （3) と同様に T I S I (一） 、 CE— 2又は CE
一 3を添加培地で培養した 2種類の T I S I ( + ) を調製した。 また MKN— 4 5は 1 0 OUZmlの I FN—ァで 37°C48時間処理した。 上記 4種の標的細As target cells, use TISI (1), CE-2 or CE as in Example 4 (3). Two types of TISI (+) were prepared by culturing one to three in a supplemented medium. MKN-45 was treated with 10 OUZml of IFN-48 for 48 hours at 37 ° C. The above four types of target
O C O C
胞は細胞傷害活性測定当日、 実施例 4の （3)記載と同様の方法により51 C r標On the day of the cytotoxic activity measurement, the cells were labeled with 51 Cr
B哉した。 B-ya.
(3) CTLによる細胞傷害活性の測定 (3) Measurement of cytotoxic activity by CTL
(1) で調製したエフ クタ一細胞及び （2) で調製した標識標的細胞を用い 実施例 1の （5)記載の方法と同様に細胞傷害活性を測定した。 結果を表 7に示 す。 表 7 ぺプチド名 Ε/Τ TISI(-) TISK+) MKN - 45 Using the effector cell prepared in (1) and the labeled target cell prepared in (2), cytotoxic activity was measured in the same manner as in the method described in (1) of Example 1. Table 7 shows the results. Table 7 Peptide name Ε / Τ TISI (-) TISK +) MKN-45
CE- 2 10 0.9 89.1 25.8 CE- 2 10 0.9 89.1 25.8
10 1.9 92.0 67.9 10 1.9 92.0 67.9
なお表 7において E / Tは標的細胞に対するエフェクター細胞の比を示す。 その結果、 C Ε— 2又は C Ε— 3いずれかのぺプチドで誘導して得られたエフ ェクタ一細胞が T I S I ( + ) 及び ΜΚΝ— 45に対して抗原ペプチド特異的な 細胞傷害性を示し、 C Ε Α特異的な C T Lであることが明らかとなつた。 実施例 6 1£尺2ノ11 6 1：抗原ぺプチド特異的( 丁 の調製 （1) In Table 7, E / T indicates the ratio of effector cells to target cells. As a result, effector cells obtained by induction with either C C-2 or CΕ-3 peptides showed antigen-peptide-specific cytotoxicity against TISI (+) and ΜΚΝ-45. However, it was revealed that it was a CΕ-specific CTL. Example 6 1 × 2 11 6 1: Antigen peptide specific (preparation of Ding (1)
本実施例における CTLは、 実施例 1と同様に SAC— Iを用いる CTL誘導 法により調製した。
( 1 ) CTL誘導用ペプチドの選択及び合成 CTL in this example was prepared by the CTL induction method using SAC-I in the same manner as in Example 1. (1) Selection and synthesis of CTL-inducing peptide
1 255アミノ酸よりなる HER 2Zn e uタンパク質のアミノ酸配列につい て、 HLA— A 24結合性モチーフ構造を有する配列を中心に、 その他の位置の アミノ酸の種類も考慮して検索し、 表 8に示した 5個のペプチドを選択し、 ぺプ チド合成機を用いて作製した。 表 8 配列番号 HER2/neu タンパク質中の位置 名称 The search was performed for the amino acid sequence of the HER2Zneu protein consisting of 1 255 amino acids, centering on the sequence having the HLA-A24 binding motif structure, and also considering the types of amino acids at other positions. Were selected and prepared using a peptide synthesizer. Table 8 SEQ ID NO: Location in HER2 / neu protein
6 8 - 1 6 HE - 1 6 8-1 6 HE-1
20 44 0 - 44 8 HE - 4 20 44 0-44 8 HE-4
2 1 78 0 - 78 8 HE - 2 2 1 78 0-78 8 HE-2
22 90 7- 9 1 5 HE - 5 22 90 7- 9 1 5 HE-5
23 95 1 - 95 9 HE— 3 23 95 1-95 9 HE— 3
なお、 表 8において配列番号の項の番号は、 各ペプチドのアミノ酸配列を示し た配列表の配列番号を示す。 また HER 2Zn e uタンパク質中の位置の項の番 号は、 HER2 n e uタンパク質中の N末端からのアミノ酸数を示す。 また名 称の項の記号は、 本発明者等が命名したぺプチドの名称を示す。 In Table 8, the numbers in the SEQ ID No. column indicate the SEQ ID numbers in the sequence listing showing the amino acid sequences of the respective peptides. The number of the term at the position in the HER2Zneu protein indicates the number of amino acids from the N-terminus in the HER2Zneu protein. The symbol in the name column indicates the name of the peptide named by the present inventors.
(2) エフェクター細胞の調製 (2) Preparation of effector cells
下記の方法で HE— 1〜HE— 5のいずれかのぺプチドで個別に刺激された 5 種類のエフェクタ一細胞を調製した。 Five kinds of effector cells individually stimulated with any one of HE-1 to HE-5 peptides were prepared in the following manner.
実施例 1の （2) で調製した保存 PBMC：、 HE- 1〜HE— 5いずれかのぺ
プチドを用い後記実施例 8の （1)記載の方法で調製した SAC— 1処理による 初回刺激用抗原提示細胞、 及び HE - 1〜HE— 5いずれかのペプチドを用い後 記実施例 8の （2)記載の方法で調製した抗原提示細胞いずれか 1種を含む 5種 のプレートを用い、 実施例 1の （3) と同様の方法で HE— 1〜HE— 5いずれ かのぺプチドで刺激された 5種のエフェク夕一細胞を調製した。 Stored PBMC prepared in (2) of Example 1 or any of HE-1 to HE-5 Using an antigen-presenting cell for initial stimulation by SAC-1 treatment prepared by the method described in Example 8 (1) described below and a peptide of any of HE-1 to HE-5 prepared in Example 8 (1) 2) Using five types of plates containing any one of the antigen-presenting cells prepared by the method described in the above, stimulated with any of HE-1 to HE-5 peptides in the same manner as (3) in Example 1. Five types of effector cells were prepared.
(3) CTL標的細胞の調製 (3) Preparation of CTL target cells
細胞傷害活性測定のための標的細胞として、 実施例 1の （4) と同様の方法で HE- 1〜HE— 5のいずれかのぺプチドを含有する培地で培養した 5種類の T I S I ( + )並びに卵巣がん細胞株 SKOV3 (HLA—八3及ぴ11£1^2 11 e u陽性） 及びその HLA— A24形質転換株 S KO V— A 24 (HLA-A2 4及び HERSZn eu陽性） を用いた。 更に上記細胞は細胞傷害活性測定当日 実施例 1の （4) と同様の方法で51 C r標識した。 As target cells for measuring cytotoxic activity, five types of TISI (+) cultured in a medium containing any of HE-1 to HE-5 peptides in the same manner as in (4) of Example 1 And the ovarian cancer cell line SKOV3 (HLA-83 and 11 £ 1 ^ 2 11 eu positive) and its HLA-A24 transformant S KO V-A24 (HLA-A24 and HERSZn eu positive) were used. . Further, the cells were labeled with 51 Cr in the same manner as in (1) of Example 1 on the day of measuring the cytotoxic activity.
