WO2008023786A1 - Method for producing virus-specific ctl - Google Patents

Method for producing virus-specific ctl Download PDF

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Publication number
WO2008023786A1
WO2008023786A1 PCT/JP2007/066439 JP2007066439W WO2008023786A1 WO 2008023786 A1 WO2008023786 A1 WO 2008023786A1 JP 2007066439 W JP2007066439 W JP 2007066439W WO 2008023786 A1 WO2008023786 A1 WO 2008023786A1
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virus
specific ctl
ctl
hcmv
specific
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PCT/JP2007/066439
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French (fr)
Japanese (ja)
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Susumu Suzuki
Kazue Watanabe
Shingo Taji
Ritsuko Kin
Yoshiki Akatsuka
Toshitada Takahashi
Kiyotaka Kuzushima
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Medical & Biological Laboratories Co., Ltd.
Aichi-Ken
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Priority to JP2008530963A priority Critical patent/JP5433825B2/en
Publication of WO2008023786A1 publication Critical patent/WO2008023786A1/en

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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • 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
    • A61K39/12—Viral 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
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • 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
    • A61K39/464838—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to cytotoxic T lymphocytes specific to viruses, particularly human cytomegalovirus (hereinafter referred to as “H CMV”). And a method for treating and / or preventing HCMV infection using HCMV-specific CTL prepared by this method.
  • H CMV human cytomegalovirus
  • HCMV has a latent infection in most healthy adults. For those with normal immunity, HCMV infection rarely appears. However, those who are immunosuppressed (for example, cancer patients, patients undergoing transplantation surgery such as hematopoietic stem cell transplantation, AIDS patients, etc.) develop HCMV infection symptoms and lethal interstitial pneumonia. Besides, it may cause retinitis, hepatitis, etc.
  • Hematopoietic stem cell transplantation is not only applied to hematopoietic tumors such as leukemia, but is also being applied to some solid tumors and metabolic diseases.
  • the origin of stem cells to be transplanted is increasing not only in HLA compatible donors but also in the number of cases using stem cells from alternative donors such as bone marrow bank 'umbilical cord blood bank and HLA incompatible donors.
  • stem cells from alternative donors such as bone marrow bank 'umbilical cord blood bank and HLA incompatible donors.
  • the risk of opportunistic infections increases because of the powerful immunosuppressive treatment given to the transplanted patient.
  • Ganciclovir is an excellent antiviral agent, and side effects caused by frequent doses, which are also widely used for HCMV infections, are frequently problematic. Therefore, a new method for controlling HCMV safely and effectively is strongly desired.
  • HCMV-specific CT L has the ability to destroy HCMV-infected cells when they are found, so if the function is activated effectively, it will lead to the development of new prevention and treatment methods for HCMV-related diseases. Na There is a high possibility of losing. Therefore, cell therapy using HCMV-specific CTLs has attracted attention as a new treatment method that replaces antiviral agents. In Europe and the United States, application research has already been conducted on this cell therapy, and the effect expected in clinical trials has been obtained (Non-patent Document 1).
  • Non-patent document 1 ELIZABETH A. WALTER et al., N Eng J Med, 1995; 333: 1038-1044
  • Non-patent document 2 N Watanabe et al., Cytotherapy, 2004; 6 (5): 514- 522
  • Non-Patent Document 3 Mark Cobbold et al., J Exp Med., 2005; 202 (3): 379-386
  • Non-Patent Document 4 Marek Cebecauer et al., J Immunol., 2005; 174 (11): 6809-6819
  • Non-Patent Document 5 Foster AE, Gottlieb DJ, Marangolo M, Bartlett A, Li YC, Barton GW, Romanogli JA , Bradstock KF.Rapid, large-scale generation of highly pure cytomeg alovirus-specific cytotoxic T cells for adoptive immunotherapy., J Hematother Stem Cell Res. 2003 Feb; 12 (l): 93- 105
  • Dendritic cells are used to further propagate induced HCMV-specific CTL (Non-patent Document 5). For this reason, the same problem as the above (1) occurs.
  • the burden on the donor is larger because a larger number of dendritic cells are required for proliferation than for induction.
  • the REM method is known as a very efficient growth method. REM method is a large amount Since it requires erythrocyte mononuclear cells and EBV-LCL, it is not a realistic method for commercialization.
  • Non-patent Document 3 MHC_t etramer is produced using a protein derived from Escherichia coli, there is a problem in that the survival rate of CTL is reduced in addition to the safety problem (Non-patent Document 4).
  • the HCMV-specific CTL culture method that has been carried out so far involves complicated steps such as preparing antigen-presenting cells, and thus must rely on open culture. Cultivation in an open system needs to be performed in a cell processing facility in order to prevent external contamination of bacteria, viruses, etc., and enormous equipment costs and management costs are required.
  • the present invention has been made in view of the above problems, and its purpose is to combine practicality and safety in order to realize the practical use of cell therapy using virus (particularly HCMV) -specific CTL. It is also necessary to provide a closed culture system for virus-specific CTL that can be implemented at low cost.
  • the CTL preparation process (manufacturing process) is composed of three processes: a CTL induction process, a CTL isolation process, and a CTL growth process.
  • the inventors have invented a method for preparing HCMV-specific CTL in a simple and inexpensive manner by obtaining appropriate conditions in these three steps. It was.
  • the method for preparing HCMV-specific CTL according to the present invention is as follows.
  • the following (1) to (3) relate to the CTL induction process.
  • One virus selected from the group consisting of HTLV_1, influenza, adenovirus, AIDS virus, hepatitis B virus, hepatitis C virus, Epstein Barr virus, human papilloma virus, and human cytomegalovirus (HCMV) (
  • a method for inducing HCMV) specific CTL which comprises culturing a mixture of peripheral blood mononuclear cells and virus-specific CTL epitope peptide.
  • a method for isolating virus-specific CTL wherein the virus-specific CTL induced by any of the induction methods described in (1) to (3) above or other methods is used for induction. Isolation characterized by isolating virus-specific CTL targeting the newly expressed CD137 antigen after restimulation by contact with the same epitope peptide as used in the medium. Method.
  • a method for isolating virus-specific CTL wherein the virus-specific CTL induced by any of the induction methods described in (1) to (3) above or other methods is used for induction. It is necessary to isolate virus-specific CTL targeting the newly expressed CD137 antigen after restimulation by contacting in the medium with antigen-presenting cells presenting the same epitope peptide as that used.
  • a method for isolating a virus-specific CTL characterized.
  • virus-specific CTL After re-stimulation by contact with the MHC molecule that forms a complex with the same epitope peptide as the one used in the medium, virus-specific CTL is simply targeted against the newly expressed CD137 antigen.
  • a method for isolating virus-specific CTL which comprises separating the virus-specific CTL.
  • virus-specific CTL refers to virus-specific CD8 + CTL and virus-specific CD4 + T cells are also included.
  • (11) A method for propagating virus-specific CTL, wherein the sputum cells are proliferated from peripheral blood mononuclear cells using OKT3 (anti-CD3 monoclonal antibody), and the above (1) to (3)! / After contacting the same epitope peptide as that used for induction in the medium, this sputum cell and any of the above (4) to (; 10) It is characterized by co-culturing the virus-specific CTL isolated by the described isolation method or (1) to (3)! /, Or the winores-specific CTL induced by any of the induction methods described above. To propagate virus-specific CTL.
  • Virus-specific CTL mixed ratio of T cells grown with OKT3; 10: 1 to 1: 3
  • ALyS505N 100 IU / mL to 1000 IU / mL IL-2, 0.1% to 1% autologous plasma
  • ALyS505N 10 ng / mL IL-15, 0 ⁇ 1% ⁇ 1% autologous plasma
  • a method for producing a virus-specific CTL comprising a step of inducing virus-specific CTL by the induction method according to any one of (1) to (3) above.
  • a method for producing virus-specific CTL comprising the step of isolating virus-specific CTL by the isolation method according to any one of (4) to (; 10) above.
  • a method for producing virus-specific CTL comprising the step of proliferating virus-specific CTL by the proliferation method described in any one of (11) to (; 13) above.
  • a therapeutic agent for a viral infection containing a virus-specific CTL produced by the method for producing a virus-specific CTL of any one of (14) to (; 17) above And / or preventive agent.
  • a method for examining the immune response ability of CTL comprising: (a) a CTL epitope peptide in a medium; (B) at least one component selected from the group consisting of antigen-presenting cells presenting this CTL epitope peptide, or MHC molecules forming a complex with said CTL epitope peptide, and (b) lymphocytes collected from a subject Alternatively, a test method characterized by determining the immune response ability by detecting a CD137 molecule newly expressed on the lymphocyte or CTL after co-culturing with the CTL in culture in contact.
  • the CTL preparation process (manufacturing process), which includes the CTL induction process, the CTL isolation process, and the CTL proliferation process, is complicated and expensive. Has become an obstacle in commercialization.
  • the method for preparing a virus (particularly, HCMV) -specific CTL of the present invention made it possible to prepare a simple and inexpensive HCMV-specific CTL.
  • the CTL induction process has been conventionally performed by co-culturing antigen-presenting cells such as dendritic cells and lymphocytes.
  • the preparation of antigen-presenting cells such as dendritic cells is complicated and requires a great deal of labor and expense.
  • the induction step according to the present invention does not require preparation of antigen-presenting cells, and thus can induce virus-specific CTLs quickly and easily.
  • the conventional method requires dendritic cells, and thus has the same problems as described above.
  • the proliferation process of virus-specific CTLs according to the present invention allows simple and inexpensive proliferation of vinores-specific CTLs by substituting T cells grown with OKT3 as antigen-presenting cells.
  • MHC-tetramer is a protein derived from Escherichia coli, so there are safety issues. Decreased survival rates have been pointed out.
  • a method for isolating CTL using CD 107 antigen or interferon gamma (IFNg) as a target is known (Leukemia (2005) 19, 707-709).
  • HCMV reacts with an almost 100% probability in patients who are immunosuppressed, such as patients after hematopoietic stem cell transplantation, and cause fatal infections. Therefore, control is an important issue that affects patient prognosis.
  • antiviral drugs such as ganciclovir have been used to control HCMV.
  • ganciclovir have been used to control HCMV.
  • HCMV-specific CTL specifically recognizes HCM V-infected cells and has extremely strong cytotoxic activity! According to the present invention, it is possible to provide a safe and effective virus-preventing drug, therapeutic drug by virus-specific CTL, and a technique for preparing this easily and inexpensively.
  • HCMV HCMV-specific CTL induction step
  • HCMV-specific CTL isolation step HCMV-specific CTL growth step
  • peripheral blood mononuclear cells and HCMV-specific CTL epitope peptide are contained in a medium containing plasma. Incubate with contact.
  • a CTL epitope peptide is a molecular chain of linear amino acids linked together by peptide bonds between the ⁇ -amino and carboxyl groups of adjacent amino acid residues, and is a human leucocyte antigen (human leucocyte).
  • HLA antigen
  • MHC_peptide complex A complex of MHC and peptide expressed on the cell (hereinafter referred to as “MHC_peptide complex”! /) Is recognized by the lower cell receptor (TCR) and B7-l on the antigen-presenting cell, This is done by binding an auxiliary molecule such as B7-2 to CD28, which is an auxiliary molecule on the T cell side.
  • HLA class I molecules and CTL epitope peptides expressed on the surface of membranes such as monocytes, B cells, and dendritic cells contained in minute amounts are contained in peripheral blood mononuclear cells. It is considered that a complex is formed and functions as an antigen-presenting cell.
  • the induction process that does not require preparation of antigen-presenting cells such as dendritic cells, which has been conventionally required, can be greatly reduced.
  • HCMV-specific CTL can be induced at low cost.
  • the binding between the MHC-peptide complex and TCR that occurs during antigen presentation is important for enhancing the antigen-presenting ability (this increases the antigen-presenting ability even if the binding ability is too strong). It can not be said). Therefore, the concentration of added CTL epitope peptide affects the effect of antigen presentation.
  • the final concentration is preferably O.OS g / mL ⁇ g / mL.
  • the final concentration of HLA-A24-restricted HCMV epitope peptide is 0.2, 1 g / mL-5, ig / mL! / ⁇ .
  • HLA differs between races and individuals. When the HLA is different, the epitope peptide is different even for the same antigen (this is the HLA restriction of the epitope peptide! /).
  • the optimal concentration in the induction process of HLA-A24 and HLA-A2 restricted HCMV epitope peptide which is the most common in the world and is the most common in the world Natsuta.
  • the basal medium RPMI1640, AIM-V containing 5% to 10% autologous plasma or Dulbecco's modified eagle medium (Dulbecco's modified eagle medium) is desirable, and peripheral blood mononuclear cells and the CTL epitope peptide are mixed for 2 days. It is still more effective to add IL-2 later to a final concentration of 10 IU / mL, and then use the medium while doubling the IL-2 concentration every 2-3 days.
  • the concentration of carbon dioxide gas, the temperature, and the number of days of culture are preferably 5%, 37 ° C, and 14-21 days, respectively.
  • the cell concentration is preferably 10 6 to 2xl0 6 cells / mL.
  • cells Incubating in close contact is important for efficient antigen presentation. For this purpose, it is preferable to culture in a U-shaped culture vessel.
  • isolation method in the present embodiment is the HCMV-specific CTL and HCMV-specific CTL epitope peptide induced by the induction method in the present embodiment.
  • the CD137 molecule newly expressed on the HCMV-specific CTL is isolated as a target by culturing in a medium containing plasma.
  • CD137 molecule (4-1BB, also referred to as ILA) is a molecule identified by Kwon BS et al. It is related to CTL differentiation and proliferation, and is known to be selectively expressed on CTL by stimulation via TCR. ing. Therefore, by adding an epitope peptide to a T cell population containing HCMV-specific CTL, a complex consisting of an HLA class I molecule and an epitope peptide is expressed on the T cells contained in the medium. This complex comes into contact with the TCR on the HCMV-specific CTL, and the CD137 molecule is selectively expressed on the HCMV-specific CTL.
  • This HCMV-specific CTL is reacted with a substance that recognizes CD 137 molecules, such as magnetic fine particles coated with CD 137 antibody, and then used with a suitable separation device (eg, magnetic separation device).
  • a suitable separation device eg, magnetic separation device.
  • a culture plate coated with CD137 antibody can be used and isolated by pan-jung method.
  • the anti-CD137 antibody may be a monoclonal antibody or a polyclonal antibody.
  • the effective final concentration of epitope peptide added to the medium is 1 ng / mL-10 ng / mL.
  • HCMV-specific CTLs are desirably isolated after 15 to 24 hours when CD137 antigen expression is highest after addition of the epitope peptide into the medium. In the isolation step, when CD137 antibody and HCMV-specific CTL come into contact with each other, stimulation is transmitted from the CD137 molecule into the cell, so that an effect of increasing the proliferation of HCMV-specific CTL is also expected.
  • proliferation method in this embodiment involves contacting an epitope peptide with T cells proliferated using OT3 (CD3 monoclonal antibody). On the T cell, a complex consisting of an HLA class I molecule and an epitope peptide.
  • a HCMV-specific CTL is proliferated by forming a body and contacting the TCR expressed on the HCMV-specific CTL prepared by the isolation method in the present embodiment.
  • CD80 and CD86 molecules which are costimulatory molecules, are highly expressed on T cells proliferated by stimulating peripheral blood mononuclear cells with OKT3 (hereinafter referred to as “OK ⁇ 3- ⁇ cells”), It can be used as an antigen-presenting cell.
  • OKT3 peripheral blood mononuclear cells
  • dendritic cells have generally been used as antigen-presenting cells in the CTL growth process, and their preparation has been expensive and complicated.
  • a large amount of dendritic cells are required in the CTL proliferation process (Non-patent Document 5), and blood must be newly collected from the donor, which puts a heavy burden on the donor.
  • a part of the blood collected at the time of CTL induction is used as the antigen-presenting cell, which is obtained by stimulating and proliferating with ⁇ 3.
  • HCMV-specific CTL can be propagated easily and at low cost.
  • the expression level of CD80 and CD86 on OKT3-T cells used as antigen-presenting cells is high. After stimulating PBMC with OKT3, it is desirable to use T cells cultured for 2 weeks to 1 month, where the expression level of CD80 and CD86 is highest.
