WO2015099134A1 - 再構成されたt細胞レセプター遺伝子を有する多能性幹細胞由来のt前駆細胞を用いる免疫細胞療法 - Google Patents
再構成されたt細胞レセプター遺伝子を有する多能性幹細胞由来のt前駆細胞を用いる免疫細胞療法 Download PDFInfo
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Definitions
- This application relates to immune cell therapy. Specifically, the present invention relates to immune cell therapy using pluripotent stem cell-derived T precursor cells having a reconstituted T cell receptor gene. The present application also relates to a method of inducing T progenitor cells from pluripotent stem cells for use in immune cell therapy.
- T-iPS cells cancer antigen-specific cytotoxic T cells
- T-iPS cells cancer antigen-specific cytotoxic T cells
- the advantages of this method are: i) All T cells made from T-iPS cells are specific for the original antigen. ii) T cells made from T-iPS cells are young and active. iii) By amplifying at the stage of iPS cells, it is possible to produce infinite desired T cells.
- T-iPS cells inherit the gene structure of the original cancer antigen-specific cytotoxic T cell in which the T cell receptor (hereinafter abbreviated as “TCR”) gene is reconstituted, and are derived from such T-iPS cells.
- TCR T cell receptor
- the specificity for this cancer antigen is inherited by T cells.
- T cells to be transferred have already been passaged for several generations, and the activity of killing cancer cells is reduced.
- the method using T-iPS cells makes it possible to provide a large amount of young and active regenerative T cells.
- T cells derived from the above-described T-iPS cells still has room for improvement. This is the following point. i) When T cells are induced from T-iPS cells, there is a possibility that TCR ⁇ -chain gene secondary reconstitution occurs in the induction process, resulting in T cells with specificity different from the original antigen specificity. is there. Such T cells may contain dangerous self-reactive T cells. ii) It is not known whether T cells regenerated in vitro are as functionally sound as naive T cells that are normally produced (in the thymus). iii) The cost is high because it takes time and effort to generate a large amount of T cells necessary for treatment of a single patient.
- iv) i an unexpected TCR chain change described that self-reactive T cells can be formed, but the original TCR can still be dangerously reactive.
- the original T cells of T-iPS cells were cells that were present without negative selection of the thymus and peripheral tolerance in the periphery, and thus were self-reactive among those who had the original T cells. It can be inferred that there is no. However, it may be a cell that survived accidentally despite being self-reactive.
- the possibility of having reactivity to attack normal tissues is further increased.
- T progenitor cells capable of differentiating into T cells
- Differentiation into T cells in the thymus of patients undergoing cell therapy eliminates regenerative T cells if they are reactive to components of the patient's body, ensuring safety Is done.
- the regenerated T cells have high quality, and a large amount of good quality T cells are produced from a small amount of progenitor cells, so that the cost can be suppressed.
- T progenitor cells for immune cell therapy, characterized in that T progenitor cells are obtained by in vitro induction of pluripotent stem cells having a reconstituted T cell receptor gene.
- T precursor cells are CD34 + CD5 + CD4-CD8- cells, CD34 + CD38-CD45RA-CD10- cells, CD45RA + CD10 + CD7-CD5- cells, CD45RA + CD10 + CD7 + CD5- cells, CD45RA + CD10
- the method according to [1] which is selected from the group consisting of + CD7 + CD5 + cells, CD3-CD4 + CD8- cells and CD4 + CD8 + cells.
- the pluripotent stem cell having a reconstituted T cell receptor gene is an iPS cell derived from a human cytotoxic T cell.
- the human cytotoxic T cell is a cytotoxic T cell induced by stimulating human peripheral blood mononuclear cells with an antigen.
- the antigen is a cancer antigen and immune cell therapy is performed to treat a cancer patient.
- Immune cell therapy comprising obtaining T progenitor cells from pluripotent stem cells having a reconstituted T cell receptor gene and administering the T progenitor cells to a subject in need of treatment.
- T precursor cells are CD34 + CD5 + CD4-CD8- cells, CD34 + CD38-CD45RA-CD10- cells, CD45RA + CD10 + CD7-CD5- cells, CD45RA + CD10 + CD7 + CD5- cells, CD45RA + CD10
- the immune cell therapy according to [6] which is selected from the group consisting of + CD7 + CD5 + cells, CD3-CD4 + CD8- cells and CD4 + CD8 + cells.
- the immune cell therapy according to [6] or [7], wherein the pluripotent stem cell having a reconstituted T cell receptor gene is an iPS cell derived from a human cytotoxic T cell.
- mode of this application is shown.
- FIG. The figure which shows the antigen-specific killer activity of LMP2-specific CTL used for iPS cell induction
- CD8 positive cells obtained by co-culturing T progenitor cells with LMP2 antigen-specific TCR with human HLA-A2402-expressing thymus tissue in Example C were obtained in the presence of antigen-presenting cells and LMP2 peptide.
- FIG. 1 schematically illustrates an immune cell therapy, which is one embodiment of the present invention, in which T precursor cells specific for a cancer antigen are induced for cancer treatment and administered to a patient.
- iPS cells are induced by a known method from cytotoxic T cells specific to cancer antigens (hereinafter sometimes abbreviated as “CTL”).
- T precursor cells are induced in vitro from the obtained iPS cells (T-iPS cells).
- This T precursor cell is administered to a cancer patient.
- T progenitor cells administered to the patient migrate to the thymus, where they are induced into mature T cells (na ⁇ ve T cells).
- the resulting mature T cells maintain a reconstituted T cell receptor gene.
- Naive T cells become activated antigen-specific cytotoxic T cells by being stimulated with the cancer antigen used to induce cytotoxic T cells before inducing T-iPS cells. Attack specifically.
- T progenitor cell includes from the stage corresponding to the hematopoietic stem cell, which is the most undifferentiated cell among the hematopoietic cells, to the stage corresponding to the cell immediately before receiving the positive selection / negative selection. . T precursor cells will be specifically described.
- Hematopoietic stem cells in human bone marrow are generally identified as CD34 + CD38 ⁇ CD45RA ⁇ CD10 ⁇ cells (+ indicates that it is expressed on the cell surface, ⁇ indicates that it is not expressed).
- Differentiation into T cells first becomes CD45RA + CD10 + cells with limited differentiation to the TB-myeloid system, and then CD7 and CD5 are expressed in turn.
- progenitor cells are biased so that they can easily differentiate into T cells.
- the most undifferentiated progenitor cells in the thymus are CD34 + CD5 + CD4 ⁇ CD8 ⁇ cells, which are differentiated in the thymus once through the CD3 ⁇ CD4 + CD8 ⁇ cell stage and then become CD4 + CD8 + cells.
