WO2012137538A1 - Composition d'induction de lymphocytes t cytotoxiques - Google Patents

Composition d'induction de lymphocytes t cytotoxiques Download PDF

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WO2012137538A1
WO2012137538A1 PCT/JP2012/053396 JP2012053396W WO2012137538A1 WO 2012137538 A1 WO2012137538 A1 WO 2012137538A1 JP 2012053396 W JP2012053396 W JP 2012053396W WO 2012137538 A1 WO2012137538 A1 WO 2012137538A1
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cells
peptide
antibody
derived
cytotoxic
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PCT/JP2012/053396
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Japanese (ja)
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直秀 山下
人三 長山
成晴 藤田
幸乃 木村
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国立大学法人東京大学
テラ株式会社
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Priority to TW101105197A priority Critical patent/TW201245224A/zh
Publication of WO2012137538A1 publication Critical patent/WO2012137538A1/fr

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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
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • A61K39/464452—Transcription factors, e.g. SOX or c-MYC
    • A61K39/464453—Wilms tumor 1 [WT1]
    • 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
    • A61K39/464454—Enzymes
    • A61K39/464457—Telomerase or [telomerase reverse transcriptase [TERT]
    • 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
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)

Definitions

  • the present invention relates to a composition for inducing cytotoxic T cells, a pharmaceutical composition containing cytotoxic T cells obtained by exposing the composition for inducing cytotoxic T cells, and a method for producing the pharmaceutical composition And about.
  • antigen-specific cytotoxic T cells are induced by the interaction of hematopoietic stem cell-derived CD8-positive cells with dendritic cells.
  • the interaction involves the association of a fragment of the antigen molecule with the HLA class I molecule on the surface of the dendritic cell and the TCR / CD3 / CD8 complex on the surface of the CD8 positive cell.
  • the CD8-positive cell specifically recognizes a cell in which an HLA class I molecule that matches the compatible type of the HLA class I molecule forms a complex with a fragment of the antigen molecule.
  • Cell to attack The interaction is also facilitated by the association of CD28 molecules on the surface of CD8 positive cells with CD80 / 86 molecules on the surface of dendritic cells.
  • cytotoxic T cells that specifically recognize virus antigens attack virus-infected cells.
  • cytotoxic T cells that recognize tumor-specific antigens attack the tumor cells. Therefore, in tumor immunotherapy, the problem is how to efficiently induce cytotoxic T cells.
  • CD8 positive cells which are precursor cells of cytotoxic T cells, from hematopoietic stem cells such as cord blood, peripheral blood, and bone marrow.
  • CD8 positive cells which are precursor cells of cytotoxic T cells
  • hematopoietic stem cells such as cord blood, peripheral blood, and bone marrow.
  • An example of such an artificial antigen-presenting cell is a technique in which a cell such as fibroblast is transfected with a gene such as an HLA class I molecule, an antigen molecule, or a CD80 / 86 molecule to substitute for a dendritic cell. is there.
  • a complex of an MHC class I molecule tetramer and an antigen peptide (hereinafter referred to as “antigen tetramer”) can bind to a plurality of T cell receptors on the same cell surface.
  • antigen tetramer In addition to detecting T cells recognizing (Non-patent document 1), it is used for antigen-specific T cell activation (Non-patent document 2).
  • CD8 positive cells can be stimulated by using anti-CD3 antibodies with agonist activity instead of a complex of molecular fragments and HLA class I molecules on the surface of dendritic cells.
  • an anti-CD28 antibody having agonist activity is used instead of the CD80 / 86 molecule on the surface of dendritic cells.
  • CD8 positive cells can be stimulated.
  • antigen-specific CTL induction is not possible with anti-CD3 / CD28 beads alone, and in order to induce antigen-specific CTL, it is necessary to use either an antigen-presenting cell derived from a living body or an artificial antigen-presenting cell in combination. It was.
  • the present invention provides a composition for inducing cytotoxic T cells, comprising an anti-CD28 antibody, a solid support on which the anti-CD28 antibody is immobilized, and a soluble peptide capable of binding to MHC class I molecules. .
  • composition for inducing cytotoxic T cells of the present invention may comprise an anti-CD28 antibody, a solid support on which the anti-CD28 antibody is immobilized, and a soluble peptide capable of binding to MHC class I molecules. .
  • the solid support may be a culture vessel or a microbead used for cell culture containing the CD8-positive cells.
  • the soluble peptide capable of binding to the MHC class I molecule is presented as an antigen by the patient's HLA-compatible HLA complex and recognized by the cytotoxic T cell. May be.
  • the present invention provides a pharmaceutical composition for treating tumors.
  • the pharmaceutical composition for tumor treatment of the present invention comprises the composition for inducing cytotoxic T cells of the present invention, and the soluble peptide capable of binding to the MHC class I molecule is an amino acid sequence of a specific antigen protein of tumor cells. Including the portion, the cytotoxic T cells recognize the tumor cells.
  • the specific antigen protein of the tumor cell may be WT-1.
  • the present invention provides a pharmaceutical composition for treating viral diseases.
  • the pharmaceutical composition for treating viral diseases of the present invention comprises the composition for inducing cytotoxic T cells of the present invention, wherein the soluble peptide capable of binding to the MHC class I molecule is an amino acid sequence of a virus-specific antigen protein. Including the portion, the cytotoxic T cells recognize the tumor cells.
  • the specific antigen protein of the virus may be a pp65 protein of cytomegalovirus.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition of the present invention is obtained by exposing the composition for inducing cytotoxic T cells of the present invention to CD8 positive cells derived from hematopoietic stem cells, and recognizes a soluble peptide capable of binding to the MHC class I molecule. Includes cytotoxic T cells.
  • the hematopoietic stem cells are any stem cell-derived hematopoietic stem cell selected from the group consisting of embryonic stem cells, adult stem cells and induced pluripotent stem (iPS) cells, and cord blood-derived hematopoietic stem cells.