(4) CTLによる細胞傷害活性の測定 (4) Measurement of cytotoxic activity by CTL
(2) のエフェクター細胞及び （3) の標的細胞を用い、 実施例 1の （5) と 同様の方法で特異的細胞傷害活性 （％) を算出した。 結果を表 9に示す。
Using the effector cells of (2) and the target cells of (3), the specific cytotoxic activity (%) was calculated in the same manner as in (5) of Example 1. Table 9 shows the results.
表 9 ぺプチド名 E/T TISI (-） TISI ( + ) SK0V3-A24 Table 9 Peptide name E / T TISI (-) TISI (+) SK0V3-A24
HE - 1 10 5.3 41.9 4.3 HE-1 10 5.3 41.9 4.3
HE - 2 10 1.4 0.0 0.4 HE-2 10 1.4 0.0 0.4
HE- 3 10 68.3 67.2 0.0 HE-3 10 68.3 67.2 0.0
HE- 4 10 0.4 0.7 0.0 HE- 4 10 0.4 0.7 0.0
HE- 5 10 0.0 9.4 0.9 HE-5 10 0.0 9.4 0.9
表 9において E ZTは標的細胞に対するェフエクタ一細胞の比を示す。 In Table 9, EZT indicates the ratio of Effecta-to-target cells.
その結果、 HE— 1で誘導して得られたエフヱクタ一細胞が HE— 1ペプチド 添加培地で培養した T I S I ( + ) に対して抗原べプチド特異的な細胞傷害性を 示した。 実施例 7 HER 2/n e u特異的 CTLの調製 （2) As a result, effector cells obtained by induction with HE-1 showed antigen-peptide-specific cytotoxicity against TIS I (+) cultured in a medium supplemented with HE-1 peptide. Example 7 Preparation of HER 2 / neu specific CTL (2)
本実施例における CTLは抗原提示用樹状細胞 （DC) を用いた誘導法により 調製した。 The CTL in this example was prepared by an induction method using dendritic cells (DC) for antigen presentation.
実施例 6の （1) で調製した HE— 1ペプチドについて、 健常人の PBMCよ り該ぺプチドを認識するェフ クタ一細胞を誘導し細胞傷害活性を測定した。 ( 1 ) エフェクター細胞の調製 With respect to the HE-1 peptide prepared in (1) of Example 6, one effector cell that recognizes the peptide was induced from PBMC of a healthy person, and the cytotoxic activity was measured. (1) Preparation of effector cells
実施例 1の （2) で調製した PBMC：、 HE- 1ペプチドを用い後記実施例 8 の （3)記載の方法で調製した DC、 及び HE - 1ペプチドを用い後記実施例 8 の （2)記載の方法で調製した抗原提示細胞を含むプレートを用い、 実施例 5の (1) と同様の方法で HE— 1ペプチドで刺激されたエフェクター細胞を調製し
た ( PBMC prepared in (2) of Example 1, DC prepared by the method described in (3) of Example 8 using HE-1 peptide, and (2) of PBMC described below using HE-1 peptide Using the plate containing the antigen-presenting cells prepared in the manner described, effector cells stimulated with HE-1 peptide were prepared in the same manner as in Example 5, (1). (
(2) CTL標的細胞の調製 (2) Preparation of CTL target cells
標的細胞として実施例 6の （3) と同様に T I S I (一） 、 HE— 1添加培地 で培養で培養した T I S I ( + ) を調製した。 また SKOV3及び SKOV3— A24は 1 0 OUZmlの I FN—ァで 37°C48時間処理した。 上記 4種の標 的細胞は細胞傷害活性測定当日、 実施例 1の （4) と同様の方法により51 C r標 5哉した。 As target cells, TISI (1) and TISI (+) cultured in a culture medium supplemented with HE-1 were prepared in the same manner as in Example 6, (3). SKOV3 and SKOV3-A24 were treated with 10 OUZml of IFN-a for 48 hours at 37 ° C. The four target cell is cytotoxic activity measured day was 51 C r mark 5 Hara in the same manner as in Example 1 (4).
(3) CTLによる細胞傷害活性の測定 (3) Measurement of cytotoxic activity by CTL
(1) で調製したエフェクター細胞及び （2) で調製した標識標的細胞を用い 実施例 1の （5)記載の方法と同様に細胞傷害活性を測定した。 結果を表 1 0に 示す。 表 1 0 Using the effector cells prepared in (1) and the labeled target cells prepared in (2), cytotoxic activity was measured in the same manner as in the method described in (5) of Example 1. The results are shown in Table 10. Table 10
E/T TISK-) TISK+) SK0V3-A24 SK0V3 E / T TISK-) TISK +) SK0V3-A24 SK0V3
45 8.4 86.2 14.4 6.2 45 8.4 86.2 14.4 6.2
15 3.0 74.6 10.4 5.6 15 3.0 74.6 10.4 5.6
5 3.6 49.1 6.8 3.5 5 3.6 49.1 6.8 3.5
なお表 1 0において、 EZTは標的細胞に対するエフヱクタ一細胞の比を示す その結果、 HE— 1ペプチドで誘導して得られたエフヱクタ一細胞が T I S I
(+ ) 及び SKOV3— A24に対して抗原べプチド特異的な細胞傷害性を示し 、 HER 2/n e u特異的な CTLであることが明らかとなった。 実施例 8 非増殖性抗原提示細胞の調製 In Table 10, EZT indicates the ratio of effector cells to target cells. As a result, effector cells obtained by induction with HE-1 peptide were (+) And SKOV3-A24 showed antigen-peptide-specific cytotoxicity, indicating that the CTL was HER2 / neu-specific CTL. Example 8 Preparation of Nonproliferative Antigen Presenting Cells
(1) SAC - I処理による抗原提示細胞の調製 (1) Preparation of antigen-presenting cells by SAC-I treatment