- T cell receptors are expressed, and after undergoing positive selection and negative selection, mature CD3 + CD4 + CD8 ⁇ cells or CD3 + CD4 ⁇ CD8 + cells. Therefore, the “T progenitor cells” defined in this application are from CD34 + CD38 ⁇ CD45RA ⁇ CD10 ⁇ cells to CD4 + CD8 + cells. T cell differentiation is described in Blood 111: 1318 (2008), Nature mm Immunology 11: 585 (2010) (these documents are incorporated herein by reference).
- pluripotent stem cells having a reconstituted T cell receptor gene are used and induced to differentiate into T precursor cells.
- inducing hematopoietic cells from pluripotent stem cells it does not necessarily accurately reproduce the differentiation process seen in normal bone marrow hematopoiesis. It is thought that there is. Therefore, even in the cells generated in the system for inducing differentiation from pluripotent stem cells, the cells corresponding to the respective differentiation stages from hematopoietic stem cells to CD4 + CD8 + cells are referred to as T precursor cells in the present specification and claims.
- CD34 + CD5 + CD4 ⁇ CD8 ⁇ cells CD34 + CD38 ⁇ CD45RA ⁇ CD10 ⁇ cells, CD45RA + CD10 + CD7 ⁇ CD5 ⁇ cells, CD45RA + CD10 + CD7 + CD5 ⁇ cells, CD45RA + CD10 + CD7 Examples include + CD5 + cells, CD3-CD4 + CD8- cells, and CD4 + CD8 + cells.
- CD45RA + CD10 + CD7 + CD5 + cells and the like may be used as preferred T precursor cells.
- a method for obtaining pluripotent stem cells having a reconstituted T cell receptor gene specific for a specific antigen for example, Vizcardo et al., Cell Stem Cell 12, 31-36 2013 (this document is incorporated herein by reference) Incorporated) is exemplified. Specifically, a method of obtaining iPS cells by obtaining cytotoxic T cells specific for a specific antigen and introducing an initialization factor into the cytotoxic T cells can be used.
- iPS cells Artificial pluripotent stem cells
- ES cells embryonic stem cells
- ES cells embryonic stem cells
- It is an artificial stem cell derived from a somatic cell having characteristics of, for example, pluripotency and proliferation ability by self-replication (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al (2007), Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol.
- the reprogramming factor is a gene specifically expressed in ES cells, its gene product or non-coding RNA, a gene that plays an important role in maintaining undifferentiation of ES cells, its gene product or non-coding RNA, or It may be constituted by a low molecular compound.
- genes included in the reprogramming factor include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15 -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, SV40 or Glis1 etc. are exemplified, and these reprogramming factors may be used alone or in combination.
- the reprogramming factors include histone deacetylase (HDAC) inhibitors (eg, small molecule inhibitors such as valproate (VPA), trichostatin A, sodium butyrate, MC 1293, M344, siRNA and shRNA against HDAC (eg , Nucleic acid expression inhibitors such as HDAC1 siRNA Smartpool (registered trademark) (Millipore), HuSH 29 mer shRNA Constructs against HDAC1 (OriGene)), MEK inhibitors (eg, PD184352, PD98059, U0126, SL327 and PD0325901) , Glycogenthsynthase kinase-3 inhibitors (for example, Bio and CHIR99021), DNA methyltransferase inhibitors (for example, 5-azacytidine), histone methyltransferase inhibitors (for example, small molecule inhibitors such as BIX-01294, Suv39hl, Suv39h2 , Nucleic acid
- the reprogramming factor may be introduced into a somatic cell by a technique such as lipofection, fusion with a cell membrane-permeable peptide (for example, HIV-derived TAT and polyarginine), or microinjection.
- a cell membrane-permeable peptide for example, HIV-derived TAT and polyarginine
- Virus vectors include retrovirus vectors, lentivirus vectors (cell, ⁇ 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007 ), Adenovirus vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, Sendai virus vectors (WO 2010/008054), and the like.
- artificial chromosome vectors examples include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC).
- HAC human artificial chromosomes
- YAC yeast artificial chromosomes
- BAC bacterial artificial chromosomes
- a plasmid a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008).
- the vector can contain regulatory sequences such as a promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site, etc. so that a nuclear reprogramming substance can be expressed.
- Selective marker sequences such as kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, thymidine kinase gene, diphtheria toxin gene, reporter gene sequences such as green fluorescent protein (GFP), ⁇ -glucuronidase (GUS), FLAG, etc. Can be included.
- the above vector has a LoxP sequence before and after the introduction of the gene into a somatic cell in order to excise the gene or promoter encoding the reprogramming factor and the gene encoding the reprogramming factor that binds to it. May be. (The literature listed in this paragraph is incorporated herein by reference)
- RNA it may be introduced into somatic cells by, for example, lipofection or microinjection, and RNA that incorporates 5-methylcytidine and pseudoouridine® (TriLink® Biotechnologies) is used to suppress degradation. (Warren L, (2010) Cell Stem Cell. 7: 618-630) (these documents are incorporated herein by reference).
- a culture solution for iPS cell induction for example, DMEM, DMEM / F12 or DME culture solution containing 10 to 15% FBS (fetal bovine serum) (these culture solutions include LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, ⁇ -mercaptoethanol, etc.) or a commercially available culture solution (for example, a culture solution for mouse ES cell culture (TX-WES culture solution, Thrombo X)) , Primate ES cell culture medium (primate ES / iPS cell culture medium, Reprocell), serum-free medium (mTeSR, Stemcell Technology).
- FBS fetal bovine serum
- the somatic cell and the reprogramming factor are contacted on DMEM or DMEM / F12 containing 10% FBS for about 4 to 7 days. Then, re-spread the cells on feeder cells (for example, mitomycin C-treated STO cells, SNL cells, etc.), and use bFGF-containing primate ES cell culture medium about 10 days after contact between the somatic cells and the reprogramming factor. Culturing and generating iPS-like colonies about 30 to about 45 days or more after the contact.
- feeder cells for example, mitomycin C-treated STO cells, SNL cells, etc.
- DMEM culture medium containing 10% FBS (including LIF, penicillin / streptomycin, etc.) on feeder cells eg, mitomycin C-treated STO cells, SNL cells, etc.
- feeder cells eg, mitomycin C-treated STO cells, SNL cells, etc.
- ES-like colonies can be formed after about 25 to about 30 days or more.
- somatic cells to be initialized themselves are used (Takahashi K, et al. (2009), PLoS One.
- iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237 -241 or WO2010 / 013845) (these documents are incorporated herein by reference).
- hypoxic conditions oxygen concentration of 0.1% or more and 15% or less
- Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237 -241 or WO2010 / 013845 these documents are incorporated herein by reference.
- the culture medium is exchanged with a fresh culture medium once a day from the second day onward.
- the number of somatic cells used for nuclear reprogramming is not limited, but may be in the range of about 5 ⁇ 10 3 to about 5 ⁇ 10 6 cells per 100 cm 2 of culture dish.
- IPS cells can be selected according to the shape of the formed colonies.