  • the hematopoietic stem cells, peripheral blood-derived hematopoietic stem cells, and bone marrow blood-derived hematopoietic stem cells may be selected.
  • the present invention provides a method for producing a pharmaceutical composition containing cytotoxic T cells.
  • the method for producing a pharmaceutical composition comprising cytotoxic T cells of the present invention comprises (1) any stem cell-derived one selected from the group consisting of embryonic stem cells, adult stem cells, and induced pluripotent stem (iPS) cells.
  • CD8 positive cells are proliferated predominantly from at least one hematopoietic stem cell selected from the group consisting of hematopoietic stem cells, cord blood-derived hematopoietic stem cells, peripheral blood-derived hematopoietic stem cells, and bone marrow blood-derived hematopoietic stem cells.
  • the soluble peptide capable of binding to the MHC class I molecule comprises an amino acid sequence of human WT-1 protein or cytomegalovirus pp65 protein. There is a case.
  • the present invention provides an immunotherapy comprising the step of transplanting a pharmaceutical composition comprising the cytotoxic T cells of the present invention.
  • the immunotherapy of the present invention includes CD8 positive cells obtained by collecting hematopoietic stem cells including, but not limited to, peripheral blood and bone marrow of a patient, or CD8 differentiated and / or amplified from the obtained hematopoietic stem cells. It may include exposing the positive cells to the cytotoxic T cell-inducing composition of the present invention in vitro and reimplanting the induced cytotoxic T cells into a patient.
  • CD8 positive cells differentiated from a patient's somatic cell-derived adult stem cell or a patient's somatic cell-derived induced pluripotent stem cell are induced in vitro in the cytotoxic T cell of the present invention. Exposing to the composition for use and reimplanting the induced cytotoxic T cells into the patient.
  • the source of the CD8 positive cells of the present invention includes peripheral blood, bone marrow, lymph node, and umbilical cord blood, but may be tissues that are not limited thereto. It is preferable that the dendritic cell or its precursor cell of the present invention is prepared from peripheral blood. However, it may be prepared from hematopoietic stem cells generated from embryonic stem cells, adult stem cells and induced pluripotent stem (iPS) cells.
  • CD8 positive cells In order for CD8 positive cells to be induced in cytotoxic T cells, simultaneously with stimulating CD28 on CD8 positive cells, a complex of MHC class I molecule and antigen epitope molecule and T cell receptor / CD3 / Antigen presentation by association with the CD8 complex needs to occur.
  • CD8 positive cells are induced in cytotoxic T cells using only the immobilized anti-CD28 antibody and soluble antigen peptide, and without using dendritic cells.
  • the composition for inducing cytotoxic T cells of the present invention is assumed by assuming the involvement of cells expressing HLA class I molecules contained in the culture of CD8 positive cells. The mechanism of action can be rationally explained.
  • CD8 positive cell 1 is subjected to CTL induction by soluble peptide 2 and anti-CD28 antibody 4 immobilized on solid support 3 such as magnetic beads.
  • the anti-CD28 antibody 4 immobilized on the solid support 3 reacts with the CD28 molecule 5 on the CD8 positive cell 1, and the CD28 molecule 5 interacts with the CD80 / 86 on the dendritic cell. Stimulation is applied to CD8 positive cells 1.
  • the soluble peptide 2 alone is not recognized by the T cell receptor / CD3 / CD8 complex 6 on the CD8 positive cell 1.
  • soluble peptide 2 binds to HLA class I molecule 7 on cell 8 expressing HLA class I molecule in the culture of said CD8 positive cell 1
  • the T cell receptor / CD3 / CD8 complex on CD8 positive cell 1 6 is presented as an antigen.
  • cytotoxic T that recognizes soluble peptide 2 restricted by HLA class I molecule 7 can be obtained simply by adding soluble peptide 2 and anti-CD28 antibody immobilized on solid support 3 to CD8-positive cell 1.
  • Cell 9 is derived from CD8 positive cell 1. According to the method of the present invention, it is possible to induce CTL against different types of antigens simply by replacing the soluble peptide. Moreover, CTL induction
  • the soluble peptide capable of binding to the MHC class I molecule of the present invention may consist of the amino acid sequences listed in SEQ ID NOs: 1 to 5 in the sequence listing attached to the present specification. Soluble peptides capable of binding to MHC class I molecules of the present invention are not limited to HLA-A restricted epitopes. It has been conventionally known that cytotoxic T cells specific for other antigens can be amplified in vitro, and that clinical effects can be obtained by transplanting such cytotoxic T cells into a patient. For example, Godet, Y. et al. (Cancer Immunol.
  • cytotoxic T cells specific for HLA-B-restricted peptides derived from melanoma-specific cancer antigen tyrosinase obtained cytotoxic T cells specific for HLA-B-restricted peptides derived from melanoma-specific cancer antigen tyrosinase.
  • Straathof, K.M. C. M. (Blood, 105: 1898-1904 (2005)) are HLA-B restricted epitopes of the EB virus-related antigen LMP2, HLA-B restricted epitopes of the EB virus-related antigen EBNA1, and HLA-of the EB virus-related antigen EBNA3.
  • the soluble peptide capable of binding to the MHC class I molecule of the present invention is not only an HLA-A restricted epitope such as a peptide derived from WT-1 protein, but also an HLA-B or HLA-C restricted epitope. Also good.
  • Disease-related antigens expressed by patient cells include WT-1, human telomerase reverse transcriptase (hTERT), survivin, tyrosinase, NY-ESO-1, CEA, NSE, PSA, gp100, MART-1 and MAGE-3.
  • WT-1 human telomerase reverse transcriptase
  • hTERT human telomerase reverse transcriptase
  • survivin tyrosinase
  • tyrosinase NY-ESO-1
  • CEA gp100
  • MART-1 and MAGE-3 Including, but not limited to, tumor-associated antigens, antigens that are (over-expressed) in cancer cells such as EB virus-related antigens such as EBER and LMP-1, and cytomegalovirus pp65 (CMVpp65) protein, etc.
