WO2008023786A1 - Procédé destiné à produire des lct virus-spécifiques - Google Patents
Procédé destiné à produire des lct virus-spécifiques Download PDFInfo
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- 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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- specific ctl
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Classifications
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- C—CHEMISTRY; METALLURGY
- 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
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
La présente invention a pour objet un système de culture fermé de LCT (lymphocytes cytotoxiques) virus-spécifiques qui présentent des caractéristiques de commodité et de sécurité et peut faire l'objet d'une utilisation économique pour l'application pratique de la thérapie cellulaire à l'aide de LCT spécifiques d'un virus (en particulier HCMV). Après mélange et incubation d'un peptide épitope LCT virus-spécifique et de cellules sanguines mononucléaires périphériques, une restimulation est exécutée avec ce peptide épitope dans un milieu donné. Ensuite, le LCT virus-spécifique est isolé par ciblage d'un antigène CD137 nouvellement exprimé, et des lymphocytes T résultant de la prolifération de cellules sanguines mononucléaires périphériques par stimulation avec OKT3 (anticorps monoclonal anti-CD3) sont mis en contact avec le peptide épitope dans le milieu. Ensuite, les lymphocytes T et les LCT virus-spécifiques sont mis en culture commune, ce qui permet la prolifération du LCT virus-spécifique.
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Cited By (6)
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WO2010050212A1 (fr) | 2008-10-31 | 2010-05-06 | 株式会社ティーセルテクノロジーズ | Trousse pour la préparation d'un lymphocyte t cytotoxique spécifique d'un antigène |
CN102618498A (zh) * | 2012-03-26 | 2012-08-01 | 时宏珍 | Hla-a0201限制性抗原特异性ctl制备方法 |
CN102719401A (zh) * | 2012-07-05 | 2012-10-10 | 时宏珍 | Hla-a0201限制性抗mage抗原特异性ctl的制备方法 |
JP2013512694A (ja) * | 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 | 養子細胞療法のための細胞を培養する方法 |
US9567565B2 (en) | 2009-12-08 | 2017-02-14 | Juan F. Vera | Methods of cell culture for adoptive cell therapy |
CN113957049A (zh) * | 2021-11-08 | 2022-01-21 | 杭州鑫瑞精准生物科技有限公司 | 一种人类γδT细胞培养方法 |
-
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Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2010050212A1 (fr) | 2008-10-31 | 2010-05-06 | 株式会社ティーセルテクノロジーズ | Trousse pour la préparation d'un lymphocyte t cytotoxique spécifique d'un antigène |
JP2010130999A (ja) * | 2008-10-31 | 2010-06-17 | T-Cell Technologies Inc | 抗原特異的細胞傷害性t細胞の調製キット |
JP2013512694A (ja) * | 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 | 養子細胞療法のための細胞を培養する方法 |
JP2015133997A (ja) * | 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 | 養子細胞療法のための細胞を培養する方法 |
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 (zh) * | 2012-03-26 | 2012-08-01 | 时宏珍 | Hla-a0201限制性抗原特异性ctl制备方法 |
CN102719401A (zh) * | 2012-07-05 | 2012-10-10 | 时宏珍 | Hla-a0201限制性抗mage抗原特异性ctl的制备方法 |
CN113957049A (zh) * | 2021-11-08 | 2022-01-21 | 杭州鑫瑞精准生物科技有限公司 | 一种人类γδT细胞培养方法 |
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