- a drug resistance gene that is expressed in conjunction with a gene that is expressed when somatic cells are initialized for example, Oct3 / 4, Nanog
- a culture solution containing the corresponding drug selection The established iPS cells can be selected by culturing with the culture medium.
- the marker gene is a fluorescent protein gene
- iPS cells are selected by observing with a fluorescence microscope, in the case of a luminescent enzyme gene, by adding a luminescent substrate, and in the case of a chromogenic enzyme gene, by adding a chromogenic substrate. You can also
- a known method may be used as a method of inducing cytotoxic T cells specific for a specific antigen.
- a cytotoxic T cell specific for a cancer antigen can be obtained by stimulating lymphocytes obtained from a human by a conventional method with a cancer antigen specific for the cancer to be treated.
- Cancer antigens have been identified for various cancers, and methods for inducing cytotoxic T cells using cancer antigens or epitope peptides thereof are well known.
- lymphocytes may be stimulated using cancer cells to be treated.
- a cytotoxic T cell specific for a cancer antigen specific for the cancer may be induced from a donor afflicted with the cancer to be treated.
- Induction of reprogramming factor for example, Yamanaka factor, into induced cytotoxic T cells to induce iPS cells.
- SV40 may be added in addition to the Yamanaka factor to increase the efficiency of initialization.
- Induced iPS cells have a reconstituted T cell receptor gene derived from cancer antigen-specific cytotoxic T cells.
- iPS cells having a reconstituted T cell receptor gene are referred to as T-iPS cells.
- T cells regenerated from T-iPS cells can be used for other patients, not the original patient.
- a cell bank of T-iPS cells obtained by inducing specific cancer antigen-specific cytotoxic T cells from lymphocytes collected in advance from a human having a specific HLA type and inducing iPS from such cells can be created.
- the donor of lymphocytes may be a patient but may be a healthy person.
- a project to construct a highly versatile iPS cell bank by using a human who has a homologous HLA haplotype as a donor CYRANOSKI, Nature vol. 488, 139 (2012) (this document) Are incorporated herein by reference).
- T-iPS cells are induced from such CTL cells to produce a T-iPS cell bank.
- T-iPS cell bank is produced from lymphocyte cells collected from the same donor.
- T-iPS cell bank donor HLA information and antigen information are registered for each cell line.
- T-iPS cell bank select appropriate T-iPS cells based on the HLA type of the subject patient and the type of cancer affected. Selected T-iPS cells are induced to differentiate into T progenitor cells and administered to the patient. Furthermore, if cells differentiated into T progenitor cells instead of T-iPS cells are cryopreserved and banked, more rapid treatment can be provided to the patient.
- the induced T progenitor cells are suspended in an appropriate medium such as physiological saline or PBS and administered to a target patient whose HLA matches a certain level or more.
- an appropriate medium such as physiological saline or PBS
- HLA haplotype homo the case where one HLA haplotype corresponds is illustrated.
- the “pluripotent stem cell having a reconstituted T cell receptor gene” is not limited to the iPS cell obtained by the above-described method, but has the ability to differentiate into a tissue or organ cell. Any pluripotent stem cell capable of proliferating indefinitely and having a reconstituted T cell receptor gene may be used.
- cells obtained by introducing a reconstituted T cell receptor gene into ES cells or iPS cells are exemplified.
- the introduction of the TCR gene into pluripotent stem cells may be performed by conventional methods, for example, according to the method described in Morgan RA et al, Science, 314: 126. 2006 (the text is incorporated herein by reference). Just do it.
- TCR genes specific for various antigens are known.
- EB virus-related antigen-specific TCR gene is disclosed in Jurgens et al, Journal of investigation, 26:22, 2006 (the text of which is incorporated herein by reference).
- the WT1 antigen-specific TCR gene is disclosed, for example, in Anticancer Research 32 (12); 5201-5209, 2012 (incorporated herein by reference).
- Pluripotent stem cells having a reconstituted T cell receptor gene are induced to differentiate into T progenitor cells.
- Examples of the method for inducing differentiation into T progenitor cells include the method described in Timmermans et al., Journal of Immunology, 2009, 182: 6879-6888 (this document is incorporated herein by reference).
- pluripotent stem cells are co-cultured with OP9 stromal cells, such as mouse OP9 stromal cell culture, to obtain blood cell progenitor cells, and the resulting blood cell progenitor cells are then co-cultured with OP9 stromal cells and DLL1 cells To do.
- T progenitor cells can be selected by confirming cell surface markers by conventional methods.
- T precursor cells CD34 + CD5 + CD4 ⁇ CD8 ⁇ cells, CD34 + CD38 ⁇ CD45RA ⁇ CD10 ⁇ cells, CD45RA + CD10 + CD7 ⁇ CD5 ⁇ cells, CD45RA + CD10 + CD7 + CD5 ⁇ cells, CD45RA + CD10 + CD7 + CD5 + cells, CD3 CD4 + CD8 ⁇ cells and CD4 + CD8 + cells.
- T progenitor cells are administered to the patient.
- T progenitor cells are administered to a target patient after suspending in an appropriate medium such as physiological saline or PBS.
- an appropriate medium such as physiological saline or PBS.
- routes of administration to the patient include intravenous injection and intrathymic injection.
- the number of cells to be administered is not particularly limited, and may be appropriately determined according to the patient's age, sex, height, weight, target disease, symptoms, and the like. If an adult male weighing approximately 70 kg is a cancer patient, 10 6 to 10 8 cells are given by intravenous injection of T precursor cells, and 10 5 to 10 7 cells are given by intrathymic administration. Examples are administering cells. The optimal number of cells to be administered may be appropriately determined by clinical trials.
- T progenitor cells administered to patients migrate to the thymus and are induced into mature T cells (na ⁇ ve T cells) in the thymus.
- the resulting mature T cells maintain a reconstituted T cell receptor gene.
- Naive T cells become activated antigen-specific cytotoxic T cells by stimulating with the cancer antigen used when inducing cytotoxic T cells prior to inducing T-iPS cells, resulting in cancer To attack specifically.
- a cancer antigen peptide may be administered to a patient.
- T precursor cells are administered to a patient
- T cells are produced in the patient's thymus.
- autoreactive T cells appear, they are eliminated by negative selection.
- Improving the quality of regenerative T cells Naive T cells produced in the thymus can be expected to be healthy with normal functions.
- Relatively few cells to be administered to patients It is known that one progenitor cell that migrates to the thymus can be expected to produce at least 10 6 immature T cells.
- immature T cells undergo positive and negative selection by the thymus, so that at most 5% can migrate to the periphery as naive T cells. Moreover, the number of T cells that can respond to a specific epitope is 1 / 10,000 or less. In contrast, when T-progenitor cells derived from T-iPS are administered, almost all T cells produced in the thymus can survive thymic selection. The reason is that these T cells already express TCR that survived thymic selection. The naive T cells thus produced maintain the antigen specificity of the original killer T cells. That is, one progenitor cell can produce 10 6 antigen-specific T cells. This means that the number of cells administered to the patient is much smaller, which reduces costs.