実施例 1の （2) で調製した保存 PBMCを融解後、 細胞濃度が 2 X I 06 個 /m 1となるように 5H— RPM Iに懸濁した。 細胞懸濁液に SAC— I (Pans orbin cells、 カルビオケム社製） を終濃度 0. 005 %、 Immunobeads (Rabb it anti-Human I M、 バイオラド社製） を終濃度 20 g/m 1、 更に I L— 4 (ジェンザィム社製） を終濃度 2 On g/m 1となるように加え、 6ゥヱル培養 プレートの各ゥエルに 5 m 1ずつ分注し 37°Cの C02 インキュベータ内で 4日 間培養した。 4日間培養した細胞を終濃度 1 %のゥシ血清アルブミン （BSA) を含む生理食塩水で洗浄後、 細胞濃度が 1 X 1 07 個/ m 1となるように 1 %B SAと 3 zg/mlの；82 ミクログロブリンを含むクェン酸緩衝液 （p H 3. 0 ) で懸濁した後、 氷中 2分間処理した。 次に、 細胞懸濁液の 5倍容量の 1 % B S Α、 β2 ミクログロブリンを 3 /g/ml及びペプチドを 1 0〃g/ml含むリ ン酸緩衝液 （PH7. 5) で細胞を洗浄後、 1 %BSAと 3 zg/mlの 2 ミ クログロプリン及び 50 g/m 1のべプチドを含むリン酸緩衝液に懸濁し 20 °Cで 4時間反応させた。 反応後、 X線照射 （5500Rad ) を行い細胞濃度が 1 x 1 06 個 Zm 1となるように 5 H— R P M Iに懸濁し初回刺激用抗原提示細胞を調 製した。 After thawing the stored PBMC prepared in Example 1 (2), were suspended as a 5H-RPM I cell concentration becomes 2 XI 0 6 cells / m 1. The final concentration of SAC-I (Pans orbin cells, Calbiochem) in the cell suspension is 0.005%, the final concentration of Immunobeads (Rabb it anti-Human IM, BioRad) is 20 g / m1, and IL- added 4 (Jenzaimu Co.) to a final concentration of 2 on g / m 1, were cultured for 4 days in a C0 2 incubator each Ueru to 5 m 1 each dispensed 37 ° C for 6 Uweru culture plates . After washing the 4 days of culture cells with physiological saline containing a final concentration of 1% © Shi serum albumin (BSA), and 1% B SA as the cell concentration becomes 1 X 1 0 7 cells / m 1 3 zg / ml of; was suspended 8 Kuen acid buffer containing 2 microglobulin (p H 3. 0), and treated for 2 minutes in ice. Then, 1% BS Alpha 5 volumes of cell suspension, beta 2 microglobulin 3 / g / ml, and peptide 1 0〃G / ml containing-phosphate buffer cells (PH7. 5) After washing, the cells were suspended in a phosphate buffer containing 1% BSA, 3 micrograms / ml of 2 microglobulins and 50 g / ml of a peptide, and reacted at 20 ° C for 4 hours. After the reaction, the cells were irradiated with X-rays (5500 Rad) and suspended in 5H-RPMI so that the cell concentration became 1 × 10 6 Zm 1 to prepare antigen-presenting cells for initial stimulation.
( 2 ) 接着細胞からの抗原提示細胞の調製 (2) Preparation of antigen-presenting cells from adherent cells
実施例 1の （2) で調製した PBMCを融解後 X線照射 （5500Rad ) を行った 。 次いで細胞濃度が 4 X 1 06 個 Zm 1となるよう 5 H— RPM Iに懸濁した細 胞懸濁液を 24ゥエル培養プレートに lml ウエルずつ分注し、 C02 インキ
ュベータ一内でし 5時間培養した。 その後、 非接着細胞を吸引除去し、 更に各 ゥエルを RPM 1 1 640で洗浄して非接着細胞を除いた。 次に、 3 n g/m 1 の /32 ミクログロプリン及び 20 u g/m 1のべプチドを含む 5 H— RPM Iを 個別に各ゥヱルに 0. 5mlずつ分注し、 C02 インキュベーター内で培養した 。 2時間後に上清を吸引除去し 5H— RPMIで 1回洗浄し、 プレートの各ゥェ ルに残った細胞を再刺激用抗原提示細胞とした。 After melting the PBMC prepared in (2) of Example 1, X-ray irradiation (5500 Rad) was performed. Then aliquoted cells suspension of the 5 H- RPM I so that the cell concentration becomes 4 X 1 0 6 or Zm 1 to 24 Ueru culture plate at lml well min, C0 2 Ink The cells were cultured in an incubator for 5 hours. Thereafter, non-adherent cells were removed by suction, and each well was washed with RPM11640 to remove non-adherent cells. Then, 3 ng / m 5 H- RPM it was dispensed by 0. 5 ml min individually each Uweru containing 1/3 2 micro glow purine and 20 ug / m 1 total peptide, cultured in C0 2 incubator did . Two hours later, the supernatant was removed by suction, washed once with 5H-RPMI, and the cells remaining in each well of the plate were used as antigen-presenting cells for restimulation.
(3) D Cリツチな抗原提示細胞の調製 (3) Preparation of DC-rich antigen presenting cells
実施例 2の （1) で調製した保存 PBMCを融解後、 細胞濃度が 2 X I 06 個 /m 1となるように 5H— RPM Iに懸濁し、 該細胞懸濁液 1 Omlを T一 25 フラスコ （ヌンク社製） に入れた。 1. 5時間 37 °Cに放置後、 非接着細胞を除 き、 残った接着細胞に、 GM— CSF (ジェンザィム社製） を 1 00 OUZml 及び I L— 4 (ジヱンザィム社製） を 2000 U/m 1含む 5 H— RPM Iを、 1 Oml加え、 37°Cの C02 インキュベーター内で培養し D C細胞を誘導した 。 7日後に浮遊細胞を回収した。 接着細胞は細胞解離バッファー （ギブコ BRL 社製） にて回収し 5H— RPMIで洗浄後、 浮遊細胞と合せた。 回収した細胞を 、 ぺプチド 40 g/m 1、 δ2 ミクログロプリン 3〃 g/m 1及びを含む 1 % ヒト血清アルブミンを加えた生理食塩水中に 3 X 1 06 個 Zm l となるように懸 濁し、 20°Cの恒温槽中で 4時間反応させた。 反応後、 X線照射 （550 ORa d) を行った後、 1 X 1 06 個 Zm 1となるように 1 %ヒト血清アルブミンを加 えた生理食塩水で希釈し抗原提示細胞とした。 After thawing the stored PBMC prepared in Example 2 (1), were suspended in manner 5H-RPM I cell concentration becomes 2 XI 0 6 cells / m 1, T one 25 the cell suspension 1 OML The flask was placed in a flask (Nunc). 1. After leaving at 37 ° C for 5 hours, remove the non-adherent cells, and add 100 OUZml of GM-CSF (Genzim) and 2000 U / m of IL-4 (Genzim) to the remaining adherent cells. the 5 H- RPM I containing 1, 1 OML added, and incubated in a C0 2 incubator 37 ° C to induce DC cells. After 7 days, the floating cells were collected. The adherent cells were collected with a cell dissociation buffer (manufactured by Gibco BRL), washed with 5H-RPMI, and combined with the floating cells. The recovered cells, peptide 40 g / m 1, δ 2 micro glow purine 3〃 g / m 1 and saline plus 1% human serum albumin containing 3 X 1 0 so that six Zm l It was suspended and reacted in a thermostat at 20 ° C for 4 hours. After the reaction, the cells were irradiated with X-rays (550 ORad), and diluted with a physiological saline containing 1% human serum albumin to 1 × 10 6 Zm 1 to obtain antigen-presenting cells.