  • the final concentration of the epitope peptide added to the medium is preferably ⁇ . ⁇ ⁇ g / mL lO g / mL.
  • the medium RPMI1640, AIM-V, or Dulbecco's modified eagle medium not containing protein components such as serum and plasma is preferable.
  • the contact time is preferably 30 minutes to 60 minutes, and the contact temperature is preferably 18 ° C to 25 ° C.
  • the antigen-presenting cells and HCMV-specific CTL (for example, those isolated by the isolation method in the present embodiment) prepared in this way are co-cultured to proliferate the HCMV-specific CTL.
  • the mixing ratio of HCMV-specific CTL and antigen-presenting cells can be arbitrarily selected between 10: 1 and 1: 3.
  • AICD Activation Induced Cell Death
  • Media used for co-culture of HCMV-specific CT L and antigen-presenting cells include RPMI1640 with 5% _10% autologous plasma, 10 IU / mL-50 IU / mL IL-2, AIM_V, or a modified Dulbecco method It is preferable to use an eagle medium (Dulbecco's modified eagle medium). It is preferable to replace the medium with ALyS505N-1000 containing 1% autologous plasma 18 to 36 hours after the start of co-culture, and then replace the medium once every two to three days.
  • HCMV-specific CTL can be induced, isolated and propagated.
  • HCMV-specific CTL means HCMV-specific CD8 + CTL.
  • PBMC Peripheral Blood Mononuclear Cells isolated from peripheral blood derived from HLA-A24 molecule holder or HLA-A2 holder 5% autologous plasma, 2-mercaptoethanol, L-glutamine, HEPES slow collision RPMI1640 medium containing penicillin and streptomycin as antibiotics (hereinafter, "CTL induction medium” hereinafter) was suspended so that cell number is 2Xl0 6 pieces in ImL, round bottom tubes polypropylene 14 mL (Becton 1 mL was dispensed into Dickinson).
  • CTL induction medium penicillin and streptomycin as antibiotics
  • IL-2 was added to 10 IU / mL, and every 3 days, 1 mL of medium was replaced with CTL induction medium containing 50 IU / mL IL-2 for 5 days.
  • the induction efficiency of HCMV-specific CTLs by the method of Example 1 was examined by comparing the number of HCMV-specific CTLs before and after induction.
  • the number of HCMV-specific CTLs was calculated by multiplying the total number of cells contained in the culture by the HCMV-specific CTL positive rate measured by HCMV-specific MHC-tetramer. Measurement of HCMV-specific CTL positive rate with HCMV-specific MHC-tetramer was performed as follows.
  • Cells used for measurement are washed with PBS containing 0.1% BSA (hereinafter referred to as ⁇ cell washing buffer solution ''), and then suspended in the cell washing buffer solution so that the cell concentration force becomes 3 ⁇ 4 ⁇ 10 6 cells / mL.
  • ⁇ cell washing buffer solution '' PBS containing 0.1% BSA
  • FIG. 1 shows an example of the measurement result.
  • the vertical axis shows CD8 fluorescence intensity
  • the horizontal axis shows the fluorescence intensity of HCMV-specific MHC-tetramer in logarithmic scale
  • each dot represents the fluorescence intensity of each cell.
  • the dot in the upper left area divided into four represents CD8 and MHC-tetramer positive cells at the same time, that is, HCMV-specific CTL.
  • the cells after induction contained 10.28% HCMV-specific CTL.
  • Table 1 shows the total number of cells before and after induction, the ratio and number of HCMV-specific CTLs, and the induction efficiency of the number of HCMV-specific CTLs after induction relative to the number of HCMV-specific CTLs before induction. Expressed as a ratio.
  • CD137 monoclonal antibody (clone 4B4) diluted to 10 ag / mL with PBS was dispensed into 6 well plates and allowed to stand at 4 ° C for 18 hours. After removing the antibody solution with an aspirator, 2 mL of PBS containing 5% human albumin was dispensed and incubated at 37 ° C. for 1 hour to block the surface of the culture plate sensitized with the antibody. The plate surface was washed 3 times with PBS and then used as a CD137 monoclonal antibody-sensitized culture plate.
  • QYD peptide was added to the HLA-A24-restricted HCMV-specific CTL line culture induced in Example 1 at a final concentration of 10 ng / mL and cultured for 18 hours. Collect a portion of the cells and double-stain using PC5-labeled CD137 monoclonal antibody and PE-labeled HCMV-specific MHC-tetramer to confirm that CD137 is selectively expressed on the HCMV-specific CTL. And analyzed with a flow cytometer. As shown in FIG. 2, selective expression of CD137 molecule was observed on HCMV-specific CTL (cell population shown as MHC_tetramer positive cells).
  • the HCMV-specific CTL expressing CD137 molecule was allowed to react at 37 ° C. and adhered to the surface of the culture plate via the CD137 monoclonal antibody. After gently shaking the culture plate, cells that did not adhere to the surface of the culture plate were removed using a pipette. Thereafter, 1 mL of washing was slowly added with HEPES buffered RPMI medium containing 0.5% human albumin, and after gently shaking, cells that did not adhere to the surface of the culture plate were removed again using a pipette. After further repeating this operation twice, 1 mL of a CTL induction medium containing 50 IU / mL of IL-2 was added and cultured at 37 ° C. for 24 hours in a 5% CO incubator. More
  • HLA-A24-restricted HCMV-specific CTL isolated and cultured were suspended in cell wash buffer at a cell concentration of 10 6 / mL, and 100 L of PE-labeled HCMV-specific MHC-tetramer 10 ⁇ L and 10 ⁇ L of FITC-labeled CD8 monoclonal antibody were mixed at the same time, and then reacted at 4 ° C. for 30 minutes. After washing twice with PBS, the cells were suspended in 500 L of cell washing buffer and measured using a flow cytometer.
  • the purity of HLA-A24-restricted HCMV-specific CTL which was 10.28% before isolation, increased to 56.03% immediately after isolation and to 97.14% after one week of culture. Immediately after isolation, 48.7% of HCMV-specific CTLs were recovered and then cultured for 1 week.
  • A24-10 A24 2 1.44 0.00 0.0002 / 0.01 0.01 0.29 / 20> 1450.0
  • HLA-A24-restricted HCMV-specific CTL line was restimulated with QYD peptide, suspended in 100 L of cell washing buffer, and then 100 g / mL CD137 monoclonal antibody. (Clone 4B4) was added to the mixture, and after stirring, the mixture was reacted at room temperature for 15 minutes.
  • cell separation buffer 80 L of PBS containing 0.5% human albumin and 2 mM EDTA (hereinafter referred to as “cell separation buffer”!), Then anti-mouse 20 ⁇ L of IgG-bound micromagnetic beads (Miltu) were added, and after stirring, the mixture was reacted at 4 ° C for 15 minutes. After washing twice with cell separation buffer and suspending in 500 L of cell separation buffer, CD137 positive cells, that is, HLA-A24-constrained cells, using an automatic cell separation device (AutoMACS: manufactured by Milteni) Sex HCMV specific CTL were isolated.
  • AutoMACS automatic cell separation device
  • HCMV-specific HLA-A24-tetramer manufactured by Beckman Coulter, Inc.
  • cell separation buffer containing 0.5% hydrogenlevamine and 2 mM EDTA, and then anti-PE antibody-conjugated micromagnetic beads ( (Mirtu Co., Ltd.) 20 ⁇ L was added, and the mixture was stirred and reacted at 4 ° C for 15 minutes.
  • HCMV-specific HLA-A24-tetramer positive cells After washing twice with cell separation buffer and suspending in 500 ⁇ L cell separation buffer, HCMV-specific HLA-A24-tetramer positive cells using an automatic cell separator (AutoMACS: manufactured by Milteni) That is, HLA-A24-restricted HCMV-specific CTLs were isolated.
  • AutoMACS automatic cell separator
  • Figure 3 shows the results of isolation using an automatic cell separator.
  • the recovery rate of HLA-A24-restricted HCMV-specific CTL was 74.1% and the purity was 80.82%.
  • the recovery rate was 34.0% and the purity was 80.15%.
  • the method using the CD137 monoclonal antibody was superior to the method using the MHC-tetramer in terms of recovery.
  • a portion of PBMC (2X10 6 ) isolated during HCMV-specific CTL induction was suspended in 10 mL of CTL induction medium containing OKT3 at a concentration of 1 ⁇ g / mL and 2 in a 5% CO incubator.
  • the cells were cultured at 37 ° C for 12 days in a batch to obtain OKT3-T cells.
  • Example 3 Six HLA-A24-restricted HCMV-specific CTL 4xl0 isolated by the method described in 1 were suspended in CTL induction medium containing 4 mL of 50IU / mL IL-2. After adding 6 x 2xl0 prepared HCM V antigen-presenting cells, 24 hours in a 5% CO incubator at 37 ° C
  • HLA-A24-restricted HCMV-specific CT L was measured with a flow cytometer in the same manner as in Example 2.
  • the number of HLA-A24-restricted HCMV-specific CTLs was determined by multiplying the total number of cells by the ratio measured using a flow cytometer.
  • FIG. 4 shows flow cytometry images before and after growth.
  • the proportion of HCMV-specific CTL was almost unchanged at 98.5% before growth and 97.68% after growth.
  • the number of HLA-A24-restricted HCMV-specific CTLs grew almost 10-fold from 6 from 4xl0 to 7 from 4.lxlO.
  • HCMV-specific CTL could be prepared easily and inexpensively.
  • induction step since preparation of antigen-presenting cells is unnecessary, HCMV-specific CTL can be induced quickly and easily.
  • HCMV-specific CTL proliferation process it was possible to proliferate HCMV-specific CTL easily and inexpensively by substituting T cells that had been propagated with OKT3 as antigen-presenting cells.
  • HCMV-specific CTL isolation process by establishing an isolation method targeting the CD137 antigen, HCMV-specific CTL can be isolated safely without reducing the survival rate.
  • Non-Patent Document 1 Non-Patent Document 3, BLO OD 2002; 99: 3916.
  • HCMV-specific CTL specifically recognizes HCMV-infected cells and has an extremely strong cytotoxic activity, so by using this, a safe and effective HCMV-preventing drug by HCMV-specific CTL, It is possible to provide a therapeutic agent and a technique for preparing it easily and inexpensively.
  • FIG. 1 is a diagram showing the results of analysis using a cytoflow meter to show examples of induction of HCMV-specific CTL.
  • Example 1 after inducing HL A-A24-restricted HCMV-specific CTLs from peripheral blood mononuclear cells derived from HLA-A24-positive healthy individuals, the positive rate of HLA-A24-restricted HCMV-specific CTLs according to Example 2 was measured. A positive rate of 10.28% was calculated.
  • FIG. 2 is a diagram showing the results of analysis by a cytoflow meter for showing an example of HCMV-specific CTL isolation using a CD137 monoclonal antibody-sensitized culture plate.
  • Example 3 After isolating HL A-A24-restricted HCMV-specific CTL from the HLA-A24-restricted HCMV-specific CTL line derived in Example 1, culture for 7 days did.
  • the HLA-A24-restricted HCMV-specific CTL positive rate which was 10.28% before isolation, increased to 56.03% after isolation, and increased to 97.14% after 7 days of culture.
  • the recovery rate after isolation was 48.7%, and the growth rate after 7 days of growth was 2.9 times.
  • FIG. 3 is a diagram showing the results of analysis using a flow cytometer to show the results of isolation using an automatic cell separator. From the HLA-A24-restricted HCMV-specific CTL line induced by the method of Example 1, HLA-A24-restricted HCMV-specific CTL was isolated according to the method shown in [Example 3] 2. Regarding purity, there was no difference between the method using CD137 and the method using MHC-tetramer, but the method using CD137 was superior to the method using MHC-tetramer in terms of recovery. ! /
  • FIG. 4 is a diagram showing the results of analysis using a flow cytometer to show the results of proliferation of HCMV-specific CTLs.
  • HLA-A24-restricted HCMV-specific CTL isolated by the method of 1. was grown according to [Example 4].
  • T e tram er / CD8 positive rate us after growth for 8 days It was almost the same as before growth. Number of HCMV-specific CTL cells proliferated almost 10 times

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Abstract

It is intended to provide a closed culture system of virus-specific CTL which has convenience and safety features and can be implemented at low cost for the purpose of achieving the practical application of cell therapy with the use of CTL specific to a virus (particularly HCMV). After virus-specific CTL epitope peptide and peripheral blood mononuclear cells are mixed and incubated, restimulation with this epitope peptide is given in a medium. Then, virus-specific CTL is isolated by targeting freshly expressed CD137 antigen, and T cells proliferated from peripheral blood mononuclear cells by the stimulation with OKT3 (anti-CD3 monoclonal antibody) are brought into contact with the epitope peptide in the medium. Then, the T cells and the virus-specific CTL are cocultured, whereby the virus-specific CTL is proliferated.

Description

明 細 書  Specification
ウィルス特異的 CTLの製造方法  Method for producing virus-specific CTL
技術分野  Technical field
[0001] 本発明は、ウィルス、特にヒトサイトメガロウィルス（human cytomegalovirus;以下「H CMV」という）に対して特異的な細胞傷害性 T細胞（cytotoxic T lymphocyte;以下「C TLjと!/、う）を調製する方法、およびこの方法により調製された HCMV特異的 CTLを用 いた HCMV感染症の治療方法及び/又は予防方法に関する。  [0001] The present invention relates to cytotoxic T lymphocytes specific to viruses, particularly human cytomegalovirus (hereinafter referred to as “H CMV”). And a method for treating and / or preventing HCMV infection using HCMV-specific CTL prepared by this method.
背景技術  Background art
[0002] HCMVは、大部分の健常な成人において潜伏感染が認められる。通常の免疫能を 有する者については、 HCMVの感染症状が出ることは希である。しかし、免疫抑制状 態にある者 (例えば、がん患者、造血幹細胞移植といった移植手術を受けた患者、ェ ィズ患者など）においては、 HCMVの感染症状が現れ、致死的な間質性肺炎のほか 、網膜炎、肝炎などを引き起すことがある。  [0002] HCMV has a latent infection in most healthy adults. For those with normal immunity, HCMV infection rarely appears. However, those who are immunosuppressed (for example, cancer patients, patients undergoing transplantation surgery such as hematopoietic stem cell transplantation, AIDS patients, etc.) develop HCMV infection symptoms and lethal interstitial pneumonia. Besides, it may cause retinitis, hepatitis, etc.
造血幹細胞移植は、白血病などの造血器腫瘍への適用にとどまらず、一部の固形 腫瘍や代謝疾患にもその適用が広がっている。移植される幹細胞の起源は、 HLA適 合ドナーのみならず、骨髄バンク'臍帯血バンクや HLA不適合ドナーといった代替ド ナ一からの幹細胞を使用した症例数も増加している。これら代替ドナーからの移植の ときには、移植患者に対して強力な免疫抑制治療を施すので、 日和見感染症のリス クが高くなる。特に、 HCMVの再活性化は移植患者のほぼ全例にみられるので、 HC MV感染症の制御は移植手術後の大きな問題点のひとつである。ガンシクロビルは 優れた抗ウィルス剤であり、 HCMV感染症にも汎用されている力 頻回投与による副 作用が度々問題となる。このため、 HCMVを安全にかつ有効に制御する新しい方法 が強く望まれている。  Hematopoietic stem cell transplantation is not only applied to hematopoietic tumors such as leukemia, but is also being applied to some solid tumors and metabolic diseases. The origin of stem cells to be transplanted is increasing not only in HLA compatible donors but also in the number of cases using stem cells from alternative donors such as bone marrow bank 'umbilical cord blood bank and HLA incompatible donors. When transplanting from these alternative donors, the risk of opportunistic infections increases because of the powerful immunosuppressive treatment given to the transplanted patient. In particular, since HCMV reactivation is seen in almost all transplant patients, control of HC MV infection is one of the major problems after transplant surgery. Ganciclovir is an excellent antiviral agent, and side effects caused by frequent doses, which are also widely used for HCMV infections, are frequently problematic. Therefore, a new method for controlling HCMV safely and effectively is strongly desired.