- OP9 cells and OP9 / DLL1 cells were obtained from RIKEN BioResource Center (Tsukuba, Ibaraki, Japan).
- Human iPS cells were prepared from cord blood hematopoietic progenitor cells at the RIKEN Center for Immunology and Allergy Science (Yokohama, Kanagawa, Japan).
- the human iPS cells used can also be prepared by the method described in (Vizcardo et al, Cell Stem Cell, 12: 31-36, 2013).
- the composition of each medium is as follows: *
- the penicillin / streptomycin solution composition is penicillin 10000 U / mL and streptomycin 10000 ⁇ g / mL, so the final concentrations are 100 U / mL and 100 ⁇ g / mL, respectively.
- penicillin / streptomycin solution is penicillin 10000 U / mL and streptomycin 10000 ⁇ g / mL, so the final concentrations are 100 U / mL and 100 ⁇ g / mL, respectively.
- the collagenase solution was aspirated and washed away with 10 mL of PBS ( ⁇ ). Thereafter, 5 mL of 0.05% trypsin / EDTA solution was added, followed by incubation at 37 ° C. for 20 minutes. After culturing, the cells peeled off in a film form, so they were physically made fine by pipetting to separate the adherent cells. 20 mL of new medium A was added thereto, and further cultured at 37 ° C. for 45 minutes.
- the supernatant containing the floating cells was collected through a 100 ⁇ m mesh. After centrifuging at 4 ° C. and 1200 rpm for 7 minutes, the pellet was suspended in 10 mL of medium B. Of these, 1/10 were seeded on newly prepared OP9 / DLL1 cells, especially for FACS analysis. Cells obtained from a plurality of dishes were pooled, redistributed to the same number as the original number, and reseeded.
- the cells were then seeded on OP9 / DLL1 cells.
- cell sorting of the CD34lowCD43 + cell fraction was not performed.
- this fraction is sorted, there are concerns that the number of cells obtained may be reduced and the cells may be damaged by sorting.
- the efficiency of inducing differentiation into T cells may be lower than when sorting is not performed.
- FACS analysis was performed to confirm the differentiation stage during the culture period, but many dead cells were observed during the culture in all periods. Therefore, at the time of FACS analysis, PI (Propidium Iodide), 7-AAD, etc. were used for analysis after removing dead cells.
- PI Propidium Iodide
- 7-AAD 7-AAD
- Day 23 Blood cell colonies started to appear. Cells that loosely adhered to OP9 / DLL1 cells were gently pipetted multiple times and collected through a 100 ⁇ m mesh into a 50 mL conical tube. Centrifugation was performed at 4 ° C. and 1200 rpm for 7 minutes, and the pellet was suspended in 10 mL of medium B.
- the cells loosely adhering to the OP9 / DLL1 cells were gently pipetted several times and collected into a 50 mL conical tube through a 100 ⁇ m mesh. After centrifuging at 4 ° C. and 1200 rpm for 7 minutes, the pellet was suspended in 10 mL of medium B. Of these, 1/10 were seeded on newly prepared OP9 / DLL1 cells, especially for FACS analysis.
- CD7 + cells which are T progenitor cells, were observed, and some cells had differentiated to the CD7 + CD5 + stage.
- FIG. 1 A diagram of the FACS analysis is shown in FIG.
- Example B Transplantation of iPS cell-derived T progenitor cells into immunodeficient mice Human T by transplanting iPS cell-derived T progenitor cells made from human umbilical cord blood CD34 + cells obtained in Example A into immunodeficient NOG mice Clarified that cells are created.
- Immunodeficient NOG mice were obtained from the Central Institute for Experimental Animals (Kawasaki City, Kanagawa Prefecture, Japan).
- T progenitor cells were isolated using a cell sorter, and 10 5 cells were intravenously injected into NOG mice.
- the administered T progenitor cells were induced into mature T cells in the thymus. At that time, autoreactive T cells were excluded by negative selection.
- Example C Organ culture in mouse thymus of T progenitor cells prepared from T-iPS cells T precursor cells prepared from iPS cells not subjected to gene rearrangement in Examples A and B were injected into mice, and T cells in mouse thymus Differentiated. The generated polyclonal T cells did not attack the mice, indicating that “negative selection” occurred.
- T-iPS cells were obtained from antigen-specific T cells, and T-progenitor cells prepared from T-iPS cells were cultured in the thymus of mice into which human HLA had been introduced.
- a schematic diagram of Example C is shown in FIG.
- LMP2-T-iPS cells were induced from killer T cells expressing HLA-A2402-restricted LMP2 antigen-specific TCR.
- EB virus is a virus that causes infectious mononucleosis in the acute phase and sometimes causes cancer such as Burkitt lymphoma.
- T cells are provided by healthy individuals who have a history of infection with EB virus.
- the donor is a so-called EB virus carrier because the virus remains in the lymphocytes for a lifetime after infection. Therefore, this provider can be regarded as a chronic virus infected person although it does not develop.
- LMP2 antigen-specific cytotoxic T lymphocytes i) The following medium was used.
- Dendritic cell culture medium CellGro (CellGenix) T cell medium: ii) The sequence of LMP2 is: LMP2 antigen peptide: TYGPVFMSL LMP2 tetramer was purchased from MBL.
- iii) Antigen-presenting cells As antigen-presenting cells, a Lymphoblastoid cell line (LCL) having HLA-A2402 established from healthy volunteers at Kyoto University Hospital Hematology Tumors Norimitsu Kadowaki Laboratory was used.
- MoDC monocyte-derived dendritic cells
- Monocytes were isolated using CD14 microbeads from the peripheral blood of healthy volunteers with HLA-A2402 who were also infected with EB virus. After washing, dendritic cell culture medium was added to adjust to 5 ⁇ 10 5 / mL. 2.
- Cytokines were added to final concentrations of GM-CSF 800 U / mL (or 50 ng / mL) and IL-4 200 U / mL (or 40 ng / mL). The cells were seeded on a 6-well plate at 5 mL / well. Incubated at 37 ° C. with 5% CO 2 . 3.
- GM-CSF was added to fresh dendritic cell medium to a concentration of 800 U / mL and IL-4 to a concentration of 200 U / mL. 5. 3 mL of new dendritic cell culture medium was added to each well. 6. On Day 6, immature MoDCs are recovered from the plate and suspended in a small amount of fresh dendritic cell medium. The cell concentration was adjusted to 5 ⁇ 10 5 / mL. 7.
- GM-CSF (hereinafter, final concentration: 800 U / mL), IL-4 (200 U / mL), TNF- ⁇ (10 ng / mL), PGE 2 (1 ⁇ g / mL), 24 holes cells were seeded at approximately 5 X 10 5/1 mL / well to the plate. 8. The cells were cultured at 37 ° C and 5% CO 2 for 24 hours. 9. Peptide was added during the last 2 hours of the culture. The final concentration of peptide was 10 ⁇ M. 10. Dendritic cells (DC) were collected and washed twice with T cell medium. The number of DC cells was counted and adjusted to 2 ⁇ 10 5 / mL with T cell medium.