(4)全 PBMCからの抗原提示細胞の調製 (4) Preparation of antigen-presenting cells from whole PBMC
実施例 2の （1) と同じ方法で分離した PBMCを、 マイトマイシン処理し 〔 PBMC 1 X 1 07 個/ m 1の懸濁液にマイトマイシン C ( I CNバイオメデ イカルズ社製） を 200 g/m 1となるように添加し、 30分間放置〕 、 RP
M I 1 64 0培地で 3回洗浄した後、 ぺプチドをに 20 /g /m 1 となるよう加 えた 5H— RPM Iを等量混合した後、 24ゥヱル培養プレートに 1 m 1 /ゥェ ルずつ分注し抗原提示細胞として使用した。 実施例 9 C E A抗原べプチド機能的誘導体の作製 The PBMC isolated in the same manner as in Example 2 (1), mitomycin treated [PBMC 1 X 1 0 7 cells / m 1 of suspension mitomycin C (I CN Baiomede Ikaruzu Co.) 200 g / m 1 and leave for 30 minutes), RP After washing three times with MI 1640 medium, add 5H-RPM I with the peptide to 20 / g / m 1 in equal volume, then add 1 ml / well to a 24-well culture plate. Each was dispensed and used as an antigen-presenting cell. Example 9 Preparation of CEA Antigen Beptide Functional Derivative
( 1 ) CE- 2改変体及び C E一 3改変体の合成 (1) Synthesis of CE-2 variant and CE-13 variant
実施例 5の （ 1 ) で合成した C E— 2を基に表 1 1に示す 1 2種の改変体を設 Based on the CE-2 synthesized in (1) of Example 5, 12 types of variants shown in Table 11 were set.
S十し 7"こ。 表 1 1 配列番号 名称 S 10 7 ". Table 1 1 Sequence No. Name
24 CE- 20 1 24 CE-20 1
25 CE- 202 25 CE- 202
39 CE- 203 39 CE- 203
40 CE- 204 40 CE- 204
4 1 CE - 205 4 1 CE-205
42 CE- 20 6 42 CE-20 6
43 CE- 207 43 CE-207
44 CE- 208 44 CE- 208
45 CE- 20 9 45 CE-20 9
4 6 CE- 2 1 0 4 6 CE- 2 1 0
47 CE- 2 1 1 47 CE- 2 1 1
48 CE- 2 1 2
同様に CE— 3を基に表 1 2に示す 1 7種の改変体を設計した < 48 CE- 2 1 2 Similarly, 17 variants shown in Table 12 were designed based on CE-3.
表 1 2 配列番号 名称 Table 1 2 Sequence number Name
2 6 CE 3 0 1 2 6 CE 3 0 1
2 7 CE 3 0 2 2 7 CE 3 0 2
2 8 CE 3 0 3 2 8 CE 3 0 3
2 9 CE 3 0 4 2 9 CE 3 0 4
3 0 CE 3 0 5 3 0 CE 3 0 5
3 1 CE 3 0 6 3 1 CE 3 0 6
3 2 CE 3 0 7 3 2 CE 3 0 7
4 9 CE 3 0 8 4 9 CE 3 0 8
5 0 CE 3 0 9 5 0 CE 3 0 9
5 1 CE 3 1 0 5 1 CE 3 1 0
5 2 CE 3 1 1 5 2 CE 3 1 1
5 3 CE 3 1 2 5 3 CE 3 1 2
54 CE 3 1 3 54 CE 3 1 3
5 5 CE 3 1 4 5 5 CE 3 1 4
5 6 CE 3 1 5 5 6 CE 3 1 5
3 3 CE 3 1 6 3 3 CE 3 1 6
CE 3 1 7 CE 3 1 7
表 1 1及び 12 に示した CE— 2 0 1〜CE— 2 1 2及び CE— 3 0 1〜CE
- 3 1 5の 27種のペプチドをペプチド合成機を用いて作製した。 CE—201 to CE—212 and CE—301 to CE shown in Tables 11 and 12 -315 peptides were produced using a peptide synthesizer.
なお表 1 2における CE— 3 1 6は、 実施例 5の （ 1 ) で作製した CE— 3 ( 2. 85 nmo 1 ) に DHCP (宝酒造社製） 水溶液 （ 1 Omg/m l ) を 1 0 0 1加え、 37°C、 1 9時間反応させた後 HPLCで精製することにより作製 した。 The CE-3 16 in Table 12 was obtained by adding 100 (mg / ml) of a DHCP (Takara Shuzo) aqueous solution to CE-3 (2.85 nmo 1) prepared in (1) of Example 5. 1 was added, reacted at 37 ° C. for 19 hours, and purified by HPLC.
また CE— 3 1 7は、 配列番号 5で表される CE— 3のアミノ酸配列において N末より 4番目のシスティンのチオール残基を介したジスルフィ ド結合により 2 分子の CE— 3が結合した CE— 3の 2量体である。 本べプチドは CE— 3を 0 . 3mMとなるよう溶解した 0. 02M NH4 H C〇 3 水溶液中で 37で、 2 4時間反応させた後 H PLCで精製することにより作製した。 Further, CE-3 17 is a CE in which two molecules of CE-3 are linked by a disulfide bond via a thiol residue of the fourth cysteine from the N-terminal in the amino acid sequence of CE-3 represented by SEQ ID NO: 5. — It is a dimer of 3. This peptide was prepared by reacting CE- 3 in a 0.32 mM aqueous solution of 0.02M NH 4 HC〇3 at 37 for 24 hours and then purifying by HPLC.
(2) CE— 2又は CE— 3の機能的誘導体の同定 (2) Identification of functional derivative of CE-2 or CE-3
〇5— 2又は0£_ 3と^：1^八ー八24分子との複合体を認識する実施例 4に おいて得られた CTLが、 表 1 1及び表 1 2に示したペプチド又はペプチド類似 体と HL A— A 24分子との複合体を認識するか確認した。 The CTL obtained in Example 4 which recognizes the complex of 〇5-2 or 0 £ _3 and ^: 1 ^ 8-824 molecules was the peptide or the peptide shown in Tables 11 and 12. It was confirmed whether it recognized a complex between the peptide analog and the HLA-A24 molecule.