  • CMVpp65 cytomegalovirus pp65
  • an antigen expressed in an infected cell such as a virus-specific antigen such as,
  • the soluble peptide capable of binding to the MHC class I molecule of the present invention may be composed of the same amino acid sequence as that of a natural protein or a partly different amino acid sequence from that of the natural protein. Alternatively, it may be a peptide containing the same amino acid sequence as that of a natural protein or an amino acid sequence that is partially different from that of a natural protein.
  • the soluble peptide has amino acid sequences 126 to 134 of WT-1 protein (SEQ ID NO: 1) and amino acids 235 to 243 of WT-1 protein.
  • Mutant amino acid sequence in which the 236th methionine residue is substituted with a tyrosine residue, amino acid sequence from 461st to 469th of hTERT (SEQ ID NO: 3), and survivin splicing variant It consists of an amino acid sequence selected from the group consisting of the amino acid sequence from the 80th to 88th amino acid sequence (SEQ ID NO: 4) of 2B and the amino acid sequence from 341th to 349th amino acid sequence (SEQ ID NO: 5) of CMVpp65 Alternatively, it may contain an amino acid sequence selected from the group.
  • the solid-phased anti-CD28 antibody of the present invention may be any antibody having agonist activity against CD28.
  • any solid support capable of accessing the surface of cultured CD8-positive cells and performing effective CTL induction is used.
  • Preferred solid supports in the present invention include, but are not limited to, latex and other commercially available microparticles and culture vessels.
  • the culture vessel includes, but is not limited to, a dish, a flask, a plate, and a multiwell plate.
  • the anti-CD28 antibody of the present invention may be bound directly to the solid support of the present invention, or may be bound via a specific binding partner that performs a specific interaction such as avidin-biotin.
  • microbeads on which streptavidin is immobilized and biotinylated anti-CD28 antibody are used.
  • the microbeads may have magnetism.
  • anti-CD28 antibody immobilized on microbeads by specific interaction between biotin and streptavidin may be referred to as anti-CD28 immunobeads.
  • the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier any solution capable of suspending living cells, such as physiological saline, phosphate buffered saline (PBS), medium, serum, and the like.
  • the schematic diagram which shows the hypothesis regarding the action mechanism of the composition for cytotoxic T cell induction
  • FITC fluorescein isothiocyanate
  • APC allophycocyanin
  • FIG. 3 is a diagram showing the result of two-dimensional flow cytometry analysis in which the intensity at the fluorescence wavelength of PE and APC is measured among cells that have been stained and passed through the CD3 + gate.
  • FIG. 6 is a diagram showing the results of two-dimensional flow cytometry analysis in which the intensity of PE and APC at the fluorescence wavelength is measured among cells that have passed through the CD3 + gate.
  • FITC-labeled anti-CD3 antibody and APC-labeled anti-CD8 antibody for umbilical cord blood-derived CD8-positive cells cultured after WT-1 peptide-specific CTL induction by WT-1 mutant peptide-added allogeneic peripheral blood-derived dendritic cells
  • FIG. 3 is a diagram showing the result of two-dimensional flow cytometry analysis in which the intensity at the fluorescence wavelength of PE and APC is measured among cells that have been stained with the CD8 antibody and passed through the CD3 + gate.
  • Umbilical cord blood-derived CD8-positive cells cultured without CTL induction treatment stained with FITC-labeled anti-CD3 antibody and APC-labeled anti-CD8 antibody, and of the cells passing through the CD3 + gate, fluorescence of PE and APC
  • Umbilical cord blood-derived CD8 positive cells cultured without CTL induction treatment, with FITC-labeled anti-CD3 antibody, PE-labeled HLA-A * 24: 02WT-1 (mu) -tetramer, and APC-labeled anti-CD8 antibody FIG.
  • FIG. 3 is a diagram showing the result of two-dimensional flow cytometry analysis in which the intensity at the fluorescence wavelength of PE and APC is measured among cells that have been stained and passed through the CD3 + gate.
  • FITC-labeled anti-CD3 antibody For peripheral CD8-positive cells cultured after stimulation with hTERT soluble peptide and anti-CD28 immunobeads, FITC-labeled anti-CD3 antibody, PE-labeled HLA-A * 24: 02hTERT-tetramer, and APC-labeled anti-CD8 antibody
  • the intensity at the fluorescence wavelength of PE and APC was measured among the cells that were stained with and passed through the CD3 + gate.
  • FIG. 3 is a diagram showing the results of two-dimensional flow cytometry analysis in which the intensity at the fluorescence wavelength of PE and APC is measured among cells that have been stained with CD3 and passed through the CD3 + gate.
  • FIG. 2 is a diagram showing the results of two-dimensional flow cytometry analysis in which the intensity at the fluorescence wavelength of PE and APC was measured among cells that were stained with 02hTERT-tetramer and an APC-labeled anti-CD8 antibody and passed through a CD3 + gate.
  • PE and APC fluorescence wavelengths of cells that passed through the CD3 + gate in a sample stained by mixing FITC-labeled anti-CD3 antibody and APC-labeled anti-CD8 antibody without mixing PE-labeled survivin-2B peptide-tetramer The result figure of the two-dimensional flow cytometry analysis in which the intensity
  • FIG. 6 is a diagram showing the results of two-dimensional flow cytometry analysis in which the intensity of PE and APC at the fluorescence wavelength is measured among cells that have passed through the CD3 + gate.
  • Two-dimensional flow cytometry in which the intensity at the fluorescence wavelength of PE and APC of cells passing through a CD3 + gate was measured with a sample stained by mixing a FITC-labeled anti-CD3 antibody and an APC-labeled anti-CD8 antibody. Analysis result diagram.
  • WT-1 peptide-specific CTL induction from umbilical cord blood (1) 1. Materials and Methods (1) Human umbilical cord blood Human umbilical cord blood samples were collected after obtaining the informed consent signature of the mother and stored frozen in a liquid nitrogen system in RIKEN BioResource Center. Blood collection methods are described in Rubinstein, P. et al. (Blood, 81: 1679-1690 (1993)) and the cryopreservation method is described in Rubinstein, P. et al. (Proc. Natl. Acad. Sci. USA, 92: 10119-10122 (1995)).