- DC Dendritic cells
- LCL LCL was collected from the culture and irradiated with 35 Gy. 2. Suspended in T cell medium and adjusted to 5 ⁇ 10 5 / mL. 3. Peptide was added at 100 nM and cultured for 2 hours. 4. LCL was collected, washed with T cell medium, and adjusted to 2 ⁇ 10 5 / mL.
- T cells stimulated with LCL and dendritic cells were suspended in T cell medium and suspended at a concentration of 2 ⁇ 10 6 cells / mL. 2.
- IL-7 final concentration 5 ng / mL
- IL-15 final concentration 1 ng / mL
- First course of peptide stimulation with LCL 4.
- LCL was further cultured for 2 hours in a medium supplemented with 100 nM peptide, and CTL was added thereto. 5.
- IL-7 final concentration 5 ng / mL
- IL-15 final concentration 1 ng / mL
- LMP2-specific killer T cells CD8 positive LMP-2 tetramer positive cells
- OUN-1 leukemia cell lines proliferated by peptide stimulation on 96-well U-bottom plates 0: 1, 1: 9, 1: 3, respectively.
- 1: 1, and 3 1, and the ratio of dead cells to target cells in the presence or absence of peptide was determined by the ratio of Annexin V and PI (Propidium Iodide) found in the CFSE positive fraction .
- Annexin V and PI Propidium Iodide
- LMP2-T-iPS cells A. Activation of LMP2-specific CTL 1. CD8 positive cells were concentrated with MACS beads. 2. All cells were suspended in T cell medium, and IL-7 (final concentration 5 ng / mL) and IL-15 (final concentration 10 ng / mL) were added. Furthermore, Dynabeads Human T-Activator CD3 / CD28 was added so that T cell: beads became 1: 1, and CD8 positive cells were activated by culturing for 2 days.
- mice Organ culture of T progenitor cells in mouse thymus Organ culture in mouse thymus was performed according to the description of Kawamoto et al, Inetrnatinal Immunology, 9: 1011, 1997 (this document constitutes a part of this application by reference).
- Mice CB6F1-Tg (HLA-A * 2402 / H2-Kb) A24.01) in which the HLA-A2402 gene was introduced into C57BL6 mice were purchased from Taconic Biosciences, Inc. and used. Normal C57BL6 mice were used as control mice.
- HLA-2402 transgenic mice or control mice were mated with Rag2KO mice.
- the fetus was removed on the 15th day of gestation, and the thymus was obtained from the fetus.
- the fetal thymus was cultured for 6 days on a filter suspended on RPMI1640 medium containing deoxyguanosine (dGuo). This treatment kills thymocytes and only the cells on the thymic environment side survive.
- dGuo deoxyguanosine
- the obtained dGuo-treated fetal thymus was put into a 96-well V bottom plate one by one, 0.2 ml of RPMI1640 medium was added to each, and DP cells purified with Max beads were added thereto. After the plate is placed in a plastic bag and sealed, the gas in the bag is replaced with a mixed gas of 5% CO 2 , 25% N 2 , and 70% O 2 , and the bag is placed in an incubator at 37 ° C. DP cells and dGuo-treated thymus tissue were co-cultured under oxygen conditions. On day 7 of the culture, the culture was taken out, crushed by being sandwiched between glass slides, and the crushed material was suspended in the medium, whereby T cells obtained as floating cells were collected and analyzed.
- T cells were subjected to flow cytometry analysis.
- the transferred cells remained at the DP cell stage.
- mature T cells were generated in co-culture with human HLA-2402 expressing thymus. Almost all T cells generated were positive for LMP2 tetramer (ie, the original TCR was released) (FIG. 6).
- Lymphoblastoid cell line collected from healthy volunteers with HLA-A2402, transferred from Kyoto University Hospital Hematology Tumors Norikami Kadowaki Laboratory (Kyoto, Japan) as antigen-presenting cells B cell blast-like cell line) was used.
- target cells THP1 cells (acute monocytic leukemia cell line) transferred from Kyoto University Hospital Hematology Tumors Norimitsu Kadowaki Laboratory (Kyoto, Japan) were used.
- LMP2 antigen peptide TYGPVFMSL (419-427)
- Medium ⁇ MEM medium containing 20% fetal calf serum (life technologies, cat # 11900-073)
- the generated CD8-positive cells were isolated and cultured for 13 days in the presence of antigen-presenting cells and LMP2 peptide.
- the THP1 leukemia cell line used as target cells was labeled with CFSE.
- the above cells (killer T cells) and THP1 cells are mixed in a 96-well U-bottom plate at 0: 1, 1: 3, 1: 1, 3: 1, and 9: 1, respectively, in the presence or absence of peptides.
- the cells were cultured for 4 hours, and the dead cell ratio of the target cells was assayed by the ratio of Annexin V positive cells found in the CFSE positive fraction (FIG. 7).
- the target cells When the peptide was not added, the target cells were hardly killed, but as the amount of peptide added increased, the target cells were killed more efficiently. The killing ability was not inferior to that of the original killer T cells (FIG. 5). That is, sufficiently strong antigen-specific cytotoxic activity was confirmed.
- Example C shows that T-iPS cell-derived progenitor cells can differentiate normally in the thymic environment where HLA molecules are present, and antigen-specific CTLs are induced by antigen stimulation.