先ず実施例 4の （2) と同様の方法で調製した CE— 2を認識する CTしが、 51C r標識 T I S I (一） 細胞、 及び CE - 20 1〜CE - 2 1 2ぺプチドのい ずれか 1つを用い実施例 4の （3) と同様の方法で調製した 1 2種の61 C r標識 T I S I (十） 細胞に対し傷害性を示すか確認した。 その結果 CE— 2を認識す る CTLは、 CE— 20 1、 CE— 203、 CE— 204、 CE— 20 6、 CE — 207、 CE— 208、 CE— 20 9、 CE— 2 1 0、 CE— 2 1 2を添加し た培地での培養により得られた51 C r標識 T I S I ( + ) に対し明らかな細胞傷 害性を示した。 なお該 CTLは、 51(： 1"標識丁 13 I (一） 細胞に対しては細胞 傷害性を示さず、 CE— 20 1、 CE— 203、 CE— 204、 CE— 20 6、 CE— 207、 CE - 208、 CE - 20 9、 CE - 2 1 0、 CE— 2 1 2が C E— 2の機能的誘導体であることが明らかとなつた。
4人の健常人 PBMCを出発材料として実施例 4の （2) と同様の方法で調製 して得られた、 CE— 3を認識する出発材料が異なる 4種の CTLが、 51Cr標 識 T I S I (—） 細胞及び表 12に示した 1 7種のペプチドのいずれか 1つを用 い実施例 4の （3) と同様の方法で調製した 1 7種の51 Cr標識 T I S I ( + ) 細胞に対し傷害性を示すか確認した。 その結果 CE— 3を認識する CTLは、 C E— 301、 CE - 302、 CE— 305、 CE— 307、 CE - 31 0、 CE — 31 1、 CE— 31 2、 CE - 314、 CE_3 1 6、 CE— 31 7いずれか のべプチドを個別に添加した培地での培養により得られた 1 0種の51 C r標識 T I S I ( + ) に対し明らかな細胞傷害性を示した。 なお該 CTLは51 C r標識 T I S I (一） 細胞に対しては細胞傷害性を示さず、 CE— 30 1、 CE- 302 、 CE - 305、 CE - 307、 CE- 31 0、 CE - 31 1、 CE - 312、 CE— 31 4、 CE— 31 6、 CE— 31 7が C E— 3の機能的誘導体であるこ とが明らかとなった。 実施例 1 0 CTLを用ぃたHLA-A24分子の検出 First, CTs recognizing CE-2 prepared in the same manner as in (2) of Example 4 were obtained from 51 Cr-labeled TISI (1) cells and CE-201 to CE-212 peptide. Using either one of them, it was confirmed whether or not it shows toxicity to 12 kinds of 61 Cr-labeled TISI (ten) cells prepared in the same manner as (3) of Example 4. As a result, the CTLs that recognize CE-2 are CE-201, CE-203, CE-204, CE-206, CE-207, CE-208, CE-209, CE-210, CE - showed clear cell injury property to 2 1 51 C r obtained by cultivation of 2 in medium supplemented with labeled TISI (+). The CTL did not show cytotoxicity against 51 (: 1 "-labeled 13 I (one) cells, CE-201, CE-203, CE-204, CE-206, CE-207. , CE-208, CE-209, CE-210, CE-212 were revealed to be functional derivatives of CE-2. Four healthy individuals PBMC were obtained by preparing in the same manner as in (2) of Example 4 as starting materials, CE- 3 recognizes the starting material four different CTL is, 51 Cr-labeled TISI (—) Cells and 17 kinds of 51 Cr-labeled TISI (+) cells prepared in the same manner as (3) in Example 4 using one of the 17 kinds of peptides shown in Table 12 It was confirmed whether or not it showed any injuries. As a result, the CTLs that recognize CE-3 are CE-301, CE-302, CE-305, CE-307, CE-310, CE-311, CE-312, CE-314, CE_316, It showed clear cytotoxicity against 10 kinds of 51 Cr-labeled TISI (+) obtained by culturing in a medium to which any of CE-317 peptides were individually added. Note The CTL did not show cytotoxicity against 51 C r-labeled TISI (I) cells, CE- 30 1, CE- 302, CE - 305, CE - 307, CE- 31 0, CE - 31 1 , CE-312, CE-314, CE-316, CE-317 were found to be functional derivatives of CE-3. Example 10 Detection of HLA-A24 Molecule Using CTL
本発明の CTLにより HLA— A24分子が検出出来るかヒト由来 PBMCを 被検細胞として検討した。 Whether or not HLA-A24 molecule can be detected by the CTL of the present invention was examined using human-derived PBMC as test cells.
従来法の抗 HL A抗体を用い、 発現している HLAタイプの判明している 8人 より実施例 1の （2) と同様の方法で PBMCを調製し、 被検細胞用 PBMC8 検体とした。 各検体を 2つに分け、 一方は 1 0 g/mlとなるように CE— 3 を溶解した 5H— RPMIに各々懸濁し、 これを CE— 3添加検体群とした。 も う一方は 5H— RPMIに各々懸濁し、 これを CE— 3無添加検体群とした。 各 群を 37でで 2時間放置後、 遠心分離操作により細胞を回収し、 上清を除去した 。 本操作により CE— 3添加群より過剰の CE— 3を除去した。 次いで上記 1 6 種の細胞を 5 H— RP Mlに懸濁後、 96ウェルマイク口プレートの各ゥエルに 各々 1 X 1 04 個づっ分注した。 次いで各ゥヱルに実施例 4において CE— 3を
用い得られた CTLを 3 x 1 04 個づっ加え、 37°C、 22時間じ02 ィンキュ ベータ一内に放置後、 各ゥヱル上清中の I FN— 7量をデュオセット 〔Du 0 s e t ;ジヱンザィム （Genzyme)社製〕 により測定した。 その結果 CE 一 3添加群の内、 HLA— A24分子を発現しているヒ卜より調製した PBMC 5検体を分注したゥエルにのみ I FN— 7の存在が確認された。 なお対照とした CE- 3無添加群のいずれのゥヱルの I F N— 7量は検出限界以下であつた。 従 つて本発明の CTLを用いることにより HLA— A24分子を検出出来ることが 明らかとなった。 Using a conventional method of anti-HLA antibody, PBMCs were prepared from eight individuals with known HLA types in the same manner as in Example 1, (2), and used as PBMC8 specimens for test cells. Each sample was divided into two, and one was suspended in 5H-RPMI in which CE-3 was dissolved so as to have a concentration of 10 g / ml, and this was used as a CE-3 added sample group. The other was suspended in 5H-RPI, and this was used as a sample group without CE-3. After leaving each group at 37 for 2 hours, the cells were collected by centrifugation, and the supernatant was removed. This operation removed excess CE-3 from the CE-3 added group. Next, after suspending the above 16 kinds of cells in 5 H-RPMl, 1 × 10 4 cells were dispensed to each well of a 96-well microphone plate. Next, CE-3 in Example 4 was added to each cell. The CTL obtained using 3 x 1 0 4 pieces Dzu' addition, 37 ° C, 22 h Ji 0 2 Inkyu after leaving the beta in one, I FN- 7 weight duo set [Du 0 The set in each Uweru supernatant ; Genzyme). As a result, among the 13 CE-added groups, the presence of IFN-7 was confirmed only in the wells into which 5 specimens of PBMC prepared from humans expressing the HLA-A24 molecule were dispensed. The amount of IFN-7 in any of the control group without CE-3 was below the detection limit. Therefore, it was revealed that HLA-A24 molecule can be detected by using the CTL of the present invention.