  • the amino acid sequence (CYTWNQMNL) of the peptide is listed as SEQ ID NO: 2 in the attached sequence listing.
  • a biotinylated anti-human CD28 mouse monoclonal antibody (clone CD28.2, BioLegend Japan Co., Ltd.) is a magnetic bead (Dynabeads TM M-280 Streptavidin, Life Technologies Japan Co., Ltd.). And then incubated at room temperature for 30 minutes and then used after binding by biotin-avidin reaction.
  • the anti-CD28 antibody bound to the magnetic beads is referred to as a complex of biotinylated CD28 antibody and beads or a solid-phased anti-CD28 antibody.
  • Umbilical cord blood obtained from RIKEN (ID No .: HCB00747, HLA-A locus compatible type: 24:02 and 02:06) 25 mL is 37 ° C
  • PBS supplemented with 5% final concentration of dextostane 40 (Terumo Japan) and 2.5% human serum albumin (Japanese Red Cross).
  • the obtained leukocytes were further washed 3 times with PBS supplemented with 5 mM EDTA and 2% human serum albumin.
  • the isolated CD14-positive monocytes were suspended at a concentration of 10 6 cells / mL in X-VIVO TM 15 medium supplemented with 5% human AB serum, and a 35 mm petri dish (EZ-BindShut TM, Asahi Glass Co., Ltd.). ), And then 50 ng / mL recombinant human granulocyte macrophage colony stimulating factor (GM-CSF, Peprotech) and 50 ng / mL recombinant human interleukin-4 (IL-4, Peprotech) was added and cultured.
  • GM-CSF granulocyte macrophage colony stimulating factor
  • IL-4 human interleukin-4
  • prostaglandin E2 (Daiichi Fine Chemical Co., Ltd.) and picibanil (OK432, Roche Chugai Pharmaceutical) with a final concentration of 10 ⁇ g / mL were added.
  • the cells were further cultured for 1 day to obtain mature dendritic cells.
  • CD4 positive cells the magnetic particle separator after being removed using (DynaMag-15), new Dynabeads (TM), Tcell Expander CD3 / CD28 (TM, Life Technologies Japan Ltd.) is mononuclear per 10 7 After adding 25 ⁇ L and stirring for 30 minutes on ice or at 4 ° C., CD3 / CD28 positive cells were concentrated using a magnetic particle separator (DynaMag-15).
  • the CD8 positive cells are suspended in a suspension of 10 6 cells / mL in X-VIVO TM 15 medium supplemented with 5% human AB type serum and 300 ng / mL recombinant human IL-15 (Peprotech). It was done.
  • the medium to which the cytokine was added was changed every 2 to 3 days after the start of the culture. At that time, the number of viable cells was measured using trypan blue staining, and a fresh medium supplemented with 300 ng / mL of IL-15 was added so that the cell concentration was 10 6 cells / mL.
  • the CD3 / CD28 positive cells were stimulated with soluble peptides and anti-CD28 immunobeads according to the procedure described below when the total number of cells reached 1 ⁇ 10 6 .
  • X-VIVO with 5% human AB serum and 10 ng / mL recombinant human IL-7 (Peprotech) added for the first week ( (Trademark) was cultured in 15 medium. On the 7th day and after, IL-7 was switched to 300 ng / mL recombinant human IL-15 (Peprotech), and the same results were obtained even when cultured for a minimum of 19 days.
  • the second centrifugation was performed under conditions of 500 ⁇ g and room temperature for 5 minutes. The supernatant was removed and the pellet was suspended in 30 mL of the diluent.
  • the third centrifugation was performed under conditions of 500 ⁇ g and room temperature for 5 minutes. The supernatant was removed, and the pellet was suspended in PBS supplemented with 2 mM EDTA and 0.1% human serum albumin so that the cell concentration was 1 ⁇ 10 7 cells / mL (hereinafter, “ Mononuclear cell suspension ").
  • Dynal (trademark) immunomagnetic beads CD14 (Life Technologies Japan, Inc.) was added to the mononuclear cell suspension by 25 ⁇ L per 10 8 mononuclear cells, and stirred at 4 ° C. for 30 minutes. After the reaction, CD14 positive monocytes were separated using a magnetic particle separator (DynaMag-15), and these monocytes were separated from 50 ng / mL GM-CSF, 50 ng / mL IL-4, and 5% human AB. Cultured in X-VIVO TM 15 medium supplemented with type serum. During the culture period of 6 days, no medium was exchanged. On the sixth day of culture, 50 ng / mL prostaglandin E2 and 10 ⁇ g / mL picibanil were added, followed by further culturing for one day to obtain mature dendritic cells.
  • HLA-A * 24: 02WT-1 (mu) CYTWNQMNL (HLA-A * 24 : 02 for donor) or HLA-a * 02: 01WT- 1 RMFPNAPYL ( for HLA-a * 02:01 donor) was added and stirred for 3 hours at 37 ° C, the peptide wherein the dendritic on the cell Of HLA class I molecules.
  • the mature dendritic cells and the CD8 positive cells were mixed in a 12-well plate at a ratio of 1: 5 to 1: 6.7, and further co-cultured for 12 days after the initial stimulation.
  • the anti-CD28 antibody used for induction is also biotinylated and immobilized on avidin-bound beads. Therefore, if the immobilized anti-CD28 antibody used for induction remains in the sample of flow cytometry analysis for detection of WT-1 peptide-specific CTL, PE-labeled HLA-A * 24: 02WT.
  • the tetramer of PE-labeled WT-1 peptide biotinylated on a part of the beads of -1 (mu) -tetramer is dropped and replaced with a biotinylated anti-CD28 antibody.
  • cells expressing CD28 also react upon detection of WT-1 peptide-specific CTL. Therefore, the possibility of such dropout / replacement was verified by the following experiment.