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Abstract
Description
i)T-iPS細胞からつくられるT細胞はすべて元の抗原に特異的である。
ii)T-iPS細胞からつくられるT細胞は若く活性がある。
iii)iPS細胞の段階で増幅させることによって、所望のT細胞を無限につくることができる。
従来から行われているin vitroで増幅させたT細胞を用いる細胞移入治療では、移入するT細胞がすでに何代か継代されたものであり、がん細胞を殺す活性が低下していることが問題とされていたが、T-iPS細胞を用いる方法により、若く活性のある再生T細胞を大量に提供することが可能となる。
i)T-iPS細胞よりT細胞を誘導すると、誘導の過程でTCRα鎖遺伝子の二次的な再構成が起こって、元の抗原特異性とは異なる特異性を示すT細胞が生じる可能性がある。そのようなT細胞の中には危険な自己反応性T細胞が含まれている可能性もある。
ii)in vitroで再生させたT細胞は、正常に(胸腺で)つくられるナイーブT細胞ほど機能的に健全であるかどうか分からない。
iii)一人の患者の治療に必要な大量のT細胞をつくるために手間がかかるためにコストが高い。
iv)i)では不測のTCR鎖の入れ替えによって自己反応性T細胞ができると述べたが、元のTCRのままでも危険な反応性を示すことはありうる。T-iPS細胞の元のT細胞は胸腺の負の選択や末梢での末梢性寛容を受けずに存在していた細胞であるからその元のT細胞を持っていた人の中では自己反応性は無いと類推できる。しかし、自己反応性であるにかかわらず偶然生き延びていた細胞である可能性もある。また、再生T細胞を他人に投与するような戦略で使う場合は、正常組織を攻撃する反応性を有している可能性はさらに高くなる。
[1] 再構成されたT細胞レセプター遺伝子を有する多能性幹細胞をin vitroで誘導してT前駆細胞を得ることを特徴とする、免疫細胞療法用T前駆細胞を得る方法。
[2] T前駆細胞がCD34+CD5+CD4-CD8-細胞、CD34+CD38-CD45RA-CD10-細胞、CD45RA+CD10+CD7-CD5-細胞、CD45RA+CD10+CD7+CD5-細胞、CD45RA+CD10+CD7+CD5+細胞、CD3-CD4+CD8-細胞およびCD4+CD8+細胞からなる群から選択される、[1]記載の方法。
[3] 再構成されたT細胞レセプター遺伝子を有する多能性幹細胞が、ヒト細胞傷害性T細胞から誘導されたiPS細胞である、[1]または[2]記載の方法。
[4] ヒト細胞傷害性T細胞が、ヒト末梢血単核球を抗原により刺激して誘導された細胞傷害性T細胞である、[3]記載の方法。
[5] 抗原ががん抗原であり、免疫細胞療法ががん患者を治療するために行われる、[4]記載の方法。
[6] 再構成されたT細胞レセプター遺伝子を有する多能性幹細胞からT前駆細胞を得、治療を必要とする対象へ当該T前駆細胞を投与することを含む、免疫細胞療法。
[7] T前駆細胞がCD34+CD5+CD4-CD8-細胞、CD34+CD38-CD45RA-CD10-細胞、CD45RA+CD10+CD7-CD5-細胞、CD45RA+CD10+CD7+CD5-細胞、CD45RA+CD10+CD7+CD5+細胞、CD3-CD4+CD8-細胞およびCD4+CD8+細胞からなる群から選択される、[6]記載の免疫細胞療法。
[8] 再構成されたT細胞レセプター遺伝子を有する多能性幹細胞が、ヒト細胞傷害性T細胞から誘導されたiPS細胞である、[6]または[7]記載の免疫細胞療法。
[9] ヒト細胞傷害性T細胞が、ヒト末梢血単核球を抗原により刺激して誘導された細胞傷害性T細胞である、[8]記載の免疫細胞療法。
[10] 抗原ががん抗原であり、治療を必要とする対象が、がんを患っている患者である、[9]に記載の免疫細胞方法。
患者へ投与されたT前駆細胞は胸腺に移住し、胸腺にて成熟T細胞(ナイーブT細胞)へと誘導される。得られる成熟T細胞は再構成されたT細胞レセプター遺伝子を維持している。ナイーブT細胞はT-iPS細胞を誘導する前に細胞傷害性T細胞を誘導した際に用いたがん抗原で刺激されることによって、活性化された抗原特異的細胞傷害性T細胞となり、がんを特異的に攻撃する。
上記培養の間には、培養開始2日目以降から毎日1回新鮮な培養液と培養液交換を行う。また、核初期化に使用する体細胞の細胞数は、限定されないが、培養ディッシュ100cm2あたり約5×103~約5×106細胞の範囲であってもよい。
頻度の高いHLAハプロタイプをホモで有するヒトをドナーとして用いることにより、汎用性の高いiPS細胞バンクを構築するプロジェクトが日本において行われている(CYRANOSKI, Nature vol. 488, 139(2012) (この文献は引用により本願に組み込まれる))。同様のドナーから採取したリンパ球細胞から、治療対象とするがんに特異的ながん抗原を用いてCTL細胞を誘導し、かかるCTL細胞からT-iPS細胞を誘導してT-iPS細胞バンクを作製すると、汎用性の高いT-iPS細胞バンクとなる。
T-iPS細胞バンクを利用する場合、対象とする患者のHLA型ならびに罹患しているがんの種類に基づき、適切なT-iPS細胞を選択する。選ばれたT-iPS細胞をT前駆細胞ヘ分化誘導し、患者へ投与する。また、T-iPS細胞ではなくT前駆細胞へ分化誘導させた細胞を凍結保存してバンク化しておけば、患者に対してより迅速な治療を提供することができる。
引用した文献はES細胞より成熟T細胞を誘導する方法を説明するものであるが、本願の方法に用いるために、成熟T細胞となる前のT前駆細胞を取り出して用いることができる。T前駆細胞は、常法により細胞表面のマーカーを確認して選別することができる。例えば、下記細胞をT前駆細胞として用いることができる:
CD34+CD5+CD4-CD8-細胞、CD34+CD38-CD45RA-CD10-細胞、CD45RA+CD10+CD7-CD5-細胞、CD45RA+CD10+CD7+CD5-細胞、CD45RA+CD10+CD7+CD5+細胞、CD3-CD4+CD8-細胞およびCD4+CD8+細胞。
i)安全性の保障
T前駆細胞を患者に投与した場合、T細胞は患者の胸腺中でつくられる。胸腺では、もし自己反応性T細胞が出現しても負の選択で排除される。
ii)再生T細胞の品質の改良
胸腺中でつくられたナイーブT細胞は、正常な機能を持つ健全なものであると期待できる。
iii)患者へ投与すべき細胞数が比較的少ない
胸腺へ移住する1個の前駆細胞は、少なくとも106個の未成熟T細胞をつくり出すことが期待できることが知られている。正常には、未成熟T細胞は胸腺による正負の選択を経るので、ナイーブT細胞として末梢に移行できるのはせいぜい5%である。しかも、特定のエピトープに反応できるT細胞は、1/10,000以下である。これに対して、T-iPSから誘導されたT前駆細胞を投与した場合、胸腺でつくられるほとんどすべてのT細胞は胸腺による選択を生き延びることができる。その理由は、これらのT細胞は、すでに胸腺の選択を生き延びたTCRを発現しているからである。そしてこのようにつくられたナイーブT細胞は、元のキラーT細胞の抗原特異性を維持している事である。すなわち、1個の前駆細胞は106個の抗原特異的T細胞をつくり出すことができる。ということは、患者に投与する細胞数は格段に少なくてすみ、コスト削減になる。