一方、 CE— 3添加群において I FN—ァの存在が認められた PBMC検体の うち、 HLA— A24ホモのヒトより調製した PBMC検体を分注したゥエルに おける I FN— 7量は、 HLA— A24ヘテロのヒトより調製した P BMC検体 を分注したゥヱルにおける I FN— 7量の約 2倍量存在した。 従って本発明の C TLを用いることにより HLA— A24発現量を推定出来ることが明らかとなつ た。 実施例 1 1 CTLを用いた HLA— A2分子の検出及び HLA— A2サブタイ プの同定 On the other hand, of the PBMC specimens in which the presence of IFN-α was observed in the CE-3 added group, the amount of IFN-7 in the well into which the PBMC specimen prepared from HLA-A24 homozygous human was dispensed was HLA- The amount of IFN-7 was about twice the amount of IFN-7 in the dispensed PBMC specimen prepared from A24 heterozygous human. Therefore, it became clear that the expression level of HLA-A24 can be estimated by using the CTL of the present invention. Example 11 Detection of HLA-A2 molecule using CTL and identification of HLA-A2 subtype
配列番号 37記載のアミノ酸配列で表される MAGE 3由来の HLA— A2拘 束性ペプチドである FLWGPRALVを用いて、 実施例 1 (3) と同様の方法 で、 HLA— A2 (A* 0201 ) を保有する健常人由来の P BMCよりエフェ クタ一細胞を調製した。 標的細胞として HLA— A 2細胞である .221 (A 2 . 1 ) 細胞を細胞傷害性測定前日に上記べプチド 1 0 / gZm 1を含む培地 （ . 221 (A2. 1) (+ ) ) 又は含まない培地 （ .221 (A2. 1) (一） ） で培養したものを使用した。 測定当日、 51 C r標識した .221 (A2. 1) ( + ) 又は .221 (A2. 1) (—） 細胞とエフェクター細胞を混和し、 5時間 後培地中に遊離された51 C r量を測定した。 その結果、 調製したエフヱクタ一細
胞がぺプチド特異的な傷害性を示す C T Lであることが明らかとなつた。 また、 . 22 1 (A2. 1 ) (+ ) 又は .2 2 1 (A2. 1 ) (―) 細胞と CTLを混 和し 1日後の上清中の I FN— 7を測定したところ、 エフヱクタ一細胞である C T Lがべプチド特異的に I F N—ァを遊離することが明らかとなった。 この C T Lを増やした後、 各種 H L A— A 2細胞に対してべプチド特異的な I F N— 遊 離作用の有無を検討した。 HLA-A2 (A * 0201) was synthesized in the same manner as in Example 1 (3) using FLWGPRALV, which is a MAGE3-derived HLA-A2-restricted peptide represented by the amino acid sequence of SEQ ID NO: 37. One effector cell was prepared from PBMCs derived from a healthy person. .221 (A2.1) cells, which are HLA-A2 cells as target cells, were cultured on the day before the cytotoxicity measurement in a medium containing the above peptide 10 / gZm1 (.221 (A2.1) (+)) or A culture cultured in a medium (.221 (A2. 1) (1)) not containing was used. Measurements day, 51 C r-labeled .221 (. A2 1) (+ ) or .221 (. A2 1) (- ) cells and effector cells were mixed, 51 C r amount liberated into the culture medium after 5 hours Was measured. As a result, the prepared effector It was revealed that the vesicles were CTL showing peptide-specific cytotoxicity. Also, .221 (A2.1) (+) or .221 (A2.1) (-) cells were mixed with CTL, and IFN-7 in the supernatant one day later was measured. It was revealed that one cell, CTL, released IFN-a in a peptide-specific manner. After increasing this CTL, the presence or absence of a peptide-specific IFN-migrating effect on various HLA-A2 cells was examined.
1 ) 株化細胞 . 22 1 (A2. 1 ) (HLA-A* 0 2 0 1) 、 CLA (HLA -A* 0206) 、 FUN (HLA-A* 0203) 、 p 81 5 (HLA-A* 1) Cell line.221 (A2. 1) (HLA-A * 0201), CLA (HLA-A * 0206), FUN (HLA-A * 0203), p815 (HLA-A *
0202) に対する反応性 0202)
上記各細胞にぺプチド 1^〇？1¾八]^¥を 1 0〃gZm 1 となるように添加 し、 37°Cで 4時間インキュベートした。 過剰のペプチドを洗浄除去後、 96ゥ エルマイクロプレートに 1 X 1 04 個 /0. 1 m l/ゥエル入れ、 これに CTL 細胞を 1 X 1 05 個 Z0. 1ml ウヱル加え、 20時間反応させた。 対照とし てペプチド無添加細胞を用意し、 同様に CTL細胞と反応させた。 反応液より上 清を取り適当に希釈後、 I FN— 7量を定量した。 その結果、 この CTLは、 . 221 (A2. 1) (HLA-A* 0201 ) に対してのみ、 ペプチド特異的なPeptide 1 ^ 〇 in each of the above cells? 1¾8] ^ ¥ was added to give 10〃gZm 1 and incubated at 37 ° C for 4 hours. After washing remove excess peptide 96 © El microplate 1 X 1 0 4 cells / 0. Put 1 ml / Ueru, to this CTL cell 1 X 1 0 5 or Z0. 1 ml Uweru added and reacted for 20 hours Was. Cells without peptide were prepared as a control and reacted with CTL cells in the same manner. The supernatant was removed from the reaction solution, diluted appropriately, and the amount of IFN-7 was quantified. As a result, this CTL was peptide-specific only for .221 (A2.1) (HLA-A * 0201).