[0003] HCMV感染細胞の活動を制御している主な免疫担当細胞は、 HCMV感染細胞を 特異的に認識する CD8+CTL、すなわち HCMV特異的 CTLである。 HCMV特異的 CT Lは、 HCMV感染細胞を発見すると、それを破壊する能力を持っているので、その機 能を有効に活性化すれば、 HCMV関連疾患の新しい予防法と治療法の開発につな がる可能性が高い。従って、抗ウィルス剤に代わる新たな治療法として、 HCMV特異 的 CTLによる細胞療法が注目されている。欧米においては、この細胞療法は既に応 用研究が行われており、臨床試験において期待された効果が得られている（非特許 文献 1)。 [0003] The main immunocompetent cells that control the activity of HCMV-infected cells are CD8 + CTLs that specifically recognize HCMV-infected cells, that is, HCMV-specific CTLs. HCMV-specific CT L has the ability to destroy HCMV-infected cells when they are found, so if the function is activated effectively, it will lead to the development of new prevention and treatment methods for HCMV-related diseases. Na There is a high possibility of losing. Therefore, cell therapy using HCMV-specific CTLs has attracted attention as a new treatment method that replaces antiviral agents. In Europe and the United States, application research has already been conducted on this cell therapy, and the effect expected in clinical trials has been obtained (Non-patent Document 1).
[0004] 非特許文献 1： ELIZABETH A. WALTER et al. , N Eng J Med, 1995; 333: 1038-1044 非特許文献 2 : N Watanabe et al. , Cytotherapy, 2004; 6(5): 514-522  [0004] Non-patent document 1: ELIZABETH A. WALTER et al., N Eng J Med, 1995; 333: 1038-1044 Non-patent document 2: N Watanabe et al., Cytotherapy, 2004; 6 (5): 514- 522
非特許文献 3 : Mark Cobbold et al. , J Exp Med. , 2005; 202(3): 379-386  Non-Patent Document 3: Mark Cobbold et al., J Exp Med., 2005; 202 (3): 379-386
非特許文献 4 : Marek Cebecauer et al. , J Immunol. , 2005; 174(11): 6809-6819 非特許文献 5 : Foster AE， Gottlieb DJ， Marangolo M， Bartlett A, Li YC， Barton GW ,Romagnoli JA, Bradstock KF. Rapid, large-scale generation of highly pure cytomeg alovirus- specific cytotoxic T cells for adoptive immunotherapy. , J Hematother Stem Cell Res. 2003 Feb; 12(l):93- 105·  Non-Patent Document 4: Marek Cebecauer et al., J Immunol., 2005; 174 (11): 6809-6819 Non-Patent Document 5: Foster AE, Gottlieb DJ, Marangolo M, Bartlett A, Li YC, Barton GW, Romanogli JA , Bradstock KF.Rapid, large-scale generation of highly pure cytomeg alovirus-specific cytotoxic T cells for adoptive immunotherapy., J Hematother Stem Cell Res. 2003 Feb; 12 (l): 93- 105
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0005] しかし、これまで報告されて!/、る HCMV特異的 CTLの調製方法は、以下に掲げた問 題があるため、事業として応用されるまでには至って!/、な!/、。 [0005] However, the methods for preparing HCMV-specific CTL that have been reported so far have the following problems.
( 1 ) HCMV特異的 CTLを誘導させるために、抗原提示細胞の調製を必要とする。抗 原提示細胞としては、樹状細胞が使われることが多いが、樹状細胞の調製は煩雑で 費用が嵩む。  (1) In order to induce HCMV-specific CTL, it is necessary to prepare antigen-presenting cells. Dendritic cells are often used as antigen-presenting cells, but the preparation of dendritic cells is complicated and expensive.
(2)誘導した HCMV特異的 CTLをさらに増殖させる場合に、樹状細胞が使われる（ 非特許文献 5)。このため、上記（1 )と同様の問題が生じる。また、増殖において使用 する樹状細胞を調製するため、再度ドナーから採血する必要がある。更に、増殖時 には誘導時に比べて、多数の樹状細胞が必要となることからドナーにかかる負担が 大きい。  (2) Dendritic cells are used to further propagate induced HCMV-specific CTL (Non-patent Document 5). For this reason, the same problem as the above (1) occurs. In addition, in order to prepare dendritic cells for use in proliferation, it is necessary to collect blood from the donor again. In addition, the burden on the donor is larger because a larger number of dendritic cells are required for proliferation than for induction.
[0006] (3)誘導した HCMV特異的 CTLをさらに増殖させる別の手段として、 OKT3ゃレクチ ンによる非特異的な刺激を用いることがある。しかし、この方法では、相対的に HCMV 特異的 CTL以外の T細胞が増殖しやすいため効率性に欠ける。  [0006] (3) As another means of further proliferating the induced HCMV-specific CTL, non-specific stimulation with OKT3 is sometimes used. However, this method is relatively inefficient because T cells other than HCMV-specific CTL are relatively proliferative.
また、非常に効率の良い増殖法として REM法が知られている。 REM法は大量の末 梢血単核球と、 EBV-LCLを必要とすることから、事業化において現実的な方法では ない。 In addition, the REM method is known as a very efficient growth method. REM method is a large amount Since it requires erythrocyte mononuclear cells and EBV-LCL, it is not a realistic method for commercialization.
(4)誘導、増殖した HCMV特異的 CTLの純度を高めるための方法として、 MHC-tet ramerを使った HCMV特異的 CTL単離法が一般化して!/、る（非特許文献 3)。 MHC_t etramerは、大腸菌由来の蛋白質を用いて作られるので、安全面での問題があること に加え、 CTLの生存率が低下するという問題点がある（非特許文献 4)。  (4) The HCMV-specific CTL isolation method using MHC-tet ramer is generalized as a method for enhancing the purity of induced and proliferated HCMV-specific CTL (Non-patent Document 3). Since MHC_t etramer is produced using a protein derived from Escherichia coli, there is a problem in that the survival rate of CTL is reduced in addition to the safety problem (Non-patent Document 4).
また、純度を高めるための究極の手段として、 HCMV特異的 CTLのクローニングを 行うことも報告されている。しかし、現在のところ、作業の効率、費用面から、この方法 を事業化することは現実的ではない。  It has also been reported that HCMV-specific CTL cloning is performed as the ultimate means to increase purity. However, at present, it is not practical to commercialize this method in terms of work efficiency and cost.
[0007] (5)これまで行われて!/、る HCMV特異的 CTL培養法は、抗原提示細胞を調製する など、複雑なステップを経るので開放系培養に頼らざるを得ない。開放系での培養は 、外部からの細菌、ウィルスなどの混入を防ぐため、細胞加工施設内で行う必要があ り、莫大な設備費、管理費を要する。 [0007] (5) The HCMV-specific CTL culture method that has been carried out so far involves complicated steps such as preparing antigen-presenting cells, and thus must rely on open culture. Cultivation in an open system needs to be performed in a cell processing facility in order to prevent external contamination of bacteria, viruses, etc., and enormous equipment costs and management costs are required.
(6)日本人に多い HLA-A24に拘束性の HCMV特異的 CTLについては、臨床応用 に向けた体外での調製方法に関する報告はなされていない。 HLA-A24拘束性 HCM V特異的 CTLが末梢血中に含まれる数は、欧米人に多い HLA-A2拘束性 HCMV特 異的 CTLのそれに比べると百分の一程度である。これは、 HLA-A24拘束性 HCMV特 異的 CTLの体外での誘導、増殖が困難であることの理由のひとつと考えられる。従つ て、 日本においては、 HLA-A24拘束性 HCMV特異的 CTLを体外で調製する方法を 工夫する必要がある。  (6) There are no reports on in vitro preparation methods for HCMV-specific CTL restricted to HLA-A24, which are common in Japanese, for clinical application. The number of HLA-A24-restricted HCM V-specific CTLs in the peripheral blood is about one-hundred compared to that of HLA-A2-restricted HCMV-specific CTLs, which are common in Westerners. This is thought to be one of the reasons that induction and proliferation of HLA-A24-restricted HCMV-specific CTLs in vitro are difficult. Therefore, in Japan, it is necessary to devise a method for preparing HLA-A24-restricted HCMV-specific CTL in vitro.
本発明は上記問題点に鑑みてなされたものであり、その目的は、ウィルス（特に、 H CMV)特異的 CTLによる細胞治療の実用化を実現するため、簡便性と安全性を兼ね 備え、し力、も低コストで実施可能なウィルス特異的 CTLの閉鎖系培養システムの提供 することである。  The present invention has been made in view of the above problems, and its purpose is to combine practicality and safety in order to realize the practical use of cell therapy using virus (particularly HCMV) -specific CTL. It is also necessary to provide a closed culture system for virus-specific CTL that can be implemented at low cost.
課題を解決するための手段  Means for solving the problem
[0008] 一般に CTLの調製工程 (製造工程）は、 CTL誘導工程、 CTL単離工程、及び CTL 増殖工程の 3工程から構成される。本発明者らは、これら 3工程において、適切な条 件を求めることにより、簡便かつ安価に HCMV特異的 CTLを調製する方法を発明し た。 [0008] In general, the CTL preparation process (manufacturing process) is composed of three processes: a CTL induction process, a CTL isolation process, and a CTL growth process. The inventors have invented a method for preparing HCMV-specific CTL in a simple and inexpensive manner by obtaining appropriate conditions in these three steps. It was.
すなわち、本発明に係る HCMV特異的 CTLの調製方法は、以下の通りである。 下記（1)〜（3)は、 CTLの誘導工程に関するものである。  That is, the method for preparing HCMV-specific CTL according to the present invention is as follows. The following (1) to (3) relate to the CTL induction process.
(1) HTLV_1、インフルエンザ、アデノウイルス、エイズウイルス、 B型肝炎ウィルス、 C型肝炎ウィルス、ェプスタインバーウィルス、ヒトパピローマウィルス、及びヒトサイト メガロウィルス（HCMV)からなる群から選択される一つのウィルス（特に、 HCMV)特 異的 CTLを誘導する方法であって、末梢血単核球とウィルス特異的 CTLェピトープ ペプチドを混合培養することを特徴とする誘導方法。  (1) One virus selected from the group consisting of HTLV_1, influenza, adenovirus, AIDS virus, hepatitis B virus, hepatitis C virus, Epstein Barr virus, human papilloma virus, and human cytomegalovirus (HCMV) ( In particular, a method for inducing HCMV) specific CTL, which comprises culturing a mixture of peripheral blood mononuclear cells and virus-specific CTL epitope peptide.
(2)前記混合培養において、ェピトープペプチドの培地中での終濃度が 0.01 g/m L-lO g/mLであることを特徴とする上記（1)に記載の誘導方法。このとき、ウィルス 力 ¾CMVである場合には、 HLA-A2拘束性ェピトープである NLVPMVATV (配列番 号 1)では 0.05 a g/mL-2 μ g/mL、 HLA-A24拘束性ェピトープである QYDPVAALF ( 配列番号 2)では 0.2 H g/mL-5 μ g/mLであることが好まし!/ヽ。  (2) The induction method according to (1) above, wherein in the mixed culture, the final concentration of the epitope peptide in the medium is 0.01 g / m L-lO g / mL. At this time, when the virus power is ¾ CMV, NLAPMVATV (SEQ ID NO: 1), which is an HLA-A2 restricted epitope, 0.05 ag / mL-2 μg / mL, and QYDPVAALF, an HLA-A24 restricted epitope (SEQ ID NO: 1) Number 2) is preferably 0.2 H g / mL-5 μg / mL! / ヽ.
(3)前記混合培養に用いる培地として、 5%-10%自己血漿を含む RPMI1640を用い 、ェピトープペプチドを添加後、 2日後に終濃度が 10 IU/mLとなるように IL-2を加え、 その後 IL-2濃度を 2-3日ごとに 2倍づっ上げながら 12日間- 19日間培養することを特 徴とする上記（1)または（2)に記載の誘導方法。  (3) Use RPMI1640 containing 5% -10% autologous plasma as the medium for the mixed culture, and add IL-2 so that the final concentration is 10 IU / mL 2 days after the addition of the epitope peptide. In addition, the induction method according to (1) or (2) above, wherein the culture is then performed for 12 to 19 days while increasing the IL-2 concentration by 2 times every 2-3 days.
下記 (4)〜（； 10)は、 CTLの単離工程に関するものである。  The following (4) to (; 10) relate to the isolation process of CTL.
(4)ウィルス特異的 CTLを単離する方法であって、上記（1)〜（3)のいずれかに記 載の誘導方法、またはその他の方法で誘導されたウィルス特異的 CTLを、誘導に用 いたェピトープペプチドと同じェピトープペプチドと培地中で接触させることにより再 刺激後、新たに発現する CD137抗原を標的として、ウィルス特異的 CTLを単離するこ とを特徴とする単離方法。  (4) A method for isolating virus-specific CTL, wherein the virus-specific CTL induced by any of the induction methods described in (1) to (3) above or other methods is used for induction. Isolation characterized by isolating virus-specific CTL targeting the newly expressed CD137 antigen after restimulation by contact with the same epitope peptide as used in the medium. Method.
(5)ウィルス特異的 CTLを単離する方法であって、上記（1)〜（3)のいずれかに記 載の誘導方法、またはその他の方法で誘導されたウィルス特異的 CTLを、誘導に用 いたェピトープペプチドと同じェピトープペプチドを提示した抗原提示細胞と培地中 で接触させることにより再刺激後、新たに発現する CD137抗原を標的として、ウィルス 特異的 CTLを単離することを特徴とするウィルス特異的 CTLの単離方法。 (6)ウィルス特異的 CTLを単離する方法であって、上記（1)〜（3)のいずれかに記 載の誘導方法、またはその他の方法で誘導されたウィルス特異的 CTLを、誘導に用 いたェピトープペプチドと同じェピトープペプチドと複合体を形成する MHC分子と培 地中で接触させることにより再刺激後、新たに発現する CD137抗原を標的として、ゥ ィルス特異的 CTLを単離することを特徴とするウィルス特異的 CTLの単離方法。 (5) A method for isolating virus-specific CTL, wherein the virus-specific CTL induced by any of the induction methods described in (1) to (3) above or other methods is used for induction. It is necessary to isolate virus-specific CTL targeting the newly expressed CD137 antigen after restimulation by contacting in the medium with antigen-presenting cells presenting the same epitope peptide as that used. A method for isolating a virus-specific CTL characterized. (6) A method for isolating virus-specific CTL, wherein the virus-specific CTL induced by any of the induction methods described in (1) to (3) above or other methods is used for induction. After re-stimulation by contact with the MHC molecule that forms a complex with the same epitope peptide as the one used in the medium, virus-specific CTL is simply targeted against the newly expressed CD137 antigen. A method for isolating virus-specific CTL, which comprises separating the virus-specific CTL.
(7)前記再刺激に用いるェピトープペプチドの培地中での終濃度が 0. lng/mL-100 Ong/mLであることを特徴とする上記（4)〜（6)に記載の単離方法。このとき、ウィルス 力 ¾CMVである場合には、 Ing/mL-lOng/mLであることが好まし!/、。  (7) The isolation according to (4) to (6) above, wherein the final concentration of the epitope peptide used for restimulation in the medium is 0.1 ng / mL-100 Ong / mL Method. At this time, when the virus power is ¾ CMV, it is preferably Ing / mL-lOng / mL! /.
[0010] (8)前記再刺激は、 5%-10%自己血漿と 10 IU/mL〜50 IU/mLの IL-2または 10ng/ mL IL-15のいずれ力、とを含む RPMI1640培地中で 8時間〜 15時間に渡って行うことを 特徴とする上記 (4)〜（7)に記載の単離方法。  [0010] (8) The restimulation is performed in RPMI1640 medium containing 5% -10% autologous plasma and either 10 IU / mL to 50 IU / mL IL-2 or 10 ng / mL IL-15. The isolation method according to any one of (4) to (7) above, which is carried out for 8 to 15 hours.
(9) CD137抗原を標的とする際に、磁気標識した抗 CD137抗体を用いることを特徴 とする上記 (4)〜（8)の!/、ずれかに記載の単離方法。  (9) The method according to any one of (4) to (8) above, wherein a magnetically labeled anti-CD137 antibody is used when targeting the CD137 antigen.