  • CD3 / CD28 positive cells were obtained from two HLA-A * 24 : 02 positive cord blood samples by the procedure described in section (4). The cells were cultured for 42 days in X-VIVO TM 15 supplemented with 1,500 IU / mL IL-2, 300 ng / mL IL-15, and 5% human AB serum. . Also, CD3 / CD28 positive cells were isolated from HLA-A * 0201 positive healthy volunteers according to the procedure described in section 7.3, and 300 IU / mL IL-2, 300 ng / mL IL-15, 5 Cultured in X-VIVO TM 15 supplemented with% human AB serum for 21 days.
  • Biotinylated anti-human CD28 mouse monoclonal antibody (clone CD28.2, BioLegend Japan Co., Ltd.) was added to the human umbilical cord blood or normal human peripheral blood-derived CD3 / CD28 positive cells at 20 ⁇ L per 10 6 cells, 0 ° C to Stir at 4 ° C for 30 minutes.
  • HLA-A * 24: 02WT- 1 (mu) CYTWNQMNL tetramer (for HLA-A * 24:02 donor) or, PE-labeled HLA-A * 02: 01WT- 1 RMFPNAPYL tetramer (HLA-A * 02:01 donor) was added and incubated at room temperature for 20 minutes to bind the tetramer on the T cell receptor of the cells.
  • 20 ⁇ L of APC-labeled anti-mouse IgG donkey monoclonal antibody (eBioscience), which is a secondary antibody for verifying that the anti-human CD28 mouse monoclonal antibody was bound to the cell surface, was added, and 0 ° C. to 4 ° C. For 20 minutes, and the binding of biotinylated CD28 antibody on the cells was verified by flow cytometry.
  • FIGS. 2A to 2H show the initial stimulation when the total number of cells reached 1 ⁇ 10 6 in any of the above three procedures, the second stimulation on the seventh day from the initial stimulation, and the two WT ⁇ Regarding cord blood-derived CD8-positive cells that have been induced to induce 1 peptide-specific CTL, HLA-A * 24: 02WT-1 (mu) ⁇ of the cells that passed the CD3 + gate on the 12th day after the initial stimulation It is a result figure of the two-dimensional flow cytometry analysis of the cell which recognizes a tetramer specifically (henceforth "tetramer positive cell").
  • FIGS. 2A to H the vertical axis represents the fluorescence intensity of the PE label, and the horizontal axis represents the fluorescence intensity of the APC label.
  • the samples of FIGS. 2A and 2B are X-VIVO TM 15 medium supplemented with 5% human AB type serum and 300 ng / mL recombinant human IL-15 without any of the above three induction treatments.
  • the cord blood-derived CD8-positive cells cultured for the same number of days as in the experiments of FIGS. 2C and 2D with medium exchange alone.
  • FIGS. 1C and D show WT-1 peptide-specific CTL induction with soluble WT-1 peptide and anti-CD28 immunobeads at a stage where the total number of cells reached 1 ⁇ 10 6 , and 7 It is umbilical cord blood-derived CD8 positive cells cultured until the 12th day after the first stimulation after being performed twice with the second stimulation on the day.
  • FIGS. 1C and D show WT-1 peptide-specific CTL induction with soluble WT-1 peptide and anti-CD28 immunobeads at a stage where the total number of cells reached 1 ⁇ 10 6 , and 7 It is umbilical cord blood-derived CD8 positive cells cultured until the 12th day after the first stimulation after being performed twice with the second stimulation on the day.
  • FIGS. 1E and 2F show WT-1 peptide-specific CTL induction by WT-1 mutant peptide-added allogeneic peripheral blood-derived dendritic cells at the time when the total number of cells reached 1 ⁇ 10 6 , and after initial stimulation It is umbilical cord blood-derived CD8-positive cells cultured twice after the second stimulation on the seventh day and then until the 12th day after the first stimulation.
  • FIGS. 1-10 show WT-1 peptide-specific CTL induction by WT-1 mutant peptide-added allogeneic peripheral blood-derived dendritic cells at the time when the total number of cells reached 1 ⁇ 10 6 , and after initial stimulation It is umbilical cord blood-derived CD8-positive cells cultured twice after the second stimulation on the seventh day and then until the 12th day after the first stimulation.
  • 2G and H show initial stimulation at the stage when WT-1 peptide-specific CTL induction by WT-1 mutant peptide-added autologous cord blood monocyte-derived dendritic cells reaches a total cell number of 1 ⁇ 10 6 ,
  • the cord blood-derived CD8-positive cells cultured twice after the second stimulation on the seventh day after stimulation and then cultured until the 12th day after the first stimulation.
  • 2A, C, E and G result samples were stained by mixing FITC-labeled anti-CD3 antibody and APC-labeled anti-CD8 antibody.
  • soluble WT-1 peptides and anti-CD28 immunobead by WT-1 peptide-specific CTL induced from umbilical cord blood that has been subjected to CD3 / CD28 positive about 8 cells passing through the CD3 + gate of the cell.
  • WT-1-specific cytotoxic T cells react with lymphoma and other malignant cells that overexpress WT-1, but are not known to attack normal cells that express only a relatively small amount of WT-1. (Gao, L.
  • HLA-a * 24:02 allyl context in cancer antigen is a peptide HLA-a * 24: 02WT- 1 (mu) did not specifically recognize the CYTWNQMNL.
  • FIG. 1 shows that
  • HLA-A * Peptide HLA-A a cancer antigen, in the context of 24:02 allele * 24: 02WT-1 (mu) CYTWNQMNL was not specifically recognized.
  • FIG. 2A, C, E, and G the sample stained without mixing with PE-labeled HLA-A * 24: 02WT-1 (mu) -tetramer and mixed with only the APC-labeled anti-CD8 antibody.
  • the number of cells exhibiting fluorescence at the emission wavelength of PE was 0.1% or less.
  • the WT-1 peptide-specific CTL induction treatment with soluble WT-1 peptide and anti-CD28 immunobeads is as follows. Compared with the induction treatment using the other two types of dendritic cells, CTLs that recognize the WT-1 peptide were induced much more.