1 FN— 7の遊離を起こしていた。 1 FN-7 was released.
2) ヒト PBMCに対する反応性 2) Reactivity to human PBMC
ボランティア健常人 3人の PBMC (A氏： HLA— A* 0207/A 1 1、 B氏： A* 0206/A 24、 C氏： A* 0201 /A* 0206 ) にべプチド 1^ 0?1¾入1^¥を1 0〃gZmlとなるように添加し、 37 °Cで 2時間イン キュペートした。 過剰のペプチドを洗浄除去後、 96ウェルマイク口プレートに 各 1 X 1 04 個 Z0. lmlZゥヱル入れ、 これに CTL細胞を 1 x 1 04 個 0. lmlZゥエル加え、 20時間反応させた。 対照としてペプチド無添加細胞 を用意し、 同様に CTL細胞と反応させた。 反応液より上清を取り適当に希釈後 、 I FN— 7量を定量した。 その結果、 この CTLは、 HLA-A* 0201を
有する C氏の PBMCに対してのみ、 ぺプチド特異的な I FN—ァの遊離を起こ していた。 3 volunteers with healthy volunteers (Mr. A: HLA—A * 0207 / A11, Mr. B: A * 0206 / A24, Mr. C: A * 0201 / A * 0206) 1 ^ 0? 1¾ 1 ^ ¥ was added so as to be 10〃gZml, and the mixture was incubated at 37 ° C for 2 hours. After washing remove excess peptide, put the 1 X 1 0 4 or Z0. LmlZ Uweru in 96-well microphone port plate, which in CTL cells 1 x 1 0 4 or 0. LmlZ Ueru added and allowed to react for 20 hours. As a control, cells without peptide were prepared and reacted with CTL cells in the same manner. The supernatant was taken from the reaction solution, diluted appropriately, and the amount of IFN-7 was quantified. As a result, this CTL has HLA-A * 0201 Only the PBMC of Mr. C had released peptide-specific IFN-α.
上記 1)、 2) の結果より、 HLA_ A 2拘束性ペプチド及びそのペプチドで HLA-A* 0201の PBMCより誘導した CTLを用いることにより、 被検 細胞について DN Aタイビングを実施することなく、 ^1 八—八2のサブタィプ まで同定できることが明らかとなつた。 配列表フリーテキスト From the results of 1) and 2) above, the use of the HLA_A2-restricted peptide and the CTL induced from the PBMC of HLA-A * 0201 with the peptide enables the test cell to be subjected to DNA typing without using ^. It became clear that it was possible to identify up to 18-82 subtypes. Sequence listing free text
配列番号 33のアミノ酸配列の 4番目のアミノ酸は、 2—ハイド口キシ— 4 ( R, S) 一 L—システィン一 S—ィル一 2—シクロペンテン一 1一オンである。 The fourth amino acid in the amino acid sequence of SEQ ID NO: 33 is 2-hydroxy-4 (R, S) -1L-cysteine-S-yl-1-2-cyclopentene-11one.
産業上の利用可能性 Industrial applicability
本発明の CTLは HLA— A24及び MA G E— 3共に陽性、 HLA— A24 及び MAGE— 1共に陽性、 HLA— A24及び CEA共に陽性、 又は HLA— A 24及び HER 2 /n e u共に陽性の腫瘍細胞を選択的に傷害することができ 、 がん治療を目的とした細胞医薬として、 また、 体外摘出試料中の HL A— A 2 4及び MA GE- 3共に陽性の腫瘍細胞、 H L A - A 24及び MA G E— 1共に 陽性、 HLA— A24及び CEA共に陽性、 又は HLA— A24及び HER2Z n e u共に陽性の腫瘍細胞の有無の検出、 更に抗原べプチドと同時に使用するこ とにより体外摘出試料中の細胞の H L Aタイピング等に有用である。 また本発明 の抗原提示細胞は該 C T Lを調製するあるいは制がん剤として有用である。 更に 本発明の抗原べプチドを有効成分とした医薬用組成物は C T Lの誘導剤更には制 がん剤として有用である。
The CTL of the present invention can be used to express 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. It can be selectively injured and used as a cell medicine for the treatment of cancer. In addition, HLA-A24 and MAGE-3 positive tumor cells, HLA-A24 and MA in extracorporeal samples Detection of the presence of tumor cells positive for both GE-1 and HLA-A24 and CEA, or HLA-A24 and HER2Z neu, and the HLA of cells in the extracorporeal sample when used together with the antigen peptide. Useful for typing, etc. Further, the antigen-presenting cell of the present invention is useful for preparing the CTL or as an anticancer agent. Further, the pharmaceutical composition containing the antigen peptide of the present invention as an active ingredient is useful as a CTL inducer and further as a carcinostatic agent.
Claims
1. 配列番号 1〜 6のいずれかに記載のァミノ酸配列で表されるヒト主要組織 適合性抗原 （HLA) - A 24拘束性抗原ペプチド及びその機能的誘導体から選 択される少なくとも一つの抗原ペプチドと HLA— A24分子との複合体を細胞 表面に提示する細胞を認識する細胞傷害性 Tリンパ球。 1. At least one antigen selected from a human major histocompatibility complex (HLA) -A24 restricted antigen peptide and a functional derivative thereof represented by the amino acid sequence of any one of SEQ ID NOs: 1 to 6 Cytotoxic T lymphocytes that recognize cells that present a complex of peptide and HLA-A24 molecule on the cell surface.
2. 請求項 1記載の細胞傷害性 Tリンパ球を有効成分として含有してなる制が ん剤。 2. An anticancer agent comprising the cytotoxic T lymphocyte according to claim 1 as an active ingredient.
3. 配列番号 1〜6のいずれかに記載のアミノ酸配列で表されるヒト主要組織 適合性抗原 （HLA) - A 24拘束性抗原ペプチド及びその機能的誘導体から選 択される少なくとも一つの抗原ペプチドを使用することを特徴とする請求項 1記 載の細胞傷害性 Tリンパ球の誘導方法。 3. At least one antigen peptide selected from human major histocompatibility antigen (HLA) -A24 restricted antigen peptide and a functional derivative thereof represented by the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 6 2. The method for inducing cytotoxic T lymphocytes according to claim 1, wherein the method is used.
4. 配列番号 1〜6のいずれかに記載のアミノ酸配列で表されるヒト主要組織 適合性抗原 （HLA) - A 24拘束性抗原ペプチド及びその機能的誘導体から選 択される少なくとも一つの抗原ペプチドを有効成分として含有してなる、 請求項 1記載の細胞傷害牲 Tリンパ球の誘導剤。 4. At least one antigen peptide selected from a human major histocompatibility complex (HLA) -A24 restricted antigen peptide and a functional derivative thereof represented by the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 6 The agent for inducing cytotoxic T lymphocytes according to claim 1, which comprises as an active ingredient.