(10) CD137抗原を標的とする際に、抗 CD137抗体を感作した固相を用いることを 特徴とする上記 (4)〜（8)の!/、ずれかに記載の単離方法。  (10) The isolation method according to any one of (4) to (8) above, wherein a solid phase sensitized with an anti-CD137 antibody is used when targeting the CD137 antigen.
また、上記（1)〜（3)の誘導方法、および (4)〜（； 10)の単離方法については、ウイ ルス特異的 CTLとは、ウィルス特異的 CD8+CTLに加えて、ウィルス特異的 CD4+T細 胞も含まれる。  In addition, for the induction methods (1) to (3) and the isolation methods (4) to (; 10) above, virus-specific CTL refers to virus-specific CD8 + CTL and virus-specific CD4 + T cells are also included.
下記（11)〜（； 13)は、 CTLの増殖工程に関するものである。  The following (11) to (; 13) relate to the proliferation process of CTL.
(11)ウィルス特異的 CTLの増殖方法であって、 OKT3 (抗 CD3モノクローナル抗体） を用いて末梢血単核球から増殖させた Τ細胞と、上記（1)〜（3)の!/、ずれかにお!/、て 誘導の際用いたェピトープペプチドと同じェピトープペプチドとを培地中で接触させ た後に、この Τ細胞と、上記 (4)〜（； 10)のいずれかに記載の単離方法で単離したゥ ィルス特異的 CTLまたは（1)〜（3)の!/、ずれかに記載の誘導方法で誘導したウイノレ ス特異的 CTLとを共培養することを特徴とするウィルス特異的 CTLの増殖方法。  (11) A method for propagating virus-specific CTL, wherein the sputum cells are proliferated from peripheral blood mononuclear cells using OKT3 (anti-CD3 monoclonal antibody), and the above (1) to (3)! / After contacting the same epitope peptide as that used for induction in the medium, this sputum cell and any of the above (4) to (; 10) It is characterized by co-culturing the virus-specific CTL isolated by the described isolation method or (1) to (3)! /, Or the winores-specific CTL induced by any of the induction methods described above. To propagate virus-specific CTL.
[0011] (12)上記（11)において ΟΚΤ3を用いて末梢血単核球から増殖させた Τ細胞を、ゥ ィルス特異的 CTLェピトープペプチドと培地中で接触させる際に、下記条件を用いる ことを特徴とする増殖方法。 (a)培地中に加えるェピトープペプチドの終濃度； 0.0001 H 8/01し〜10 g/mL[0011] (12) The following conditions are used when the sputum cells grown from peripheral blood mononuclear cells using sputum 3 in (11) above are contacted with a virus-specific CTL epitope peptide in the medium. Proliferation method characterized by the above. (a) the final concentration of E pitot flop peptide added to the culture medium; 0.0001 H 8/01 teeth to 10 g / mL
(b)接触時間； 30分間〜 60分間 (b) Contact time; 30 minutes to 60 minutes
(c)培養温度； 18°C〜25°C  (c) Culture temperature: 18 ° C-25 ° C
(d)培地；血清、血漿など蛋白成分を含まな!/、RPMI1640。  (d) Medium; free of protein components such as serum and plasma! /, RPMI1640.
(13)上記（11)において、以下の共培養条件を特徴とする増殖方法。  (13) In the above (11), the proliferation method characterized by the following co-culture conditions:
(e)ウィルス特異的 CTL： OKT3を用いて増殖させた T細胞の混合比； 10: 1〜1:3 (e) Virus-specific CTL: mixed ratio of T cells grown with OKT3; 10: 1 to 1: 3
(f)培地組成 共培養開始後 1日目- 3日目まで； RPMI1640 (5%〜10%自己血漿、 1 0 IU/mL〜50 IU/mL IL-2)または RPMI1640(5%〜10%自己血漿、 10ng/mL IL-15)(f) Medium composition From day 1 to day 3 after initiation of co-culture; RPMI1640 (5% -10% autologous plasma, 10 IU / mL-50 IU / mL IL-2) or RPMI1640 (5% -10% (Autologous plasma, 10ng / mL IL-15)
、共培養開始後 1日目 3日目以降； ALyS505N (100 IU/mL〜1000 IU/mL IL- 2、 0.1 %〜1%自己血漿）または ALyS505N (10ng/mL IL-15, 0· 1%〜1%自己血漿）。 , 1st day after coculture start 3rd day and later; ALyS505N (100 IU / mL to 1000 IU / mL IL-2, 0.1% to 1% autologous plasma) or ALyS505N (10 ng / mL IL-15, 0 · 1% ~ 1% autologous plasma).
[0012] (14)上記（1)〜（3)の!/、ずれかに記載の誘導方法によりウィルス特異的 CTLを誘 導する工程を含むウィルス特異的 CTLの製造方法。 [0012] (14) A method for producing a virus-specific CTL comprising a step of inducing virus-specific CTL by the induction method according to any one of (1) to (3) above.
(15)上記（4)〜（； 10)の!/、ずれかに記載の単離方法によりウィルス特異的 CTLを 単離する工程を含むウィルス特異的 CTLの製造方法。  (15) A method for producing virus-specific CTL, comprising the step of isolating virus-specific CTL by the isolation method according to any one of (4) to (; 10) above.
(16)上記（11)〜（； 13)の!/、ずれかに記載の増殖方法によりウィルス特異的 CTLを 増殖する工程を含むウィルス特異的 CTLの製造方法。  (16) A method for producing virus-specific CTL, comprising the step of proliferating virus-specific CTL by the proliferation method described in any one of (11) to (; 13) above.
(17)上記（1)〜（3)の!/、ずれかに記載のウィルス特異的 CTLの誘導方法、上記 (4 )〜（10)の!/、ずれかに記載のウィルス特異的 CTLの単離方法、上記（11)〜（； 14)の いずれかに記載のウィルス特異的 CTLの増殖方法をそれぞれ組み合わせることを特 徴とするウィルス特異的 CTLの製造方法。  (17) The method for inducing a virus-specific CTL according to (1) to (3) above, or any of the above, or the method of inducing the virus-specific CTL according to any of the above (4) to (10)! An isolation method and a method for producing a virus-specific CTL characterized by combining the virus-specific CTL growth method according to any one of (11) to (; 14) above.
(18)上記（14)〜（； 17)の!/、ずれかに記載のウィルス特異的 CTLの製造方法によつ て製造されたウィルス特異的 CTL。  (18) A virus-specific CTL produced by the method for producing a virus-specific CTL of any one of (14) to (; 17) above.
[0013] (19)上記（14)〜（； 17)の!/、ずれかに記載のウィルス特異的 CTLの製造方法によつ て製造されたウィルス特異的 CTLを含むウィルス感染症の治療剤及び/または予防 剤。  [0013] (19) A therapeutic agent for a viral infection containing a virus-specific CTL produced by the method for producing a virus-specific CTL of any one of (14) to (; 17) above And / or preventive agent.
(20)上記（19)に記載のウィルス感染症の治療剤及び/または予防剤を用いるこ とを特徴とするウィルス感染症の治療方法及び/または予防方法。  (20) A method for treating and / or preventing a viral infection, characterized by using the therapeutic and / or preventive agent for a viral infection described in (19) above.
(21) CTLの免疫応答能の検査方法であって、培地中で、（a)CTLェピトープぺプチ ド、この CTLェピトープペプチドを提示した抗原提示細胞、または前記 CTLェピトー プペプチドと複合体を形成する MHC分子からなる群から選択される少なくとも一つの 成分と、（b)被験者から採取したリンパ球または培養中の CTLとを接触させながら共培 養した後、前記リンパ球または CTL上に新たに発現する CD137分子を検出することに より免疫応答能を判断することを特徴とする検査方法。 (21) A method for examining the immune response ability of CTL, comprising: (a) a CTL epitope peptide in a medium; (B) at least one component selected from the group consisting of antigen-presenting cells presenting this CTL epitope peptide, or MHC molecules forming a complex with said CTL epitope peptide, and (b) lymphocytes collected from a subject Alternatively, a test method characterized by determining the immune response ability by detecting a CD137 molecule newly expressed on the lymphocyte or CTL after co-culturing with the CTL in culture in contact.
なお、上記については、ウィルスのェピトープペプチドに対して応用できる製造方 法等を説明したが、本発明に関する技術は、その他のペプチド (例えば、腫瘍抗原） についても応用できる。  In the above description, production methods applicable to viral epitope peptides have been described. However, the technology relating to the present invention can also be applied to other peptides (for example, tumor antigens).
発明の効果  The invention's effect
[0014] CTL誘導工程、 CTL単離工程、及び CTL増殖工程の各工程からなる CTLの調製ェ 程 (製造工程）は、いずれも煩雑で高コストであるため、 CTLを用いた細胞治療の実 用化において障害となってきた。本発明のウィルス（特に、 HCMV)特異的 CTLの調 製方法は、簡便で安価な HCMV特異的 CTLの調製を可能とした。例えば、 CTLの誘 導工程は従来、樹状細胞などの抗原提示細胞とリンパ球を共培養することにより行わ れてきた。樹状細胞などの抗原提示細胞の調製は煩雑で、その調製には多大な労 力と経費が必要であった。これに対し、本発明による誘導工程は、抗原提示細胞の 調製が不要であるため、迅速かつ簡便にウィルス特異的 CTLの誘導を行える。  [0014] The CTL preparation process (manufacturing process), which includes the CTL induction process, the CTL isolation process, and the CTL proliferation process, is complicated and expensive. Has become an obstacle in commercialization. The method for preparing a virus (particularly, HCMV) -specific CTL of the present invention made it possible to prepare a simple and inexpensive HCMV-specific CTL. For example, the CTL induction process has been conventionally performed by co-culturing antigen-presenting cells such as dendritic cells and lymphocytes. The preparation of antigen-presenting cells such as dendritic cells is complicated and requires a great deal of labor and expense. In contrast, the induction step according to the present invention does not require preparation of antigen-presenting cells, and thus can induce virus-specific CTLs quickly and easily.
[0015] また、 CTL増殖工程においても、従来の方法では樹状細胞を必要とするため、前記 と同様の問題点があった。本発明によるウィルス特異的 CTLの増殖工程は、抗原提 示細胞として OKT3で増殖させた T細胞に代替することによって簡便で安価なウイノレ ス特異的 CTLの増殖を可能とした。さらに、 CTL単離工程においては MHC-tetramer を用いることが一般的となっている力 MHC- tetramerは大腸菌由来の蛋白質である ことから安全面での問題があることに加え、 CTL単離後の生存率の低下が指摘され ている。また、 CD 107抗原やインターフェロンガンマ（IFNg)を標的として、 CTLを単離 する方法が知られている（Leukemia (2005) 19， 707-709)。しかしながら、この方法で は、 CTLを単離したときの収率が悪いという欠点が知られている。本発明においては 、 CD137抗原を標的とした単離方法を樹立することにより、このような問題点を克服し た。すなわち、 CD137抗原を標的とすれば、生存率を低下させることなく安全にウイ ノレス特異的 CTLの単離が可能となる。 [0015] Also in the CTL growth step, the conventional method requires dendritic cells, and thus has the same problems as described above. The proliferation process of virus-specific CTLs according to the present invention allows simple and inexpensive proliferation of vinores-specific CTLs by substituting T cells grown with OKT3 as antigen-presenting cells. Furthermore, it is common to use MHC-tetramer in the CTL isolation process. MHC-tetramer is a protein derived from Escherichia coli, so there are safety issues. Decreased survival rates have been pointed out. In addition, a method for isolating CTL using CD 107 antigen or interferon gamma (IFNg) as a target is known (Leukemia (2005) 19, 707-709). However, this method has a known disadvantage that the yield when CTL is isolated is poor. In the present invention, such a problem has been overcome by establishing an isolation method targeting the CD137 antigen. In other words, targeting the CD137 antigen can safely lead to a decrease in survival rate. Allows isolation of Nores-specific CTL.
[0016] HCMVは免疫抑制状態にある患者、例えば造血幹細胞移植後の患者にお!/、ては 、ほぼ 100%の確率で再活性化し、致死的な感染症を引き起こす。従って、その制御 は患者の予後を左右する重要な問題である。従来 HCMVの制御にはガンシクロビル 等の抗ウィルス薬が使用されている力 頻回投与による副作用の問題があり、より安 全で効果的な、予防薬、治療薬の開発が望まれていた。 HCMV特異的 CTLは、 HCM V感染細胞を特異的に認識し、かつ極めて強力な細胞傷害活性を有して!/、る。 本発明によれば、安全で効果的なウィルス特異的 CTLによるウィルス予防薬、治療 薬、及び、これを簡便で安価に調製する技術を提供することができる。 [0016] HCMV reacts with an almost 100% probability in patients who are immunosuppressed, such as patients after hematopoietic stem cell transplantation, and cause fatal infections. Therefore, control is an important issue that affects patient prognosis. Conventionally, antiviral drugs such as ganciclovir have been used to control HCMV. There is a problem of side effects due to frequent administration, and the development of safer and more effective preventive and therapeutic drugs has been desired. HCMV-specific CTL specifically recognizes HCM V-infected cells and has extremely strong cytotoxic activity! According to the present invention, it is possible to provide a safe and effective virus-preventing drug, therapeutic drug by virus-specific CTL, and a technique for preparing this easily and inexpensively.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0017] 次に、本発明の実施形態について、図面を参照しつつ詳細に説明するが、本発明 の技術的範囲は、下記の実施形態によって限定されるものではなぐその要旨を変 更することなぐ様々に改変して実施することができる。また、本発明の技術的範囲は 、均等の範囲にまで及ぶものである。  Next, embodiments of the present invention will be described in detail with reference to the drawings. However, the technical scope of the present invention is not limited by the following embodiments, but changes the gist thereof. Various modifications can be made. Further, the technical scope of the present invention extends to an equivalent range.
以下、本発明の実施形態については、ウィルスの代表として HCMVを例にとって説 明する力 その他のウィルス（HTLV_1、インフルエンザ、アデノウイルス、エイズウィル ス、 B型肝炎ウィルス、 C型肝炎ウィルス、ェプスタインバーウィルス、ヒトパピローマウ ィルス）についても同様に実施することができる。 CTLの製造について、（l) HCMV特 異的 CTLの誘導工程、（2) HCMV特異的 CTLの単離工程、および（3) HCMV特異的 CTLの増殖工程の三つに分けて説明する。  In the following, embodiments of the present invention will be described by taking HCMV as an example of a virus. Other viruses (HTLV_1, influenza, adenovirus, AIDS virus, hepatitis B virus, hepatitis C virus, Epstein Barr) The same can be done for viruses and human papillomaviruses. The production of CTL will be described in three parts: (1) HCMV-specific CTL induction step, (2) HCMV-specific CTL isolation step, and (3) HCMV-specific CTL growth step.
[0018] まず HCMV特異的 CTLの誘導工程につ!/、て説明する。  [0018] First, the process of inducing HCMV-specific CTL will be described.
本実施形態における HCMV特異的 CTLの誘導方法 (以下、「本実施形態の誘導方 法」という。）では、末梢血単核球と HCMV特異的 CTLェピトープペプチドを血漿を含 む培地中で接触させながら培養する。「CTLェピトープペプチド」とは、隣接するァミノ 酸残基の α -ァミノ基とカルボキシル基間のペプチド結合により相互に結合した線状 のアミノ酸の分子鎖であって、ヒト白血球抗原 (human leucocyte antigen,以下「HLA」 という。）クラス I分子と複合体を形成し、 HCMV特異的 CTL上に発現する T細胞レセプ ターと結合性を有するペプチドを意味する。 T細胞に対する抗原提示は、抗原提示 細胞上に発現する MHCとペプチドとの複合体（以下、「MHC_ペプチド複合体」と!/、う )が下細胞受容体（TCR)に認識され、かつ抗原提示細胞上の B7-l、 B7-2等の補助 分子が T細胞側の補助分子である CD28等と結合することによってなされる。本実施 形態の誘導方法においては、末梢血単核球に含まれる単球、 B細胞、微量に含まれ る樹状細胞等の膜表面上に発現する HLAクラス I分子と CTLェピトープペプチドが複 合体を形成し、抗原提示細胞として機能するものと考えられる。 In the method for inducing HCMV-specific CTL in this embodiment (hereinafter referred to as “the method for inducing this embodiment”), peripheral blood mononuclear cells and HCMV-specific CTL epitope peptide are contained in a medium containing plasma. Incubate with contact. A CTL epitope peptide is a molecular chain of linear amino acids linked together by peptide bonds between the α-amino and carboxyl groups of adjacent amino acid residues, and is a human leucocyte antigen (human leucocyte). antigen (hereinafter referred to as “HLA”) A peptide that forms a complex with a class I molecule and has a binding property to a T cell receptor expressed on an HCMV-specific CTL. Antigen presentation to T cells is antigen presentation A complex of MHC and peptide expressed on the cell (hereinafter referred to as “MHC_peptide complex”! /) Is recognized by the lower cell receptor (TCR) and B7-l on the antigen-presenting cell, This is done by binding an auxiliary molecule such as B7-2 to CD28, which is an auxiliary molecule on the T cell side. In the induction method of the present embodiment, HLA class I molecules and CTL epitope peptides expressed on the surface of membranes such as monocytes, B cells, and dendritic cells contained in minute amounts are contained in peripheral blood mononuclear cells. It is considered that a complex is formed and functions as an antigen-presenting cell.