  • WT-1 peptide-specific CTL induction from umbilical cord blood (2) 1. Materials and Methods CD8 positive cells derived from umbilical cord blood (ID number: HCB00751, HLA-A locus compatible type: 24:02 and 33:03) different from Example 1 were subjected to the same three types of induction treatment as in Example 1. And subjected to flow cytometry analysis of tetramer positive cells.
  • FIG. 3 shows the initial stimulation when the total number of cells reached 1 ⁇ 10 6 with soluble WT-1 peptide and anti-CD28 immunobeads, the second stimulation on the seventh day from the initial stimulation, and the two WT- Results of two-dimensional flow cytometry analysis of tetramer positive cells out of cells that passed the CD3 + gate on the 12th day after initial stimulation for cord blood-derived CD8 positive cells subjected to the induction treatment of 1 peptide-specific CTL
  • FIG. The vertical axis of the results in FIG. 3 is the fluorescence intensity of the PE label, and the horizontal axis is the fluorescence intensity of the APC label.
  • FIG. 3 shows the initial stimulation at the stage where the total number of cells reached 1 ⁇ 10 6 by soluble WT-1 peptide and anti-CD28 immunobeads, and 7 days after the initial stimulation.
  • umbilical cord blood-derived CD3 / CD28 which was subjected to WT-1 peptide-specific CTL induction by soluble WT-1 peptide and anti-CD28 immunobeads also in cord blood-derived CD8 positive cells different from Example 1.
  • About 9.43% of the positive cells that passed the CD3 + gate were tetramer positive.
  • the same umbilical cord blood was subjected to WT-1 peptide-specific CTL induction by autologous umbilical cord blood-derived dendritic cells, and WT-1 peptide-specific CTL induction by allogeneic HLA-matched peripheral blood-derived dendritic cells.
  • X-VIVO TM 15 medium supplemented with 5% human type AB serum and 300 ng / mL IL-15, a complex of biotinylated CD28 antibody and beads, and cancer antigen WT-1 specific CD3 / CD28 thawed in a medium supplemented with the genetically mutated soluble peptide HLA-A * 24: 02WT-1 (mu) CYTWNQMNL or CMVpp65 specific peptide HLA-A * 24: 02CMVpp65 QYDPVAALF (SEQ ID NO: 5) Positive cells were cultured for 10 days.
  • the cells on day 10 is recovered in PBS, PE-labeled HLA-A * 24: 02WT- 1 (mu) CYTWNQMNL or tetramer, PE-labeled HLA-A * 24: 02CMVpp65 QYDPVAALF tetramer or not 106 cells per 20 ⁇ L added And after standing at room temperature for 20 minutes, FITC-labeled anti-human CD3 monoclonal mouse IgG2a antibody (clone HIT3a, BioLegend Japan) and APC-labeled anti-CD8 antibody (clone RPA-T8, BioLegend Japan) 20 ⁇ L each was added and stirred at 0 ° C. to 4 ° C. for 20 minutes before being subjected to flow cytometry analysis.
  • CD3 + gate Among the cells that passed through, CMVpp65 peptide tetramer positive cells were about 3.39% (not shown). In a sample in which cord blood-derived CD3 positive cells in a control experiment in which peptide-specific CTL induction was not performed were stained with a mixture of PE-labeled WT-1 peptide tetramer and PE-labeled CMVpp65 peptide tetramer, a CD3 + gate was used.
  • Cord blood-derived CD3 positive cells subjected to CMVpp65 peptide-specific CTL induction passed through CD3 + gate in samples stained with only APC-labeled anti-CD8 antibody without mixing PE-labeled CMVpp65 peptide tetramer Among the cells, 0.27% showed fluorescence at the fluorescence wavelength of PE (not shown).
  • Umbilical cord blood-derived CD3 positive cells in a control experiment in which peptide-specific CTL induction was not performed were mixed with only APC-labeled anti-CD8 antibody without mixing PE-labeled WT-1 peptide tetramer or PE-labeled CMVpp65 peptide tetramer. Of the cells that passed through the CD3 + gate, 0.14% of the cells showed fluorescence at the fluorescence wavelength of PE (not shown).
  • the amino acid sequence of a peptide presented as an antigen is determined according to the HLA compatible type of the patient, this treatment induces many cytotoxic T cells regardless of whether they are tumor antigens or viral antigens. It is possible to do.
  • the antigen can be easily changed even when tumor cells or viruses are mutated to cause an escape mutation that acquires vaccine resistance.
  • CTL induction was effective once, it can be expected that it can be performed with a simpler culture protocol than the conventional CTL induction method.
  • Example 1 WT-1 peptide-specific CTL induction from peripheral blood Materials and Methods
  • Peripheral blood was collected from the same healthy volunteer subjects as in Example 1 (7.1) (HLA-A locus adapted: HLA-A * 24:02 and 26:01). Blood was collected in the same procedure as in Example 1 (7.1), a mononuclear cell suspension was prepared in the same procedure as in Example 1 (7.2), and CD14 positive monocytes were removed from the mononuclear cell suspension. It was. CD8 positive cells were purified from the remaining CD14 negative fraction using a flow comp CD8 kit (Life Technologies Japan). Specifically, biotinylated anti-CD8 antibody and human peripheral blood mononuclear cell suspension CD14 negative fraction were reacted on ice (0 ° C.
  • the reaction was performed at (0 ° C. to 4 ° C.) for 15 minutes, and CD8 positive cells were separated using a magnetic particle separator (DynaMag-15). Thereafter, a release buffer was added at room temperature and incubated for 10 minutes to release the avidin-conjugated magnetic immunobeads from CD8 positive lymphocytes.
  • the recovered CD8-positive cells were cultured in X-VIVO TM 15 medium supplemented with 5% human AB type serum and 300 ng / mL IL-15 at 37 ° C. and 5% CO 2 .