5. 配列番号 1〜 6のいずれかに記載のァミノ酸配列で表されるヒト主要組織 適合性抗原 （HLA) - A 24拘束性抗原ペプチド及びその機能的誘導体から選 択される少なくとも一つの抗原べプチドを有効成分として含有してなる制がん剤 5. Human major histocompatibility antigen (HLA) represented by the amino acid sequence of any one of SEQ ID NOS: 1 to 6-at least one antigen selected from A24-restricted antigen peptides and functional derivatives thereof Anticancer drug containing peptide as an active ingredient
6. 配列番号 1〜6のいずれかに記載のアミノ酸配列で表されるヒト主要組織
適合性抗原 （HLA) -A24拘束性抗原ペプチド及びその機能的誘導体から選 択される少なくとも一つの抗原ペプチドと HLA— A24分子との複合体を細胞 表面に提示してなる抗原提示細胞。 6. Human major tissue represented by the amino acid sequence of any of SEQ ID NOs: 1 to 6 Compatible antigen (HLA)-An antigen presenting cell comprising a complex of at least one antigen peptide selected from an A24-restricted antigen peptide and a functional derivative thereof and an HLA-A24 molecule on the cell surface.
7. 請求項 6記載の抗原提示細胞を有効成分として含有してなる細胞傷害性 T リンパ球の誘導剤。 7. An inducer of cytotoxic T lymphocytes comprising the antigen-presenting cell according to claim 6 as an active ingredient.
8. 請求項 6記載の抗原提示細胞を有効成分として含有してなる制がん剤。 8. An anticancer agent comprising the antigen-presenting cell according to claim 6 as an active ingredient.
9. 請求項 1記載の細胞傷害性 Tリンパ球を被検細胞と接触させた際に生じる 変化を指標とすることを特徴とする請求項 1記載の細胞傷害性 Tリンパ球に感受 性の細胞の検出方法。 9. A cell susceptible to the cytotoxic T lymphocyte according to claim 1, wherein a change produced when the cytotoxic T lymphocyte according to claim 1 is brought into contact with a test cell is used as an index. Detection method.
1 0. 請求項 1記載の細胞傷害性 Tリンパ球を有効成分として含有してなる、 請求項 1記載の細胞傷害性 Tリンパ球に感受性の細胞の検出剤。 10. An agent for detecting cells sensitive to the cytotoxic T lymphocytes according to claim 1, comprising the cytotoxic T lymphocytes according to claim 1 as an active ingredient.
1 1. ヒト主要組織適合性抗原 （HL A)拘束性抗原べプチドから選択される 少なくとも 1つの抗原ペプチド存在下、 該抗原ペプチドと HL A分子との複合体 を認識する細胞傷害性 Tリンパ球を被検細胞と接触させることを特徴とするヒト 主要組織適合性抗原 （HLA)分子の検出方法。
1 1. Cytotoxic T lymphocytes that recognize a complex of an antigen peptide and an HLA molecule in the presence of at least one antigen peptide selected from human major histocompatibility antigen (HLA) -restricted antigen peptides A method for detecting a human major histocompatibility antigen (HLA) molecule, comprising contacting a test cell with a test cell.
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| KR10-2000-7000503A KR100480546B1 (en) | 1997-07-15 | 1998-07-13 | Cytotoxic t lymphocytes |
| JP50689899A JP3840268B2 (en) | 1997-07-15 | 1998-07-13 | Cytotoxic T lymphocytes |
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| JP9/203900 | 1997-07-15 | ||
| JP20390097 | 1997-07-15 | ||
| JP9/203917 | 1997-07-15 | ||
| JP20391797 | 1997-07-15 | ||
| JP1473698 | 1998-01-12 | ||
| JP10/14736 | 1998-01-12 |
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| PCT/JP1998/003143 WO1999003972A1 (en) | 1997-07-15 | 1998-07-13 | Cytotoxic t lymphocytes |
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| JP (1) | JP3840268B2 (en) |
| KR (1) | KR100480546B1 (en) |
| CN (1) | CN1268738C (en) |
| WO (1) | WO1999003972A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000052045A2 (en) * | 1999-02-26 | 2000-09-08 | Fondazione Centro San Raffaele Del Monte Tabor | Mage-3 derived immunogenic peptides presented by mhc of class ii and the use thereof |
| JPWO2004029248A1 (en) * | 2002-09-27 | 2006-01-26 | 昇志 佐藤 | Tumor antigen protein and use thereof |
| JP2006508664A (en) * | 2002-12-04 | 2006-03-16 | ベイラ、リサーチ、インスティテュート | A rapid one-step method for generating antigen-loaded dendritic cell vaccines from precursors |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997034617A1 (en) * | 1996-03-21 | 1997-09-25 | Cytel Corporation | Hla binding peptides and their uses |
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1998
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- 1998-07-13 CN CNB988073293A patent/CN1268738C/en not_active Expired - Fee Related
- 1998-07-13 KR KR10-2000-7000503A patent/KR100480546B1/en not_active IP Right Cessation
- 1998-07-13 WO PCT/JP1998/003143 patent/WO1999003972A1/en not_active Application Discontinuation
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO1997034617A1 (en) * | 1996-03-21 | 1997-09-25 | Cytel Corporation | Hla binding peptides and their uses |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000052045A2 (en) * | 1999-02-26 | 2000-09-08 | Fondazione Centro San Raffaele Del Monte Tabor | Mage-3 derived immunogenic peptides presented by mhc of class ii and the use thereof |
| WO2000052045A3 (en) * | 1999-02-26 | 2001-01-04 | San Raffaele Centro Fond | Mage-3 derived immunogenic peptides presented by mhc of class ii and the use thereof |
| JPWO2004029248A1 (en) * | 2002-09-27 | 2006-01-26 | 昇志 佐藤 | Tumor antigen protein and use thereof |
| JP4484707B2 (en) * | 2002-09-27 | 2010-06-16 | 昇志 佐藤 | Tumor antigen protein and use thereof |
| JP2006508664A (en) * | 2002-12-04 | 2006-03-16 | ベイラ、リサーチ、インスティテュート | A rapid one-step method for generating antigen-loaded dendritic cell vaccines from precursors |
| JP2011046717A (en) * | 2002-12-04 | 2011-03-10 | Baylor Research Inst | Rapid one step method for generating antigen-loaded dendritic cell vaccine from precursor |
| JP4662776B2 (en) * | 2002-12-04 | 2011-03-30 | ベイラー リサーチ インスティテュート | A rapid one-step method for generating antigen-loaded dendritic cell vaccines from precursors |
| JP2013224313A (en) * | 2002-12-04 | 2013-10-31 | Baylor Research Inst | Prompt one step method for generating antigen-loaded dendritic cell vaccine from precursor |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3840268B2 (en) | 2006-11-01 |
| CN1268738C (en) | 2006-08-09 |
| KR20010021938A (en) | 2001-03-15 |
| KR100480546B1 (en) | 2005-04-06 |
| CN1264423A (en) | 2000-08-23 |
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