[0019] 従って、本実施形態における誘導方法においては、従来必要とされた樹状細胞な どの抗原提示細胞を調製する必要がなぐ誘導工程を大幅に縮小することができる ので、簡便、迅速、かつ安価に HCMV特異的 CTLを誘導することが可能となる。抗原 提示の際に起こる MHC-ペプチド複合体と TCRとの結合は、適度な結合力によって 起こることが、抗原提示能を高めるために重要である (結合力が強すぎても抗原提示 能が高まるとは言えない）。このため、加える CTLェピトープペプチドの濃度は、抗原 提示の効果を左右する。 HLA-A2拘束性の HCMVェピトープペプチド（NLVPMVAT V：配列番号 1)においては、終濃度が O.OS g/mL ^ g/mLであることが望ましい。ま た、 HLA-A24拘束性の HCMVェピトープペプチド（QYDPVAALF:配列番号 2)にお V、ては、終濃度が 0.2 ,1 g/mL-5 ,i g/mLであることが望まし!/ヽ。  [0019] Therefore, in the induction method according to the present embodiment, the induction process that does not require preparation of antigen-presenting cells such as dendritic cells, which has been conventionally required, can be greatly reduced. HCMV-specific CTL can be induced at low cost. The binding between the MHC-peptide complex and TCR that occurs during antigen presentation is important for enhancing the antigen-presenting ability (this increases the antigen-presenting ability even if the binding ability is too strong). It can not be said). Therefore, the concentration of added CTL epitope peptide affects the effect of antigen presentation. In the HLA-A2-restricted HCMV epitope peptide (NLVPMVAT V: SEQ ID NO: 1), the final concentration is preferably O.OS g / mL ^ g / mL. In addition, it is desirable that the final concentration of HLA-A24-restricted HCMV epitope peptide (QYDPVAALF: SEQ ID NO: 2) is 0.2, 1 g / mL-5, ig / mL! / ヽ.
[0020] HLAは、人種間、個人間によつて異なっている。 HLAが異なると、同じ抗原であって もェピトープペプチドが異なる（これをェピトープペプチドの HLA拘束性と!/、う）。 日本 人の約 80%が保有し、かつ世界的に最も頻度の高い HLA-A24型、および HLA-A2 型に拘束性の HCMVェピトープペプチドの誘導工程における至適濃度が明ら力、とな つた。  [0020] HLA differs between races and individuals. When the HLA is different, the epitope peptide is different even for the same antigen (this is the HLA restriction of the epitope peptide! /). The optimal concentration in the induction process of HLA-A24 and HLA-A2 restricted HCMV epitope peptide, which is the most common in the world and is the most common in the world Natsuta.
次に、本実施形態における誘導方法において、培養条件の詳細を説明する。基礎 培地としては、 5%〜10%自己血漿を含む RPMI1640, AIM-Vまたはダルベッコ変法 イーグル培地 (Dulbecco's modified eagle medium)が望ましく、末梢血単核球と該 CTL ェピトープペプチドを混合 2日後に終濃度が 10 IU/mLになるように IL-2を加え、その 後、 2-3日おきに IL-2の濃度を 2倍づっ高めながら前記培地を用いると尚効果的であ る。培養中の炭酸ガス濃度、温度及び培養日数はそれぞれ 5%、 37°C、 14-21日間が 好ましい。また、細胞濃度としては、 106〜2xl06個/ mLが好ましい。さらに細胞同士を 密着させながら培養することが効率的な抗原提示にとって重要であり、そのためには 、底が U字状となった培養容器中で培養することが好ましレ、。 Next, details of the culture conditions in the induction method according to the present embodiment will be described. As the basal medium, RPMI1640, AIM-V containing 5% to 10% autologous plasma or Dulbecco's modified eagle medium (Dulbecco's modified eagle medium) is desirable, and peripheral blood mononuclear cells and the CTL epitope peptide are mixed for 2 days. It is still more effective to add IL-2 later to a final concentration of 10 IU / mL, and then use the medium while doubling the IL-2 concentration every 2-3 days. The concentration of carbon dioxide gas, the temperature, and the number of days of culture are preferably 5%, 37 ° C, and 14-21 days, respectively. Further, the cell concentration is preferably 10 6 to 2xl0 6 cells / mL. Furthermore, cells Incubating in close contact is important for efficient antigen presentation. For this purpose, it is preferable to culture in a U-shaped culture vessel.
[0021] 次に、 HCMV特異的 CTLの単離工程について説明する。本発明における HCMV特 異的 CTLの単離方法（以下、「本実施形態における単離方法」という。）は、本実施形 態における誘導方法により誘導した HCMV特異的 CTLと HCMV特異的 CTLェピトー プペプチドを血漿を含む培地中で接触させながら培養することにより HCMV特異的 C TL上に新たに発現する CD137分子を標的として単離することを特徴とする。 [0021] Next, an isolation process of HCMV-specific CTL will be described. The method for isolating HCMV-specific CTL in the present invention (hereinafter referred to as “isolation method in the present embodiment”) is the HCMV-specific CTL and HCMV-specific CTL epitope peptide induced by the induction method in the present embodiment. The CD137 molecule newly expressed on the HCMV-specific CTL is isolated as a target by culturing in a medium containing plasma.
CD137分子 (4-1BB, ILAともいう)は Kwon BSらにより同定された分子で、 CTLの分 化、増殖に関連し、 TCRを介した刺激により CTL上に選択的に発現することが知られ ている。従って HCMV特異的 CTLを含む T細胞集団にェピトープペプチドを加えるこ とにより、培地中に含まれる T細胞上には、 HLAクラス I分子とェピトープペプチドから 成る複合体が発現する。この複合体と HCMV特異的 CTL上の TCRが接触し、 HCMV 特異的 CTL上に選択的に CD137分子が発現する。この HCMV特異的 CTLは、例え ば CD 137抗体をコートした磁性微粒子のように、 CD 137分子を認識する物質と反応さ せた後、適当な分離装置 (例えば、磁気分離装置）を用いて、 HCMV特異的 CTLを 単離すること力 Sでさる。  CD137 molecule (4-1BB, also referred to as ILA) is a molecule identified by Kwon BS et al. It is related to CTL differentiation and proliferation, and is known to be selectively expressed on CTL by stimulation via TCR. ing. Therefore, by adding an epitope peptide to a T cell population containing HCMV-specific CTL, a complex consisting of an HLA class I molecule and an epitope peptide is expressed on the T cells contained in the medium. This complex comes into contact with the TCR on the HCMV-specific CTL, and the CD137 molecule is selectively expressed on the HCMV-specific CTL. This HCMV-specific CTL is reacted with a substance that recognizes CD 137 molecules, such as magnetic fine particles coated with CD 137 antibody, and then used with a suitable separation device (eg, magnetic separation device). Use force S to isolate HCMV-specific CTL.
[0022] なお、 CD137抗体をコートした磁性微粒子の代わりに、 CD137抗体をコートした培 養プレートを用い、パンユング法により単離することもできる。抗 CD137抗体としては モノクローナル抗体であってもポリクロナル抗体であっても良い。培地中に添加する ェピトープペプチドの終濃度としては 1 ng/mL - 10 ng/mLが効果的である。 HCMV 特異的 CTLは、ェピトープペプチドを培地中に加えた後に最も CD137抗原の発現が 高まる 15時間〜 24時間後に単離することが望ましい。なお、単離工程において、 CD1 37抗体と HCMV特異的 CTLが接触する際、 CD137分子から刺激が細胞内に伝達さ れるので、 HCMV特異的 CTLの増殖を高める効果も期待される。  [0022] Instead of magnetic fine particles coated with CD137 antibody, a culture plate coated with CD137 antibody can be used and isolated by pan-jung method. The anti-CD137 antibody may be a monoclonal antibody or a polyclonal antibody. The effective final concentration of epitope peptide added to the medium is 1 ng / mL-10 ng / mL. HCMV-specific CTLs are desirably isolated after 15 to 24 hours when CD137 antigen expression is highest after addition of the epitope peptide into the medium. In the isolation step, when CD137 antibody and HCMV-specific CTL come into contact with each other, stimulation is transmitted from the CD137 molecule into the cell, so that an effect of increasing the proliferation of HCMV-specific CTL is also expected.
[0023] 次に、 HCMV特異的 CTLの増殖工程について説明する。本実施形態における HC MV特異的 CTLの増殖方法（以下、「本実施形態における増殖方法」という。）は、 O T3(CD3モノクローナル抗体)を用いて増殖させた T細胞にェピトープペプチドを接触 させることにより、この T細胞上に HLAクラス I分子とェピトープペプチドから成る複合 体を形成させ、これと本実施形態における単離方法により調製した HCMV特異的 CT L上に発現する TCRを接触させることにより、 HCMV特異的 CTLを増殖させることを特 徴とする。末梢血単核球を OKT3で刺激することにより増殖させた T細胞（以下、「OK Τ3-Τ細胞」という。）上には副刺激分子である CD80及び、 CD86分子が高発現するの で、抗原提示細胞として用いることができる。従来、 CTL増殖工程において抗原提示 細胞として樹状細胞が用いられることが一般的であり、その調製には高価で、煩雑な 操作が必要であった。また、 CTL増殖工程においては、大量の樹状細胞が必要で（ 非特許文献 5)、新たにドナーから採血しなければならず、ドナーにかける負担が大き 力、つた。本実施形態による増殖方法においては、 CTL誘導の際に採血した血液の一 部を使って、 ΟΚΤ3で刺激し、増殖させて得られる Τ細胞を抗原提示細胞とするため、 従来法に比べて、簡便かつ低コストで HCMV特異的 CTLを増殖させることができる。 抗原提示細胞として用いる OKT3-T細胞上の CD80、 CD86の発現量は多レ、ほど良!/ヽ 。 PBMCを OKT3で刺激後、 CD80、 CD86の発現量が最も高くなる 2週間〜 1ヶ月間培 養した T細胞を用いることが望ましレ、。 [0023] Next, the growth process of HCMV-specific CTL will be described. The method for proliferating HC MV-specific CTL in this embodiment (hereinafter referred to as “proliferation method in this embodiment”) involves contacting an epitope peptide with T cells proliferated using OT3 (CD3 monoclonal antibody). On the T cell, a complex consisting of an HLA class I molecule and an epitope peptide. A HCMV-specific CTL is proliferated by forming a body and contacting the TCR expressed on the HCMV-specific CTL prepared by the isolation method in the present embodiment. Since CD80 and CD86 molecules, which are costimulatory molecules, are highly expressed on T cells proliferated by stimulating peripheral blood mononuclear cells with OKT3 (hereinafter referred to as “OK Τ3-Τ cells”), It can be used as an antigen-presenting cell. Conventionally, dendritic cells have generally been used as antigen-presenting cells in the CTL growth process, and their preparation has been expensive and complicated. In addition, a large amount of dendritic cells are required in the CTL proliferation process (Non-patent Document 5), and blood must be newly collected from the donor, which puts a heavy burden on the donor. In the proliferation method according to the present embodiment, a part of the blood collected at the time of CTL induction is used as the antigen-presenting cell, which is obtained by stimulating and proliferating with ΟΚΤ3. HCMV-specific CTL can be propagated easily and at low cost. The expression level of CD80 and CD86 on OKT3-T cells used as antigen-presenting cells is high. After stimulating PBMC with OKT3, it is desirable to use T cells cultured for 2 weeks to 1 month, where the expression level of CD80 and CD86 is highest.
[0024] 次に、 OKT3-T細胞（OKT3を用いて増殖させた T細胞；抗原提示細胞）とェピトー プペプチドを培地中で接触させる際の条件について以下に記す。培地中に加えるェ ピトープペプチドの終濃度は、 ο. Ι ^ g/mL lO g/mLが好ましい。培地としては、血 清、血漿などの蛋白成分を含まない RPMI1640, AIM-V,またはダルベッコ変法ィー グル培地 (Dulbecco's modified eagle medium)が好ましい。接触時間は 30分間〜 60分 間、接触温度は 18°C〜25°Cが好ましい。この条件で OKT3-T細胞とェピトープぺプ チドを接触させた後は、遊離のェピトープペプチドを除去するため、 RPMI1640などで 複数回（例えば、 3回程度）に渡って洗浄することが好ましい。このようにして調製され た抗原提示細胞と HCMV特異的 CTL (例えば、本実施形態における単離方法により 単離したもの）を共培養することにより、 HCMV特異的 CTLを増殖させる。 HCMV特異 的 CTLと抗原提示細胞の混合比は、 10: 1〜1:3の間で任意に選択できる。  [0024] Next, conditions for contacting OKT3-T cells (T cells proliferated using OKT3; antigen-presenting cells) and epitopic peptides in the medium are described below. The final concentration of the epitope peptide added to the medium is preferably ο.Ι ^ g / mL lO g / mL. As the medium, RPMI1640, AIM-V, or Dulbecco's modified eagle medium not containing protein components such as serum and plasma is preferable. The contact time is preferably 30 minutes to 60 minutes, and the contact temperature is preferably 18 ° C to 25 ° C. After contacting the OKT3-T cells with the epitope peptide under these conditions, it is preferable to wash with RPMI1640 several times (for example, about 3 times) in order to remove the free epitope peptide. . The antigen-presenting cells and HCMV-specific CTL (for example, those isolated by the isolation method in the present embodiment) prepared in this way are co-cultured to proliferate the HCMV-specific CTL. The mixing ratio of HCMV-specific CTL and antigen-presenting cells can be arbitrarily selected between 10: 1 and 1: 3.
[0025] なお、抗原提示細胞が HCMV特異的 CTLに対して過剰に混合された場合には、 H CMV特異的 CTLに対して Activation Induced Cell Death (AICD)による細胞死が誘導 されるので注意を要する。 AICDを防止するために、カスパーゼ阻害剤である Z-VAD -FMKを終濃度が 20 M〜40 Mとなるように加えることができる。 HCMV特異的 CT Lと抗原提示細胞との共培養に用いられる培地としては、 5%_10%自己血漿、 10 IU/ mL - 50 IU/mLの IL-2を含む RPMI1640、 AIM_V、またはダルベッコ変法イーグル培 地 (Dulbecco's modified eagle medium)を用いること力 S好ましい。共培養開始後、 18時 間〜 36時間後に、培地を 1%自己血漿を含む ALyS505N-1000に入れ替え、その後、 同培地を 2〜3日に一度、全体の半分を入れ替えることが好ましい。 [0025] Note that if antigen-presenting cells are mixed in excess with respect to HCMV-specific CTL, cell death due to Activation Induced Cell Death (AICD) is induced against HCMV-specific CTL. Cost. Z-VAD, a caspase inhibitor, to prevent AICD -FMK can be added to a final concentration of 20 M to 40 M. Media used for co-culture of HCMV-specific CT L and antigen-presenting cells include RPMI1640 with 5% _10% autologous plasma, 10 IU / mL-50 IU / mL IL-2, AIM_V, or a modified Dulbecco method It is preferable to use an eagle medium (Dulbecco's modified eagle medium). It is preferable to replace the medium with ALyS505N-1000 containing 1% autologous plasma 18 to 36 hours after the start of co-culture, and then replace the medium once every two to three days.