  • First stimulation was performed in a medium supplemented with CD28 immunobeads, and the same stimulation was performed on the 7th day after the initial stimulation, and the culture was continued until the 11th day after the initial stimulation. Every 2-3 days, the cells were diluted with fresh medium supplemented with IL-15 to a cell concentration of 10 6 cells / mL. On the 11th day after the initial stimulation, the tetramer positive rate was analyzed by flow cytometry.
  • WT-1 peptide tetramer positive cells accounted for about 0.15% of the cells that passed the CD3 + gate on the 11th day of culture. (Not shown).
  • peripheral blood-derived CD8-positive cells were stimulated twice with the CMVpp65 soluble peptide and anti-CD28 immunobeads on the first day of culture and the seventh day, cells that passed the CD3 + gate on the first day of culture Of these, WT-1 peptide tetramer positive cells accounted for about 4.53% (not shown).
  • the fluorescence wavelength of PE among the cells that passed through the CD3 + gate The number of cells exhibiting fluorescence was 0.13% or less (not shown).
  • telomerase hTERT-derived peptide-specific CTL induction from umbilical cord blood Materials and Methods From different umbilical cord blood (ID number: HCB01100, HLA-A locus adapted type: 24:02 and 26:01) that were confirmed to be HLA-A * 24: 02 positive, Example 1 ( According to the procedure described in 3), CD14 negative CD4 negative CD3 / CD28 positive cells were separated using magnetic immunobeads. The cells were diluted to a concentration of 10 6 cells / mL and added at 37 ° C., 5 ° C. in X-VIVO TM 15 medium supplemented with 10 ng / mL IL-7 and 5% human type AB serum.
  • X-VIVO TM 15 medium supplemented with 300 ng / mL IL-15 and 5% human AB type serum from the 7th day after amplification for the first week after culturing at% CO 2 concentration.
  • the cells were diluted once every 2 to 3 days so that the optimal cell concentration was 10 6 cells / mL.
  • initial stimulation is performed with a medium supplemented with hTERT-derived soluble peptide HLA-A * 24: 02 VYGFVRACL (SEQ ID NO: 3) and CD28 immunobeads, and further 7 days after the initial stimulation After similar stimulation to the eyes, the cells were cultured until the 14th day after the initial stimulation.
  • the complex of biotinylated CD28 antibody and beads was added at a bead: cell ratio of 4: 1.
  • Cells were harvested with PBS on day 14 post-priming, PE-labeled HLA-A * 24 of 10 6 cells per 20 ⁇ L: 02hTERT VYGFVRACL tetramer is added, after being allowed to stand at room temperature for 20 minutes, FITC-labeled anti-human 20 ⁇ L each of CD3 monoclonal mouse IgG2a antibody (clone HIT3a, BioLegend Japan) and APC-labeled anti-CD8 antibody (clone RPA-T8, BioLegend Japan) were added and stirred at 0 ° C. to 4 ° C. for 20 minutes. And subjected to flow cytometry analysis.
  • FIGS. 4A to 4D are results of two-dimensional flow cytometry analysis using FITC-labeled anti-CD3 antibody, PE-labeled HLA-A * 24: 02hTERT VYGFVRACL tetramer, and APC-labeled human anti-CD8 antibody.
  • the vertical axis represents the fluorescence intensity of the PE label
  • the horizontal axis represents the fluorescence intensity of the APC label.
  • cord blood-derived CD8-positive cells that had been subjected to induction of hTERT-derived peptide-specific CTL in a medium supplemented with hTERT soluble peptide HLA-A * 24: 02VYGFVRRACL and CD28 immunobeads, CD3 on the 14th day after initial stimulation.
  • hTERT peptide tetramer positive cells were about 0.74% (FIG. 4C).
  • the cord blood-derived CD3 positive cells in the control experiment in which peptide-specific CTL induction was not performed of the cells that passed the CD3 + gate on the 11th day of the start of culture, hTERT peptide tetramer positive cells were about 0.06%.
  • FIG. 4B PE-labeled hTERT peptide-tetramer is not mixed with cord blood-derived CD8-positive cells that have been subjected to hTERT-derived peptide-specific CTL induction treatment in a medium supplemented with hTERT soluble peptide HLA-A * 24: 02VYGFVRRACL and CD28 immunobeads
  • HLA-A * 24: 02VYGFVRRACL 02VYGFVRRACL and CD28 immunobeads
  • 0.08 cells showed fluorescence at the emission wavelength of PE. % Or less (FIG. 4D).
  • the X-VIVO TM 15 medium supplemented with 300 ng / mL IL-15 and 5% human AB serum was amplified for the first week after culturing at the CO 2 concentration.
  • the cells were diluted once every 2-3 days so that the appropriate cell concentration was 10 6 cells / mL.
  • first stimulation is performed with a medium supplemented with survivin-2B-derived soluble peptide HLA-A * 24: 02 survivin-2B AYACNTSTL (SEQ ID NO: 4) and CD28 immunobeads, The same stimulation was performed on the 7th day after the initial stimulation, and then cultured until the 10th day after the initial stimulation.
  • a complex of biotinylated CD28 antibody and beads was added at a bead: cell ratio of 4: 1.
  • PE-labeled HLA-A * 24:02 survivin-2B AYACNTSTL tetramer 106 cells per 20 ⁇ L was added and allowed to stand at room temperature for 20 minutes, FITC 20 ⁇ L of labeled anti-human CD3 monoclonal mouse IgG2a antibody (clone HIT3a, BioLegend Japan) and APC-labeled anti-CD8 antibody (clone RPA-T8, BioLegend Japan) were added at 0 ° C to 4 ° C. Stirred for 20 minutes and subjected to flow cytometry analysis.
  • FIGS. 5A to 5D show that among the cells that stained with FITC-labeled anti-CD3 antibody, PE-labeled HLA-A * 24: 02 survivin-2B AYACNTSTL tetramer and APC-labeled anti-CD8 antibody, and passed through the CD3 + gate, PE and It is a result figure of the two-dimensional flow cytometry analysis by APC. 5A to 5D, the vertical axis represents the fluorescence intensity of the PE label, and the horizontal axis represents the fluorescence intensity of the APC label.