iPS細胞からT前駆細胞の誘導
材料 OP9細胞、OP9/DLL1細胞はいずれも理研バイオリソースセンター(日本国茨城県つくば市)より入手した。
ヒトiPS細胞は理研免疫・アレルギー科学総合研究センター(日本国神奈川県横浜市)にて臍帯血造血前駆細胞から作製したものを用いた。なお、使用したヒトiPS細胞は(Vizcardo et al, Cell Stem Cell, 12:31-36, 2013)に記載の方法で作製することもできる。
100mmディッシュ(Falcon)へ6mLの0.1%ゼラチンを投入し、37℃にて30分間インキュベートした後、ゼラチン溶液を捨て、medium Aを10mL投入した。
コンフルエントなOP9培養細胞よりディッシュへ細胞を播種した。
4日後に10mLのmedium Aを投入した(最終量20mL)。
Day 0 (iPS細胞とOP9細胞の共培養開始)
OP9細胞培養物の培地を新しいmedium Aに交換した。
100mmディッシュ(Falcon)中で培養したコンフルエントなヒトiPS細胞培養物より培地を除き、新しいmedium A10mLを添加した。
ディッシュの底に塊状に張り付いて生育しているヒトiPS細胞をEZ-passageローラーで剥がした。得られたiPS細胞塊をピペッティングすることで分散させ、培地中に浮遊させた。目視でおよそ600個のiPS細胞塊(およそ1×106細胞)をOP9細胞上に播種した。
培地を新しいmedium A 20mLに交換した。
半量分の培地を新しいmedium A 10mLに交換した。
半量分の培地を新しいmedium A 10mLに交換した。
Day 13 (13日経過後) ここまでの培養で、iPS細胞は中胚葉細胞へと分化している。誘導された中胚葉細胞を、OP9細胞上から下記手順にてOP9/DLL1細胞上へ移しかえた。
培地を吸引し、HBSS(+Mg+Ca)で細胞表面上の培地を洗い流した。
その後250U collagenase IV/HBSS(+Mg+Ca)溶液10mLを加え、37℃で45分間培養した。
OP9細胞に緩く接着している細胞を、穏やかに複数回ピペッティングし、100μmのメッシュを通して50mLコニカルチューブに回収した。4℃、1200rpmで7分間遠心し、ペレットを10mLのmedium Bに懸濁させた。これらの細胞を新たに用意したOP9/DLL1細胞上に播種した。
OP9/DLL1細胞に緩く接着している細胞を、穏やかに複数回ピペッティングし、100μmのメッシュを通して50mLコニカルチューブに回収した。4℃、1200rpmで7分間遠心し、ペレットを10mLのmedium Bに懸濁させた。
OP9/DLL1細胞は底面に接着し、密集しているためグレーに見えた。T前駆細胞はOP9上に緩く結合して増えるため、OP9に対して色が明るくみえた。ただし細胞が凝集したようなはっきりとしたコロニーとして見えるわけではなく、丸く明るい粒のようなものが複数個集まっていた。
iPS細胞由来T前駆細胞の免疫不全マウスへの移植
実施例Aで得たヒト臍帯血中のCD34+細胞からつくったiPS細胞由来のT前駆細胞を、免疫不全のNOGマウスに移植することでヒトT細胞がつくられることを明らかにした。
免疫不全NOGマウスは公益財団法人実験動物中央研究所(日本国神奈川県川崎市)より入手した。
セルソーターを用いてCD7+T前駆細胞を分離し、105個をNOGマウスに静注した。
移植8週間後にマウスを殺し、解析した。成熟型のCD4+T細胞とCD8+T細胞が胸腺と脾臓中に検出された。
レシピエントマウスでは、GVH反応はみられなかった。ヒトの抹消血T細胞をマウスに移植するとマウスは死亡することが知られているので、何の症状も出なかったということは、レシピエントマウスの体の分子に反応性のあるT細胞は負の選択によって排除されていることを意味するものであった。本試験の手順と結果を図3にまとめた。
T-iPS細胞から作製したT前駆細胞のマウス胸腺における器官培養
実施例AおよびBでは遺伝子再構成をしていないiPS細胞から作製したT前駆細胞をマウスに注射し、マウス胸腺の中でT細胞を分化させた。生成したポリクローナルなT細胞はマウスを攻撃することがなかったことから、「負の選択」が起こった事を示した。
本実施例においては抗原特異的T細胞からT-iPS細胞を得、T-iPS細胞から作製したT前駆細胞をヒトのHLAが遺伝子導入されているマウスの胸腺にて、器官培養を行った。実施例Cの概略図を図4に示す。
HLA-A2402拘束性のLMP2抗原特異的なTCRを発現するキラーT細胞からiPS細胞(LMP2-T-iPS細胞)を誘導した。
i)以下の培地を用いた。
樹状細胞用培地: CellGro (CellGenix)
T細胞用培地:
LMP2抗原ペプチド:TYGPVFMSL
LMP2テトラマーはMBLより購入した。
iii) 抗原提示細胞
抗原提示細胞としては京都大学病院血液腫瘍内科門脇則光研究室にて健常人ボランティアから樹立されたHLA-A2402を有するLymphoblastoid cell line (LCL)を用いた。
1. HLA-A2402を有しかつEBウイルス既感染者でもある健常人ボランティアの末梢血よりCD14 microbeadsを用いて単球を単離した。洗浄後、樹状細胞用培地を加え、5 x 105/mLに調整した。
2. 最終濃度GM-CSF 800 U/mL (or 50 ng/mL)、IL-4 200 U/mL (or 40 ng/mL)になるようサイトカインを加えた。6-well plateに5 mL/wellで播いた。37℃、5% CO2でインキュベートした。
3. インキュベートを3日間 (以下”Day 3”様に記載する) した後に、培養上清を2.5 mL/wellずつ、静かに取って捨てた。
4. 新しい樹状細胞用培地にGM-CSFを800 U/mL、IL-4を200 U/mLの濃度になるように加えた。
5. 各wellに3 mLずつ、新しい樹状細胞用培地を加えた。
6. Day 6に未成熟MoDCをプレートから回収し、少量の新しい樹状細胞用培地に浮遊させ。5 X 105/mLになるように細胞濃度を調整した。
7. GM-CSF (以下、最終濃度: 800 U/mL)、IL-4 (200 U/mL)、TNF-α (10 ng/mL)、PGE2 (1 μg/mL)を加え、24穴プレートに約5 X 105/1 mL/wellで細胞を播種した。
8. 37℃、5% CO2で24時間培養した。
9. 上記培養の最後の2時間にペプチドを加えた。ペプチドの最終濃度は10μMとした。
10. 樹状細胞(DC)を回収し、T細胞用培地で2回洗浄した。DCの細胞数を数え、T細胞用培地で2 X 105/mLに調製した。
1. Aと同一の健常人ボランティアの末梢血より、CD3 microbeadsを用いてMACSにてT細胞を単離した。洗浄後、T細胞用培地を加え、2 x 106/mLに調整した。この時一部の細胞をフローサイトメトリー解析のために取り分けた。