上記の工程を経ることにより、 HCMV特異的 CTLを誘導、単離、及び増殖することが できる。次に、実施例を説明することにより、本発明を更に詳細に説明する。  Through the above steps, HCMV-specific CTL can be induced, isolated and propagated. Next, the present invention will be described in more detail by describing examples.
実施例 Example
以下に実施例を参照しつつ、本発明を具体的に説明するが、本発明はこれらに限 定されるものではない。また、本発明の技術的範囲は均等の範囲にまで及ぶもので ある。  Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples. Further, the technical scope of the present invention extends to an equivalent range.
なお、本実施例においては、 HCMV特異的 CTLとは、 HCMV特異的 CD8+CTLを意 味している。  In this example, HCMV-specific CTL means HCMV-specific CD8 + CTL.
〔実施例 1〕 HCMV特異的 CTLの誘導工程  [Example 1] Induction process of HCMV-specific CTL
HLA-A24分子保持者、または HLA-A2保持者由来の末梢血より分離した末梢血単 核球 (PBMC: Peripheral Blood Mononuclear Cell)を 5%自己血漿、 2-メルカプトエタノ ール、 L-グルタミン、抗生物質としてペニシリンとストレプトマイシンを含む HEPES緩 衝 RPMI1640培地 (以下、「CTL誘導用培地」という) ImL中に細胞数が 2xl06個となる ように縣濁させ、ポリプロピレン製 14mLの丸底チューブ（Becton Dickinson社製）に 1 mL分注した。 4 g/mLの HLA-A24拘束性 HCMV特異的 CTLェピトープペプチド QY D (配列番号 2 :以下、「QYDペプチド」という)、若しくは 2 8/½しの HLA-A2拘束性 HC MV特異的 CTLェピトープペプチド NLV (配列番号 1：以下、「NLVペプチド」と!/、う）を 含む CTL誘導用培地 1 mLを添加後、 2日間、 5%COインキュベータ内において、 37 Peripheral Blood Mononuclear Cells (PBMC) isolated from peripheral blood derived from HLA-A24 molecule holder or HLA-A2 holder 5% autologous plasma, 2-mercaptoethanol, L-glutamine, HEPES slow collision RPMI1640 medium containing penicillin and streptomycin as antibiotics (hereinafter, "CTL induction medium" hereinafter) was suspended so that cell number is 2Xl0 6 pieces in ImL, round bottom tubes polypropylene 14 mL (Becton 1 mL was dispensed into Dickinson). 4 g / mL HLA-A24-restricted HCMV-specific CTL epitope peptide QY D (SEQ ID NO: 2; hereinafter referred to as “QYD peptide”), or 28 / ½ HLA-A2-restricted HC MV-specific After adding 1 mL of CTL induction medium containing CTL epitope peptide NLV (SEQ ID NO: 1; “NLV peptide” and! /, U), 37 days in a 5% CO incubator.
2  2
°Cで培養した。その後、 IL-2を 10 IU/mLとなるように添加した後、 3日おきに、培地 1 mLを、 50 IU/mLの IL-2を含む CTL誘導用培地と入れ替えながら 12日間、 5% CO  Cultivated at ° C. After that, IL-2 was added to 10 IU / mL, and every 3 days, 1 mL of medium was replaced with CTL induction medium containing 50 IU / mL IL-2 for 5 days. CO
2 インキュベータ内において、 37°Cで培養した。こうして、 HCMV特異的 CTLを誘導し、 HCMV特異的 CTLラインとした。 [0027] 〔実施例 2〕 HCMV特異的 CTLの誘導効率の検討 2 The cells were cultured at 37 ° C in an incubator. In this way, HCMV-specific CTL was induced to obtain an HCMV-specific CTL line. [Example 2] Examination of induction efficiency of HCMV-specific CTL
実施例 1の方法による HCMV特異的 CTLの誘導効率を誘導前後の HCMV特異的 C TLの数を比較することにより検討した。 HCMV特異的 CTLの数は培養中に含まれる 総細胞数と HCMV特異的 MHC-tetramerにより測定した HCMV特異的 CTL陽性率を 乗ずることにより算出した。 HCMV特異的 CTL陽性率の HCMV特異的 MHC-tetramer による測定は、以下のように行った。  The induction efficiency of HCMV-specific CTLs by the method of Example 1 was examined by comparing the number of HCMV-specific CTLs before and after induction. The number of HCMV-specific CTLs was calculated by multiplying the total number of cells contained in the culture by the HCMV-specific CTL positive rate measured by HCMV-specific MHC-tetramer. Measurement of HCMV-specific CTL positive rate with HCMV-specific MHC-tetramer was performed as follows.
測定に用いる細胞を、 0.1%BSAを含む PBS (以下、「細胞洗浄緩衝液」という)で洗 浄後、細胞濃度力 ¾χ106個/ mLとなるように細胞洗浄緩衝液に縣濁させ、その 100 L にフィコエリトリン（Phycoerythrin；以下「PE」と!/、う）を標識した HCMV特異的 MHC-tet ramer 10 Lと PC5標識した CD8モノクローナル抗体 10 Lを同時に混合させた後、 4 °Cで 30分間反応させた。 PBSで 2回洗浄後、細胞を 500 Lの細胞洗浄緩衝液に縣濁 させ、フローサイトメーターを用いて測定した。 Cells used for measurement are washed with PBS containing 0.1% BSA (hereinafter referred to as `` cell washing buffer solution ''), and then suspended in the cell washing buffer solution so that the cell concentration force becomes ¾χ10 6 cells / mL. After mixing 10 L of HCMV-specific MHC-tet ramer 10 L labeled with phycoerythrin (hereinafter referred to as “PE”!), 100 L with 10 L of PC5-labeled CD8 monoclonal antibody 30 at 4 ° C Reacted for 1 minute. After washing twice with PBS, the cells were suspended in 500 L of cell washing buffer and measured using a flow cytometer.
[0028] 図 1には、測定結果の一例を示した。縦軸は CD8蛍光強度、横軸は HCMV特異的 MHC- tetramerの蛍光強度を対数尺で示し、個々の打点は各細胞の蛍光強度を表 す。 4分割した左上の領域の打点は、 CD8と MHC-tetramerが同時に陽性細胞、すな わち、 HCMV特異的 CTLを表す。誘導後の細胞中に、 10.28%の HCMV特異的 CTL が含まれていた。後に示す表 1には、誘導前後における、総細胞数、 HCMV特異的 C TLの割合及び数を示し、さらに誘導効率を誘導前の HCMV特異的 CTL数に対する、 誘導後の HCMV特異的 CTL数の比として表現した。  FIG. 1 shows an example of the measurement result. The vertical axis shows CD8 fluorescence intensity, the horizontal axis shows the fluorescence intensity of HCMV-specific MHC-tetramer in logarithmic scale, and each dot represents the fluorescence intensity of each cell. The dot in the upper left area divided into four represents CD8 and MHC-tetramer positive cells at the same time, that is, HCMV-specific CTL. The cells after induction contained 10.28% HCMV-specific CTL. Table 1 below shows the total number of cells before and after induction, the ratio and number of HCMV-specific CTLs, and the induction efficiency of the number of HCMV-specific CTLs after induction relative to the number of HCMV-specific CTLs before induction. Expressed as a ratio.
[0029] 〔実施例 3〕 HCMV特異的 CTLの単離、及び濃縮  [Example 3] Isolation and concentration of HCMV-specific CTL
1. CD137モノクローナル抗体を感作した培養プレートによる単離  1. Isolation by culture plate sensitized with CD137 monoclonal antibody
(1) CD137モノクローナル抗体感作培養プレートの作製  (1) Preparation of CD137 monoclonal antibody-sensitized culture plate
PBSで 10 a g/mLに希釈した CD137モノクローナル抗体（クローン 4B4)を 6ゥエルプ レートに 1 mL分注し、 4°Cで 18時間、静置反応させた。抗体溶液をァスピレーターで 除去後、 5%ヒトアルブミンを含む PBSを 2 mL分注し、 37°Cで 1時間インキユーペートし 、抗体が感作された培養プレート表面をブロッキングした。 PBSで 3回プレート表面を 洗浄した後、 CD137モノクローナル抗体感作培養プレートとして用いた。  1 mL of CD137 monoclonal antibody (clone 4B4) diluted to 10 ag / mL with PBS was dispensed into 6 well plates and allowed to stand at 4 ° C for 18 hours. After removing the antibody solution with an aspirator, 2 mL of PBS containing 5% human albumin was dispensed and incubated at 37 ° C. for 1 hour to block the surface of the culture plate sensitized with the antibody. The plate surface was washed 3 times with PBS and then used as a CD137 monoclonal antibody-sensitized culture plate.
[0030] (2) HCMV特異的 CTLェピトープペプチドによる HCMV特異的 CTLラインの再刺激 と CD137分子の発現解析 [0030] (2) Restimulation of HCMV-specific CTL line by HCMV-specific CTL epitope peptide And expression analysis of CD137 molecule
実施例 1で誘導した HLA-A24拘束性 HCMV特異的 CTLライン培養中に QYDぺプ チドを終濃度 10 ng/mLで加え、 18時間培養した。細胞の一部を採取し、 HCMV特異 的 CTL上に選択に CD137が発現していることを確認するため、 PC5標識 CD137モノク ローナル抗体と PE標識 HCMV特異的 MHC-tetramerを用いて二重染色を行い、フロ 一サイトメーターにより解析した。図 2に示すように、 HCMV特異的 CTL(MHC_tetram er陽性細胞として示された細胞集団)上に CD 137分子の選択的発現を認めた。  QYD peptide was added to the HLA-A24-restricted HCMV-specific CTL line culture induced in Example 1 at a final concentration of 10 ng / mL and cultured for 18 hours. Collect a portion of the cells and double-stain using PC5-labeled CD137 monoclonal antibody and PE-labeled HCMV-specific MHC-tetramer to confirm that CD137 is selectively expressed on the HCMV-specific CTL. And analyzed with a flow cytometer. As shown in FIG. 2, selective expression of CD137 molecule was observed on HCMV-specific CTL (cell population shown as MHC_tetramer positive cells).
[0031] (3) HCMV特異的 CTLの単離、及び培養 [0031] (3) Isolation and culture of HCMV-specific CTL
QYDペプチドにより再刺激した HLA-A24拘束性 HCMV特異的 CTLラインを、 CD13 7モノクローナル抗体を感作した培養プレートに移し、 1時間、 5% COインキュベータ  Transfer HLA-A24-restricted HCMV-specific CTL line restimulated with QYD peptide to culture plate sensitized with CD13 7 monoclonal antibody for 1 hour, 5% CO incubator
2  2
内において、 37°Cで反応させ、 CD137分子を発現した HCMV特異的 CTLを CD137モ ノクローナル抗体を介して、培養プレート表面に接着させた。培養プレートを軽く揺ら した後、ピペットを用いて、培養プレート表面に接着していない細胞を除去した。その 後、 0.5%ヒトアルブミンを含む HEPES緩衝 RPMI培地で洗浄をゆっくりと lmL加え、軽 く揺らした後、再度、ピペットを用いて、培養プレート表面に接着していない細胞を除 去した。さらにこの操作を 2回繰り返した後、 50 IU/mLの IL-2を含む CTL誘導用培 地を 1 mL加え、 5% COインキュベータ内において、 37°Cで 24時間培養した。さらに  The HCMV-specific CTL expressing CD137 molecule was allowed to react at 37 ° C. and adhered to the surface of the culture plate via the CD137 monoclonal antibody. After gently shaking the culture plate, cells that did not adhere to the surface of the culture plate were removed using a pipette. Thereafter, 1 mL of washing was slowly added with HEPES buffered RPMI medium containing 0.5% human albumin, and after gently shaking, cells that did not adhere to the surface of the culture plate were removed again using a pipette. After further repeating this operation twice, 1 mL of a CTL induction medium containing 50 IU / mL of IL-2 was added and cultured at 37 ° C. for 24 hours in a 5% CO incubator. More
2  2
ALyS505N-1000培地を 1 mL添加し、 5% COインキュベータ内において、 37°Cで 6  Add 1 mL of ALyS505N-1000 medium and in a 5% CO incubator at 37 ° C.
2  2
日間培養した。  Cultured for days.
[0032] (4)単離、及び培養した HCMV特異的 CTLの純度と回収率の解析  [0032] (4) Analysis of purity and recovery of isolated and cultured HCMV-specific CTL
単離、及び培養した HLA-A24拘束性 HCMV特異的 CTLを、 106/mLの細胞濃度で 細胞洗浄緩衝液に浮遊させ、その 100 Lに PEを標識した HCMV特異的 MHC-tetra mer 10 μ Lと FITC標識した CD8モノクローナル抗体 10 μ Lを同時に混合させた後、 4 °Cで 30分間反応させた。 PBSで 2回洗浄後、細胞を 500 Lの細胞洗浄緩衝液に縣濁 させ、フローサイトメーターを用いて測定した。 HLA-A24-restricted HCMV-specific CTL isolated and cultured were suspended in cell wash buffer at a cell concentration of 10 6 / mL, and 100 L of PE-labeled HCMV-specific MHC-tetramer 10 μ L and 10 μL of FITC-labeled CD8 monoclonal antibody were mixed at the same time, and then reacted at 4 ° C. for 30 minutes. After washing twice with PBS, the cells were suspended in 500 L of cell washing buffer and measured using a flow cytometer.
図 2に示すように、単離前 10.28%であった HLA-A24拘束性 HCMV特異的 CTLの 純度が、単離直後では 56.03%に、また、培養一週間後 97.14%に上昇した。また、単 離直後において 48.7%の HCMV特異的 CTLが回収され、その後 1週間の培養で、 2. As shown in FIG. 2, the purity of HLA-A24-restricted HCMV-specific CTL, which was 10.28% before isolation, increased to 56.03% immediately after isolation and to 97.14% after one week of culture. Immediately after isolation, 48.7% of HCMV-specific CTLs were recovered and then cultured for 1 week.
Figure imgf000017_0001
Figure imgf000017_0001
A2- A2 2 6.0 0.0056 / 0.28 1.69/28.1 301.8  A2- A2 2 6.0 0.0056 / 0.28 1.69 / 28.1 301.8
A24-1 A24 2 2.87 <0.0002/<0.01 0.95133.2 >4750.0 A24-1 A24 2 2.87 <0.0002 / <0.01 0.95133.2> 4750.0
A24-2 A24 2 1.32 ぐ 0.0002 /<0.01 0.15/11. >750.0A24-2 A24 2 1.32 round 0.0002 /<0.01 0.15 / 11.> 750.0
A24-10 A24 2 1.44 ぐ 0.0002 /く 0.01 0.29/20 >1450.0 A24-10 A24 2 1.44 0.00 0.0002 / 0.01 0.01 0.29 / 20> 1450.0
0033 [0034] 2.細胞自動分離装置を用いた単離 0033 [0034] 2. Isolation using automatic cell separator
( 1 ) CD 137モノクローナル抗体を用レ、た単離  (1) Isolation using CD137 monoclonal antibody
上記 1 (2)の方法に従い、 HLA-A24拘束性 HCMV特異的 CTLラインを QYDぺプチ ドにより再刺激した後、細胞洗浄緩衝液 100 Lに懸濁した後、 100 g/mL CD137モ ノクローナル抗体 (クローン 4B4)を 10 し加え、攪拌の後、室温で 15分間反応させた。 細胞洗浄緩衝液で 2回洗浄し、細胞を 0.5%ヒトアルブミン、 2 mM EDTAを含む PBS ( 以下、「細胞分離緩衝液」と!、う） 80 Lに細胞を懸濁させた後、抗マウス IgG結合マイ クロ磁性ビーズ (ミルテュ社製)を 20 ^ L加え、攪拌後、 4°Cで 15分間反応させた。細胞 分離緩衝液で 2回洗浄後、 500 Lの細胞分離緩衝液に懸濁させた後、細胞自動分 離装置 (AutoMACS:ミルテニ社製)を用いて、 CD137陽性細胞、すなわち HLA-A24拘 束性 HCMV特異的 CTLを分離した。  In accordance with method 1 (2) above, HLA-A24-restricted HCMV-specific CTL line was restimulated with QYD peptide, suspended in 100 L of cell washing buffer, and then 100 g / mL CD137 monoclonal antibody. (Clone 4B4) was added to the mixture, and after stirring, the mixture was reacted at room temperature for 15 minutes. After washing twice with cell washing buffer, the cells were suspended in 80 L of PBS containing 0.5% human albumin and 2 mM EDTA (hereinafter referred to as “cell separation buffer”!), Then anti-mouse 20 μL of IgG-bound micromagnetic beads (Miltu) were added, and after stirring, the mixture was reacted at 4 ° C for 15 minutes. After washing twice with cell separation buffer and suspending in 500 L of cell separation buffer, CD137 positive cells, that is, HLA-A24-constrained cells, using an automatic cell separation device (AutoMACS: manufactured by Milteni) Sex HCMV specific CTL were isolated.