  • HLA-A * 24 02 survivin-2B AYACNTSTL soluble peptide and anti-CD28 immunobeads were used for initial stimulation when the total number of cells reached 1 ⁇ 10 6 cells, and after the same stimulation on day 7 after the initial stimulation
  • the cord blood-derived CD8 positive cells cultured until the 10th day after the initial stimulation were stained with FITC-labeled anti-CD3 antibody, PE-labeled HLA-A * 24: 02hTERT-tetramer, and APC-labeled anti-CD8 antibody, and CD3 + Among the cells that passed through the gate, survivin-2B peptide tetramer positive cells were about 1.16% (FIG. 5C).
  • priming was performed in a medium supplemented with the following four peptides restricted to HLA-A * 24 : 02 and CD28 immunobeads.
  • the added peptides were (1) WT-1 specific mutant peptide CYTWNQMNL, (2) CMVpp65 peptide QYDPVAALF, (3) hTERT specific peptide VYGFVRACL, and (4) survivin-2B specific peptide AYACNTSTL. Met.
  • the same stimulation was performed on the 7th day after the initial stimulation, the cells were cultured until the 12th day after the initial stimulation.
  • a complex of biotinylated CD28 antibody and beads was added at a concentration of 4: 1 bead: cell ratio.
  • the cells were cultured and amplified for 12 days from the initial stimulation while being diluted once every 2 to 3 days so that the optimal cell concentration was 10 6 cells / mL.
  • the cells were collected in PBS and either PE-labeled HLA-A * 24: 02WT-1 (mu) -tetramer, PE-labeled HLA-A * 24: 02CMVpp65 tetramer, or PE-labeled HLA-A * 24: 02hTERT or tetramer, PE-labeled HLA-A * 24: 02survivin- 2B or tetramer, are added cells 10 per six 20 [mu] L, after being allowed to stand at room temperature for 20 minutes, FITC-labeled anti-human CD3 monoclonal mouse IgG2a antibody (clone HIT3a BioLegend Japan Co., Ltd.) and APC-labeled anti-CD8 antibody (clone RPA-T8, BioLegend Japan Co., Ltd.) were added in an amount of 20 ⁇ L each and stirred at 0 ° C. to 4 ° C. for 20 minutes for flow cytometry analysis. It was done.
  • FIGS. 6A to 6D show cells stained with FITC-labeled anti-CD3 antibody, PE-labeled HLA-A * 24: 02WT-1 (mu) -tetramer, and APC-labeled anti-CD8 antibody, and passed through the CD3 + gate. Among these, it is a result figure of the two-dimensional flow cytometry analysis by PE and APC. 6A to 6D, the vertical axis represents the fluorescence intensity of the PE label, and the horizontal axis represents the fluorescence intensity of the APC label.
  • priming is performed with WT-1-specific mutant peptide CYTWNQMNL and anti-CD28 immunobeads, and the same stimulation is performed on the 7th day after the priming, Umbilical cord blood-derived CD8-positive cells cultured until the 12th day after the initial stimulation were stained with FITC-labeled anti-CD3 antibody-derived PE-labeled HLA-A * WT-1-tetramer and APC-labeled anti-CD8 antibody. . Of the cells that passed the CD3 + gate, WT-1 peptide-specific tetramer positive cells accounted for about 0.89% (FIG. 6A).
  • WT1 peptide-specific tetramer positive cells were about 0.58% (FIG. 6E).
  • the fluorescence at the emission wavelength of PE was 0.05% (FIG. 6F).
  • WT-1 peptide-specific CTL induction with soluble WT-1 peptide and anti-CD28 immunobeads is applicable not only to CD8 positive cells derived from cord blood but also to CD8 positive cells derived from peripheral blood at least. It has been shown. Similarly, it was shown that WT-1 peptide-specific CTL induction by soluble CMVpp65 peptide and anti-CD28 immunobeads can be applied not only to CD8-positive cells derived from cord blood but also to CD8-positive cells derived from at least peripheral blood.
  • this technique is not limited to WT-1 and CMVpp65, and it has been shown that this technique can be used for a wide variety of other cancer antigens such as hTERT and survivin-2B.
  • the composition for inducing cytotoxic T cells of the present invention using a soluble peptide and an immobilized anti-CD28 antibody in combination is applicable to any hematopoietic stem cell-derived CD8-positive cell.
  • composition for inducing cytotoxic T cells of the present invention can be used for in vivo hematopoietic stem cells other than peripheral blood, for example, CD8 positive cells derived from bone marrow, lymph node and other tissue-derived hematopoietic stem cells, embryonic stem cells, It can also be applied to CD8 positive cells derived from hematopoietic stem cells differentiated from adult stem cells and induced pluripotent stem (iPS) cells.
  • iPS induced pluripotent stem

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Abstract

La présente invention concerne le développement d'une nouvelle technologie qui est capable de réaliser facilement l'induction de lymphocytes T cytotoxiques spécifiques d'un antigène pour tout antigène donné. La présente invention concerne une composition d'induction de lymphocytes T cytotoxiques contenant: un anticorps anti-CD28 ; un corps de support solide au niveau duquel l'anticorps anti-CD28 a été converti en phase solide ; et un peptide soluble qui peut se lier à une molécule MHC de classe 1. Dans la composition d'induction de lymphocytes T cytotoxiques, il y a des cas où le peptide soluble peut se lier à une molécule MHC de classe 1 présentée sous la forme d'un antigène au moyen d'un complexe HLA conforme au système HLA du patient et reconnu par les lymphocytes T cytotoxiques. La présente invention fournit une composition de traitement de tumeurs. La composition de traitement de tumeurs contient la composition d'induction de lymphocytes T cytotoxiques, le peptide soluble qui peut se lier à une molécule MHC de classe 1 contient une partie de la séquence d'acides aminés d'une protéine antigénique spécifique de cellules tumorales, et les lymphocytes T cytotoxiques identifient les cellules tumorales. La protéine antigénique spécifique des cellules tumorales peut être la protéine WT-1.
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