[0035] (2) MHC_tetramerを用いた単離 [0035] (2) Isolation using MHC_tetramer
HLA-A24拘束性 HCMV特異的 CTLラインを細胞洗浄緩衝液 100 μ Lに懸濁した後 、 HCMV特異的 HLA-A24-tetramer ((株)ベックマンコールター社製）を 10 L加え、 攪拌の後、室温で 15分間反応させた。細胞洗浄緩衝液で 2回洗浄し、細胞を 0.5%ヒ トァノレブミン、 2 mM EDTAを含む PBS (以下細胞分離緩衝液という） 80 Lに細胞を懸 濁させた後、抗 PE抗体結合マイクロ磁性ビーズ (ミルテュ社製)を 20 ^ L加え、攪拌後 、 4°Cで 15分間反応させた。細胞分離緩衝液で 2回洗浄後、 500 ^ Lの細胞分離緩衝 液に懸濁させた後、細胞自動分離装置 (AutoMACS:ミルテニ社製)を用いて、 HCMV 特異的 HLA-A24-tetramer陽性細胞、すなわち HLA-A24拘束性 HCMV特異的 CTL を分離した。  After suspending HLA-A24-restricted HCMV-specific CTL line in 100 μL of cell washing buffer, add 10 L of HCMV-specific HLA-A24-tetramer (manufactured by Beckman Coulter, Inc.), and after stirring, The reaction was allowed to proceed for 15 minutes at room temperature. The cells were washed twice with cell washing buffer, and the cells were suspended in 80 L of PBS (hereinafter referred to as “cell separation buffer”) containing 0.5% hydrogenlevamine and 2 mM EDTA, and then anti-PE antibody-conjugated micromagnetic beads ( (Mirtu Co., Ltd.) 20 ^ L was added, and the mixture was stirred and reacted at 4 ° C for 15 minutes. After washing twice with cell separation buffer and suspending in 500 ^ L cell separation buffer, HCMV-specific HLA-A24-tetramer positive cells using an automatic cell separator (AutoMACS: manufactured by Milteni) That is, HLA-A24-restricted HCMV-specific CTLs were isolated.
図 3に細胞自動分離装置を用レ、た単離の結果を示した。 CD 137モノクローナル抗 体を用いた単離においては、 HLA-A24拘束性 HCMV特異的 CTLの回収率 74.1%、 純度 80.82%であった。一方、 MHC-tetramerを用いた単離においては回収率 34.0 %、純度 80.15%であった。純度については両者の間で差はみられなかったが、回収 率の点では CD137モノクローナル抗体を用いた方法が MHC-tetramerを用いた方 法に比べて優れていた。  Figure 3 shows the results of isolation using an automatic cell separator. In the isolation using the CD137 monoclonal antibody, the recovery rate of HLA-A24-restricted HCMV-specific CTL was 74.1% and the purity was 80.82%. On the other hand, in the isolation using MHC-tetramer, the recovery rate was 34.0% and the purity was 80.15%. Although there was no difference between the two in terms of purity, the method using the CD137 monoclonal antibody was superior to the method using the MHC-tetramer in terms of recovery.
[0036] 〔実施例 4〕 HCMV特異的 CTLの増殖 1. OKT3-T細胞の調製 [Example 4] Proliferation of HCMV-specific CTL 1. Preparation of OKT3-T cells
HCMV特異的 CTLの誘導の際に分離した PBMCの一部 (2X106個)を 1 μ g/mLの濃 度で OKT3を含む CTL誘導用培地 10mLに懸濁させ、 5% COインキュベータ内で 2 A portion of PBMC (2X10 6 ) isolated during HCMV-specific CTL induction was suspended in 10 mL of CTL induction medium containing OKT3 at a concentration of 1 μg / mL and 2 in a 5% CO incubator.
2  2
日間、 37°Cで培養した。 ALyS505N-1000培地を 10 mL添加後、 5% COインキュベー  Incubated at 37 ° C for one day. After adding 10 mL of ALyS505N-1000 medium, 5% CO incubation
2  2
タ内で 12日間、 37°Cで培養し、 OKT3-T細胞を得た。  The cells were cultured at 37 ° C for 12 days in a batch to obtain OKT3-T cells.
[0037] 2. OKT3-T細胞を用いた HCMV抗原提示細胞の調製 [0037] 2. Preparation of HCMV antigen-presenting cells using OKT3-T cells
上記 1で調製した OKT3-T細胞 107個を細胞洗浄緩衝液で 2回洗浄の後、 1 g/mL HCMV特異的 CTLェピトープペプチドを含む 1 mL HEPES緩衝 RPMI1640培地に懸 濁させ、 25°Cで 30分間反応させた。細胞洗浄緩衝液で 2回洗浄し、 HCMV抗原提示 細胞とした。 10 7 OKT3-T cells prepared in 1 above were washed twice with cell washing buffer, and then suspended in 1 mL HEPES buffer RPMI1640 medium containing 1 g / mL HCMV-specific CTL epitope peptide. The reaction was performed at ° C for 30 minutes. The cells were washed twice with cell washing buffer to obtain HCMV antigen-presenting cells.
3. HCMV抗原提示細胞との共培養による HCMV特異的 CTLの増殖  3. Proliferation of HCMV-specific CTL by co-culture with HCMV antigen-presenting cells
〔実施例 3〕 1に示した方法で単離した HLA-A24拘束性 HCMV特異的 CTL 4xl06個 を 4 mLの 50IU/mL IL-2を含む CTL誘導用培地に懸濁させ、上記 2で調製した HCM V抗原提示細胞 2xl06個を添加した後、 5% COインキュベータ内で 24時間、 37°Cで [Example 3] Six HLA-A24-restricted HCMV-specific CTL 4xl0 isolated by the method described in 1 were suspended in CTL induction medium containing 4 mL of 50IU / mL IL-2. After adding 6 x 2xl0 prepared HCM V antigen-presenting cells, 24 hours in a 5% CO incubator at 37 ° C
2  2
培養した。 ALyS505N-1000培地を 4 mL加えた後、さらに 8日間培養した。培養後、一 部を採取し、総細胞数を血球計算版にて測定し、 HLA-A24拘束性 HCMV特異的 CT Lの割合を実施例 2と同様に、フローサイトメーターにより計測した。また、 HLA-A24 拘束性 HCMV特異的 CTLの数は総細胞数とフローサイトメーターを用いて計測した 割合を掛けることにより求めた。  Cultured. After 4 mL of ALyS505N-1000 medium was added, the cells were further cultured for 8 days. After culturing, a portion was collected, the total cell number was measured with a hemocytometer, and the ratio of HLA-A24-restricted HCMV-specific CT L was measured with a flow cytometer in the same manner as in Example 2. The number of HLA-A24-restricted HCMV-specific CTLs was determined by multiplying the total number of cells by the ratio measured using a flow cytometer.
図 4には、増殖前後のフローサトメトリー像を示した。 HCMV特異的 CTLの割合は、 増殖前 98.5%、増殖後 97.68%とほとんど変わらなかった。一方、 HLA-A24拘束性 H CMV特異的 CTL数は、 4xl06個から、 4. lxlO7個とほぼ 10倍に増殖した。 FIG. 4 shows flow cytometry images before and after growth. The proportion of HCMV-specific CTL was almost unchanged at 98.5% before growth and 97.68% after growth. On the other hand, the number of HLA-A24-restricted HCMV-specific CTLs grew almost 10-fold from 6 from 4xl0 to 7 from 4.lxlO.
[0038] このように本実施形態によれば、簡便かつ安価に、 HCMV特異的 CTLの調製を行う ことができた。 HCMV特異的 CTL誘導工程では、抗原提示細胞の調製が不要である ため、迅速かつ簡便に HCMV特異的 CTLの誘導を行えた。また、 HCMV特異的 CTL 増殖工程では、抗原提示細胞として OKT3で増殖させた T細胞に代替することによつ て、簡便で安価な HCMV特異的 CTLの増殖が可能であった。さらに、 HCMV特異的 CTL単離工程においては、 CD137抗原を標的とした単離方法を樹立することにより、 生存率を低下させることなく安全に HCMV特異的 CTLの単離が可能となった。 [0038] Thus, according to the present embodiment, HCMV-specific CTL could be prepared easily and inexpensively. In the HCMV-specific CTL induction step, since preparation of antigen-presenting cells is unnecessary, HCMV-specific CTL can be induced quickly and easily. In addition, in the HCMV-specific CTL proliferation process, it was possible to proliferate HCMV-specific CTL easily and inexpensively by substituting T cells that had been propagated with OKT3 as antigen-presenting cells. Furthermore, in the HCMV-specific CTL isolation process, by establishing an isolation method targeting the CD137 antigen, HCMV-specific CTL can be isolated safely without reducing the survival rate.
HCMV特異的 CTLをヒト CMV患者に投与することにより、症状の改善が認められる ことが複数の論文によって示されている（例えば、非特許文献 1、非特許文献 3、 BLO OD 2002;99:3916-3922)。 HCMV特異的 CTLは、 HCMV感染細胞を特異的に認識し 、かつ極めて強力な細胞傷害活性を有しているので、これを用いることにより、安全 で効果的な HCMV特異的 CTLによる HCMV予防薬、治療薬、及びこれを簡便で安価 に調製する技術を提供することができる。  Several papers have shown that HCMV-specific CTLs can be improved in human CMV patients (eg, Non-Patent Document 1, Non-Patent Document 3, BLO OD 2002; 99: 3916). -3922). HCMV-specific CTL specifically recognizes HCMV-infected cells and has an extremely strong cytotoxic activity, so by using this, a safe and effective HCMV-preventing drug by HCMV-specific CTL, It is possible to provide a therapeutic agent and a technique for preparing it easily and inexpensively.
図面の簡単な説明 Brief Description of Drawings
[図 1]HCMV特異的 CTLの誘導例を示すためのサイトフローメーターによる解析結果 を示す図である。実施例 1に従い、 HLA-A24陽性健常人由来末梢血単核球から HL A-A24拘束性 HCMV特異的 CTLを誘導した後、実施例 2に従って、 HLA-A24拘束性 HCMV特異的 CTLの陽性率を測定した。陽性率 10.28%と算定された。 FIG. 1 is a diagram showing the results of analysis using a cytoflow meter to show examples of induction of HCMV-specific CTL. According to Example 1, after inducing HL A-A24-restricted HCMV-specific CTLs from peripheral blood mononuclear cells derived from HLA-A24-positive healthy individuals, the positive rate of HLA-A24-restricted HCMV-specific CTLs according to Example 2 Was measured. A positive rate of 10.28% was calculated.
[図 2]CD137モノクロナル抗体感作培養プレートによる HCMV特異的 CTL単離例を示 すためのサイトフローメーターによる解析結果を示す図である。実施例 1で誘導した H LA-A24拘束性 HCMV特異的 CTLラインから [実施例 3]1.に示した方法に従い、 HL A-A24拘束性 HCMV特異的 CTLを単離した後、 7日間培養した。単離前では 10.28 %であった HLA-A24拘束性 HCMV特異的 CTL陽性率が単離後、 56.03%、さらに 7 日間培養後においては 97.14%に上昇した。単離後の回収率は 48.7%、 7日間増殖 後の増殖率は 2.9倍であった。 FIG. 2 is a diagram showing the results of analysis by a cytoflow meter for showing an example of HCMV-specific CTL isolation using a CD137 monoclonal antibody-sensitized culture plate. [Example 3] After isolating HL A-A24-restricted HCMV-specific CTL from the HLA-A24-restricted HCMV-specific CTL line derived in Example 1, culture for 7 days did. The HLA-A24-restricted HCMV-specific CTL positive rate, which was 10.28% before isolation, increased to 56.03% after isolation, and increased to 97.14% after 7 days of culture. The recovery rate after isolation was 48.7%, and the growth rate after 7 days of growth was 2.9 times.
[図 3]細胞自動分離装置を用いた単離の結果を示すためのフローサイトメーターによ る解析結果を示す図である。実施例 1の方法で誘導した HLA-A24拘束性 HCMV特 異的 CTLラインから [実施例 3]2.に示した方法に従い、 HLA-A24拘束性 HCMV特 異的 CTLを単離した。純度については CD137を用いた方法と MHC-tetramerを用い た方法の間で差はみられなかったが、回収率の点では CD137を用いた方法が MHC- tetramerを用いた方法に比べて優れて!/、た。  FIG. 3 is a diagram showing the results of analysis using a flow cytometer to show the results of isolation using an automatic cell separator. From the HLA-A24-restricted HCMV-specific CTL line induced by the method of Example 1, HLA-A24-restricted HCMV-specific CTL was isolated according to the method shown in [Example 3] 2. Regarding purity, there was no difference between the method using CD137 and the method using MHC-tetramer, but the method using CD137 was superior to the method using MHC-tetramer in terms of recovery. ! /
[図 4]HCMV特異的 CTLの増殖の結果を示すためのフローサイトメーターによる解析 結果を示す図である。 [実施例 3] 1.の方法で単離した HLA-A24拘束性 HCMV特異 的 CTLを [実施例 4]に従い増殖させた。 Tetramer/CD8陽性率は 8日間増殖後にお いて増殖前とほぼ同様であった。 HCMV特異的 CTL細胞数は、ほぼ 10倍に増殖した FIG. 4 is a diagram showing the results of analysis using a flow cytometer to show the results of proliferation of HCMV-specific CTLs. [Example 3] HLA-A24-restricted HCMV-specific CTL isolated by the method of 1. was grown according to [Example 4]. T e tram er / CD8 positive rate us after growth for 8 days It was almost the same as before growth. Number of HCMV-specific CTL cells proliferated almost 10 times

Claims

請求の範囲 The scope of the claims
[1] HTLV-1、インフルエンザ、アデノウイルス、エイズウイルス、 B型肝炎ウィルス、 C型肝 炎ゥイノレス、ェプスタインバーゥイノレス、ヒトパピローマウイノレス、及びヒトサイトメガロウ ィルスからなる群から選択される一つのウィルス特異的 CTLを誘導する方法であって 、末梢血単核球とウィルス特異的 CTLェピトープペプチドを混合培養することを特徴 とするウィルス特異的 CTLの誘導方法。  [1] One selected from the group consisting of HTLV-1, influenza, adenovirus, AIDS virus, hepatitis B virus, hepatitis C winoreth, Epstein Barwinoles, human papilloma winoles, and human cytomegalovirus A method for inducing a virus-specific CTL, which comprises culturing a mixture of peripheral blood mononuclear cells and a virus-specific CTL epitope peptide.
[2] 前記混合培養において、ェピトープペプチドの培地中での終濃度が 0.01 g/mL-10  [2] In the mixed culture, the final concentration of the epitope peptide in the medium is 0.01 g / mL-10.
a g/mLであることを特徴とする請求項 1に記載のウィルス特異的 CTLの誘導方法。  2. The method for inducing a virus-specific CTL according to claim 1, which is a g / mL.
[3] 前記混合培養に用いる培地として、 5% - 10%自己血漿を含む RPMI1640を用い、ェ ピトープペプチドを添加後、 2日後に終濃度が 10 IU/mLとなるように IL-2を加え、その 後 IL-2濃度を 2-3日ごとに 2倍づっ上げながら 12日間- 19日間培養することを特徴と する請求項 1または 2に記載のウィルス特異的 CTLの誘導方法。 [3] RPMI1640 containing 5% -10% autologous plasma is used as the medium for the mixed culture, and IL-2 is added so that the final concentration becomes 10 IU / mL 2 days after the addition of the epitope peptide. 3. The method for inducing virus-specific CTL according to claim 1 or 2, further comprising culturing for 12 to 19 days while increasing the IL-2 concentration by 2 times every 2-3 days.
[4] ウィルス特異的 CTLを単離する方法であって、請求項 1〜3のいずれかに記載の誘 導方法、またはその他の方法で誘導されたウィルス特異的 CTLを、誘導に用いたェ ピトープペプチドと同じェピトープペプチドと培地中で接触させることにより再刺激後  [4] A method for isolating a virus-specific CTL, wherein the virus-specific CTL induced by the induction method according to any one of claims 1 to 3 or other methods is used for induction. After restimulation by contacting in the medium with the same epitope peptide as the pitope peptide
、新たに発現する CD137抗原を標的として、ウィルス特異的 CTLを単離することを特 徴とするウィルス特異的 CTLの単離方法。 A method for isolating a virus-specific CTL, which comprises isolating a virus-specific CTL using a newly expressed CD137 antigen as a target.
[5] ウィルス特異的 CTLを単離する方法であって、請求項 1〜3のいずれかに記載の誘 導方法、またはその他の方法で誘導されたウィルス特異的 CTLを、誘導に用いたェ ピトープペプチドと同じェピトープペプチドを提示した抗原提示細胞と培地中で接触 させることにより再刺激後、新たに発現する CD137抗原を標的として、ウィルス特異的 CTLを単離することを特徴とするウィルス特異的 CTLの単離方法。  [5] A method for isolating a virus-specific CTL, wherein the virus-specific CTL induced by the induction method according to any one of claims 1 to 3 or other methods is used for induction. It is characterized by isolating virus-specific CTL targeting the newly expressed CD137 antigen after re-stimulation by contacting with antigen-presenting cells presenting the same epotope peptide as the pitope peptide in the medium. Isolation method of virus-specific CTL.
[6] ウィルス特異的 CTLを単離する方法であって、請求項 1〜3のいずれかに記載の誘 導方法、またはその他の方法で誘導されたウィルス特異的 CTLを、誘導に用いたェ ピトープペプチドと同じェピトープペプチドと複合体を形成する MHC分子と培地中で 接触させることにより再刺激後、新たに発現する CD137抗原を標的として、ウィルス特 異的 CTLを単離することを特徴とするウィルス特異的 CTLの単離方法。  [6] A method for isolating a virus-specific CTL, wherein the virus-specific CTL induced by the induction method according to any one of claims 1 to 3 or other methods is used for induction. After re-stimulation by contacting in medium with an MHC molecule that forms a complex with the same epitope peptide as the pitope peptide, isolation of the virus-specific CTL targeting the newly expressed CD137 antigen A method for isolating a virus-specific CTL characterized.
[7] 前記再刺激に用いるェピトープペプチドの培地中での終濃度が 0.1ng/mL-1000ng/ mLであることを特徴とする請求項 4〜6のいずれかに記載のウィルス特異的 CTLの単 離方法。 [7] The final concentration of the epitope peptide used for restimulation in the medium is 0.1 ng / mL-1000 ng / The method for isolating virus-specific CTL according to any one of claims 4 to 6, which is mL.
[8] 前記再刺激は、 5%-10%自己血漿と 10 IU/mL〜50 IU/mLの IL-2または 10ng/mL IL -15のいずれかとを含む RPMI1640培地中で 8時間〜 15時間に渡って行うことを特徴と する請求項 4〜7のいずれかに記載のウィルス特異的 CTLの単離方法。  [8] The restimulation is performed for 8-15 hours in RPMI 1640 medium containing 5% -10% autologous plasma and either 10 IU / mL to 50 IU / mL IL-2 or 10 ng / mL IL-15. The method for isolating virus-specific CTL according to any one of claims 4 to 7, wherein
[9] CD137抗原を標的とする際に、磁気標識した抗 CD137抗体を用いることを特徴とする 請求項 4〜8のいずれかに記載のウィルス特異的 CTLの単離方法。  [9] The method for isolating virus-specific CTL according to any one of claims 4 to 8, wherein a magnetically labeled anti-CD137 antibody is used when targeting the CD137 antigen.
[10] CD137抗原を標的とする際に、抗 CD137抗体を感作した固相を用いることを特徴とす る請求項 4〜8のいずれかに記載のウィルス特異的 CTLの単離方法。  [10] The method for isolating virus-specific CTL according to any one of [4] to [8], wherein a solid phase sensitized with an anti-CD137 antibody is used when targeting the CD137 antigen.
[11] ウィルス特異的 CTLの増殖方法であって、抗 CD3モノクローナル抗体である OKT3を 用いて末梢血単核球から増殖させた T細胞と、請求項;!〜 3の!/、ずれかにお!/、て誘 導の際に用いたェピトープペプチドと同じェピトープペプチドとを培地中で接触させ た後に、この T細胞と、請求項 4〜； 10のいずれかに記載の単離方法で単離したウイ ノレス特異的 CTLまたは請求項 1〜3のいずれかに記載の誘導方法で誘導したウィル ス特異的 CTLとを共培養することを特徴とするウィルス特異的 CTLの増殖方法。  [11] A method of proliferating virus-specific CTL, wherein T cells are proliferated from peripheral blood mononuclear cells using OKT3, an anti-CD3 monoclonal antibody, and any of claims;! 11. After contacting the same epitope peptide as that used for induction in the medium, this T cell and the single peptide according to claim 4 to 10; A method for proliferating virus-specific CTL, comprising co-cultured with wiores-specific CTL isolated by a separation method or virus-specific CTL induced by the induction method according to any one of claims 1 to 3. .
[12] 請求項 11において OKT3を用いて末梢血単核球から増殖させた T細胞を、ウィルス 特異的 CTLェピトープペプチドと培地中で接触させる際に、下記条件を用いることを 特徴とする増殖方法。  [12] The method according to claim 11, wherein the following conditions are used when the T cells expanded from peripheral blood mononuclear cells using OKT3 are contacted with a virus-specific CTL epitope peptide in a medium. Proliferation method.
(a)培地中に加えるェピトープペプチドの終濃度； 0.0001 μ 8/01し〜10 g/mL(a) the final concentration of E pitot flop peptide added to the culture medium; 0.0001 μ 8/01 teeth to 10 g / mL
(b)接触時間； 30分間〜 60分間 (b) Contact time; 30 minutes to 60 minutes
(c)培養温度； 18°C〜25°C  (c) Culture temperature: 18 ° C-25 ° C
(d)培地；血清、血漿など蛋白成分を含まな!/、RPMI1640。  (d) Medium; free of protein components such as serum and plasma! /, RPMI1640.
[13] 請求項 11において、以下の共培養条件を特徴とする増殖方法。 13. The growth method according to claim 11, characterized by the following co-culture conditions.
(e)ウィルス特異的 CTL： OKT3を用いて増殖させた T細胞の混合比； 10: 1〜1:3 (e) Virus-specific CTL: mixed ratio of T cells grown with OKT3; 10: 1 to 1: 3
(f)培地組成 共培養開始後 1日目- 3日目まで； RPMI1640 (5%〜10%自己血漿、 1 0 IU/mL〜50 IU/mL IL-2)または RPMI1640(5%〜10%自己血漿、 10ng/mL IL-15)(f) Medium composition From day 1 to day 3 after initiation of co-culture; RPMI1640 (5% -10% autologous plasma, 10 IU / mL-50 IU / mL IL-2) or RPMI1640 (5% -10% (Autologous plasma, 10ng / mL IL-15)
、共培養開始後 3日目以降； ALyS505N (100 IU/mL〜1000 IU/mL IL-2, 0.1%〜1% 自己血漿）または ALyS505N (10ng/mL IL-15, 0· 1%〜1%自己血漿）。 3rd day after start of co-culture; ALyS505N (100 IU / mL to 1000 IU / mL IL-2, 0.1% to 1% autologous plasma) or ALyS505N (10 ng / mL IL-15, 0.1% to 1% Autologous plasma).
[14] 請求項 1〜3のいずれかに記載の誘導方法によりウィルス特異的 CTLを誘導するェ 程を含むウィルス特異的 CTLの製造方法。 [14] A method for producing virus-specific CTL, comprising a step of inducing virus-specific CTL by the induction method according to any one of claims 1 to 3.
[15] 請求項 4〜； 10のいずれかに記載の単離方法によりウィルス特異的 CTLを単離するェ 程を含むウィルス特異的 CTLの製造方法。 [15] A method for producing virus-specific CTL, comprising the step of isolating virus-specific CTL by the isolation method according to any one of claims 4 to 10.
[16] 請求項 11〜； 13のいずれかに記載の増殖方法によりウィルス特異的 CTLを増殖する 工程を含むウィルス特異的 CTLの製造方法。 [16] A method for producing virus-specific CTL, comprising a step of proliferating virus-specific CTL by the growth method according to any one of [11] to [13].
[17] 請求項 1〜3のいずれかに記載のウィルス特異的 CTLの誘導方法、請求項 4〜； 10の いずれかに記載のウィルス特異的 CTLの単離方法、請求項;！;!〜 14のいずれかに記 載のウィルス特異的 CTLの増殖方法をそれぞれ組み合わせることを特徴とするウイ ノレス特異的 CTLの製造方法。 [17] The method for inducing a virus-specific CTL according to any one of claims 1 to 3, the method for isolating a virus-specific CTL according to any one of claims 4 to 10; A method for producing a wineless-specific CTL characterized by combining the virus-specific CTL growth methods described in any one of!
[18] 請求項 14〜； 17のいずれかに記載のウィルス特異的 CTLの製造方法によって製造さ れたウィルス特異的 CTL。 [18] A virus-specific CTL produced by the method for producing a virus-specific CTL according to any one of claims 14 to 17;
[19] 請求項 14〜； 17のいずれかに記載のウィルス特異的 CTLの製造方法によって製造さ れたウィルス特異的 CTLを含むウィルス感染症の治療剤及び/または予防剤。 [19] A therapeutic and / or prophylactic agent for a viral infection comprising a virus-specific CTL produced by the method for producing a virus-specific CTL according to any one of claims 14 to 17;
[20] 請求項 19に記載のウィルス感染症の治療剤及び/または予防剤を用いることを特 徴とするウィルス感染症の治療方法及び/または予防方法。 [20] A method for treating and / or preventing a viral infection, which comprises using the therapeutic and / or preventive agent for a viral infection according to claim 19.
[21] CTLの免疫応答能の検査方法であって、培地中で、（a)CTLェピトープペプチド、この[21] A method for examining the immune response ability of CTL, comprising: (a) a CTL epitope peptide,
CTLェピトープペプチドを提示した抗原提示細胞、または前記 CTLェピトープぺプチ ドと複合体を形成する MHC分子からなる群から選択される少なくとも一つの成分と、（ b)被験者力 採取したリンパ球、培養中の CTL、とを接触させながら共培養した後、 前記リンパ球または CTL上に新たに発現する CD137分子を検出することにより免疫 応答能を判断することを特徴とする検査方法。 At least one component selected from the group consisting of an antigen-presenting cell presenting a CTL epitope peptide, or an MHC molecule that forms a complex with the CTL epitope peptide, and (b) subject lymphocytes collected, A test method characterized by determining the immune response ability by co-culturing with CTL in culture in contact with each other and then detecting CD137 molecule newly expressed on the lymphocyte or CTL.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010050212A1 (en) 2008-10-31 2010-05-06 株式会社ティーセルテクノロジーズ Kit for preparation of antigen-specific cytotoxic t lymphocyte
CN102618498A (en) * 2012-03-26 2012-08-01 时宏珍 Preparation method for HLA-A0201 limited antigen specificity CTL (cytotoxic T lymphocyte)
CN102719401A (en) * 2012-07-05 2012-10-10 时宏珍 Preparation method of HLA-A0201 (Human Leukocyte Antigen-A0201) restrictive anti-MAGE (MicroArray and Gene Expression) antigenic specificity CTL (Cytotoxic T Lymphocyte)
JP2013512694A (en) * 2009-12-08 2013-04-18 ウィルソン ウォルフ マニュファクチャリング コーポレイション Methods of culturing cells for adoptive cell therapy
US9567565B2 (en) 2009-12-08 2017-02-14 Juan F. Vera Methods of cell culture for adoptive cell therapy
CN113957049A (en) * 2021-11-08 2022-01-21 杭州鑫瑞精准生物科技有限公司 Human gamma delta T cell culture method

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CELIS E. ET AL.: "Induction of anti-tumor cytotoxic T lymphocytes in normal humans using primary cultures and synthetic peptide epitopes", PROC. NATL. ACAD. SCI. U.S.A, vol. 91, no. 6, 15 March 1994 (1994-03-15), pages 2105 - 2109, XP000578285 *
FUJIE Y. ET AL.: "Induction of antitumor cytotoxic T lymphocytes from the peripheral blood mononuclear cells of cancer patients using HLA-A2-restricted MAGE-3 peptide in vitro", CLIN. CANCER RES., vol. 3, no. 12, PART 1, December 1997 (1997-12-01), pages 2425 - 2430, XP003020283 *
HO W.Y. ET AL.: "In vitro methods for generating CD8+ T-cell clones for immunotherapy from the naive repertoire", J. IMMUNOL. METHODS, vol. 310, no. 1-2, 20 March 2006 (2006-03-20), pages 40 - 52, XP005334487 *
OSTANKOVITCH M. ET AL.: "Generation of Melan-A/MART-1-specific CD8+ cytotoxic T lymphocytes from human naive precursors: helper effect requirement for efficient primary cytotoxic T lymphocyte induction in vitro", INT. J. CANCER, vol. 72, no. 6, 17 September 1997 (1997-09-17), pages 987 - 994, XP003020281 *
PLATTS K.E. ET AL.: "Phenotypic and cell cycle analysis of human peripheral blood mononuclear cells activated with interleukin-2 and/or OKT3", EXP. CELL RES., vol. 208, no. 1, September 1993 (1993-09-01), pages 154 - 160, XP003020284 *
SHUFORD W.W. ET AL.: "4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses", J. EXP. MED., vol. 186, no. 1, 7 July 1997 (1997-07-07), pages 47 - 55, XP002110608 *
TAN J.T. ET AL.: "4-1BB costimulation is required for protective anti-viral immunity after peptide vaccination", J. IMMUNOL., vol. 164, no. 5, 1 March 2000 (2000-03-01), pages 2320 - 2325, XP003020282 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010050212A1 (en) 2008-10-31 2010-05-06 株式会社ティーセルテクノロジーズ Kit for preparation of antigen-specific cytotoxic t lymphocyte
JP2010130999A (en) * 2008-10-31 2010-06-17 T-Cell Technologies Inc Kit for preparation of antigen specific cytotoxic t cell
JP2013512694A (en) * 2009-12-08 2013-04-18 ウィルソン ウォルフ マニュファクチャリング コーポレイション Methods of culturing cells for adoptive cell therapy
JP2015133997A (en) * 2009-12-08 2015-07-27 ウィルソン ウォルフ マニュファクチャリング コーポレイションＷｉｌｓｏｎ Ｗｏｌｆ Ｍａｎｕｆａｃｔｕｒｉｎｇ Ｃｏｒｐｏｒａｔｉｏｎ Methods of cell culture for adoptive cell therapy
US9567565B2 (en) 2009-12-08 2017-02-14 Juan F. Vera Methods of cell culture for adoptive cell therapy
US10533156B2 (en) 2009-12-08 2020-01-14 Baylor College Of Medicine Methods of cell culture for adoptive cell therapy
US11268066B2 (en) 2009-12-08 2022-03-08 Wilson Wolf Manufacturing Methods of cell culture for adoptive cell therapy
US11999969B2 (en) 2009-12-08 2024-06-04 Wilson Wolf Manufacturing Methods of cell culture for adoptive cell therapy
CN102618498A (en) * 2012-03-26 2012-08-01 时宏珍 Preparation method for HLA-A0201 limited antigen specificity CTL (cytotoxic T lymphocyte)
CN102719401A (en) * 2012-07-05 2012-10-10 时宏珍 Preparation method of HLA-A0201 (Human Leukocyte Antigen-A0201) restrictive anti-MAGE (MicroArray and Gene Expression) antigenic specificity CTL (Cytotoxic T Lymphocyte)
CN113957049A (en) * 2021-11-08 2022-01-21 杭州鑫瑞精准生物科技有限公司 Human gamma delta T cell culture method

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