2. 24穴プレートに、DC浮遊液(2 X 105/mL)を0.5mL/well、T細胞浮遊液(2 X 105/mL)を0.5mL/wellとなるように加えた。(DC: T = 1 X 105/well: 1 X 106/well = 1:10)
3. 3日目にIL-7(最終濃度5 ng/mL)、IL-15(最終濃度 10 ng/mL)を加えた。
4. 14日目に細胞を回収した。
1. LCLを培養中から回収し、35Gyの放射線照射を行った。
2. T細胞用培地に浮遊させ、5 X 105/mLとなるように調整した。
3. ペプチドを100nMで添加し2時間培養した。
4. LCLを回収し、T細胞用培地で洗浄後、2 X 105/mLとなるように調整した。
1. 樹状細胞で刺激したT細胞をT細胞用培地に浮遊させ、2 X 106 cells/mLの濃度に懸濁した。
2. 24穴プレートに、ペプチドを加えて培養したLCL浮遊液(2 X 105/mL)を0.5mL/well、T細胞浮遊液(2 X 105/mL)を0.5mL/wellとなるように加えた(LCL: T = 1 X 105/well: 1 X 106/well = 1:10)。同時にペプチドを100nMとなるように添加した。
3. 3日目にIL-7(最終濃度5 ng/mL)、IL-15(最終濃度 1 ng/mL)を加えた。1週間ごとにサイトカインを添加したT細胞用培地で培地交換しながら2週間培養を行った。(LCLによるペプチド刺激1クール目)
4. 再度100nMのペプチドと加えた培地でLCLを2時間培養し、ここに CTLを加えた。
5. 3日目にIL-7(最終濃度5 ng/mL)とIL-15(最終濃度 1 ng/mL)を加えた。1週間ごとにサイトカインを添加したT細胞用培地で培地交換しながら2週間培養を行った。(LCLによるペプチド刺激2クール目)
4. フローサイトメトリー解析により、CD8陽性T細胞中にCD8陽性LMP-2テトラマー陽性細胞が80%以上の割合で検出されることを確認した。
1. 標的細胞として用いるOUN-1白血病細胞株をCFSEラベルし、T細胞用培地に懸濁後、LMP2ペプチド1nM存在下で2時間培養を行った。
2. 96穴U底プレートに、ペプチド刺激によって増殖したLMP2特異的キラーT細胞(CD8陽性LMP-2テトラマー陽性細胞)とOUN-1白血病細胞株をそれぞれ0:1、1:9、1:3、1:1、3:1となるように混合し、ペプチド存在下もしくは非存在下において標的細胞の死細胞比率をCFSE陽性分画中に見られるAnnexinVとPI(Propidium Iodide)の比率によって検定した。図5に示す。
3. LMP2特異的キラーT細胞は標的細胞に対し、抗原特異的キラー活性を示すことを確認した。
A. LMP2特異的CTLの活性化
1. MACS beadsによりCD8陽性細胞を濃縮した。
2. 全ての細胞をT細胞用培地に浮遊させ、IL-7(最終濃度5 ng/mL)、IL-15(最終濃度 10 ng/mL)を加えた。さらにDynabeads Human T-Activator CD3/CD28をT細胞:beadsが1:1となるように添加し、2日間培養することでCD8陽性細胞を活性化した。
1. 活性化させたLMP2特異的CTLをT細胞用培地に浮遊させ、山中4因子とSV40が組み込まれたセンダイウィルスを培地中に添加し、そのまま2日間培養した。
2. T細胞用培地で洗浄し、さらにIL-7(最終濃度5 ng/mL)、IL-15(最終濃度 1ng/mL)を加えたT細胞用培地で2日間培養した。
3. 全ての細胞を回収後、IL-7(最終濃度5 ng/mL)、IL-15(最終濃度 1ng/mL)を添加したT細胞用培地でT細胞を懸濁し、フィーダー細胞上に播種した。
4. 2日目にiPS細胞用培地にて半量交換し、翌日から毎日iPS細胞用培地への半量交換を行い続け、培養を続けた。
1. 3週間後にiPS細胞コロニーを目視により確認した。
2. 200ulチップによりコロニーを物理的に拾い上げた。
3. 各クローンを個別に樹立し、そのうちのひとつをLMP2-T-iPS細胞として以下の実験に用いた。
LMP2-T-iPS細胞を実施例Aの手順にしたがって処理し、培養35-40日間にCD4CD8共陽性細胞(double positive: DP)を得た。
マウス胸腺における器官培養はKawamoto et al, Inetrnatinal Immunology, 9:1011, 1997(本文献は引用により本願の一部を構成する)の記載に従って行った。
C57BL6マウスにHLA-A2402遺伝子を導入したマウス(CB6F1-Tg(HLA-A*2402/H2-Kb)A24.01)をTaconic Biosciences, Inc.から購入して用いた。コントロールマウスには正常なC57BL6マウスを用いた。
免疫不全のレシピエントマウスを得るためHLA-2402遺伝子導入マウスまたはコントロールマウスとRag2KOマウスを交配した。交配により妊娠したマウスから、胎齢15日目に胎仔を取り出し、その胎仔から胸腺を得た。胎仔胸腺を6日間デオキシグアノシン(dGuo)を含むRPMI1640培地上に浮遊させたフィルターの上に置いて培養した。この処理により胸腺細胞は死滅し、胸腺環境側の細胞だけが生き残る。
培養7日目に培養物を取り出し、スライドガラスの間に挟んですりつぶして破砕し、破砕したものを培地中へ浮遊させることによって、浮遊細胞として得られるT細胞を回収、解析した。
得られたT細胞をフローサイトメトリー解析に供した。コントロールのマウス胸腺との共培養では移入した細胞はDP細胞段階にとどまっていた。一方ヒトHLA-2402発現胸腺との共培養では、成熟T細胞が生成した。生成したT細胞はほぼすべてLMP2テトラマー陽性(すなわち、元のTCRを出していた)であった(図6)。
抗原提示細胞として京都大学病院血液腫瘍内科門脇則光研究室(日本国京都府京都市)より譲渡された、HLA-A2402を有する健常人ボランティアから採取されたLymphoblastoid cell line (LCL)(B細胞芽球様細胞株)を用いた。
標的細胞として、京都大学病院血液腫瘍内科門脇則光研究室(日本国京都府京都市)より譲渡されたTHP1細胞(急性単球性白血病細胞株)を用いた。
LMP2抗原ペプチド:TYGPVFMSL(419-427)
培地:20%ウシ胎仔血清含有αMEM培地(life technologies, cat #11900-073)
標的細胞として用いるTHP1白血病細胞株をCFSEラベルした。96穴U底プレートに上記細胞(キラーT細胞)とTHP1細胞をそれぞれ0:1、1:3、1:1、3:1、9:1となるように混合し、ペプチド存在下もしくは非存在下において4時間培養し、標的細胞の死細胞比率をCFSE陽性分画中に見られるAnnexinV陽性細胞の比率によって検定した(図7)。
ペプチドを添加していない時は殆ど標的細胞を殺していないのに対して、ペプチドの添加量の増加に応じて、より効率よく殺傷した。殺傷能力は元になったキラーT細胞(図5)と比べて遜色がなかった。すなわち、十分強い抗原特異的細胞傷害活性が確認された。
Claims (10)
- 再構成されたT細胞レセプター遺伝子を有する多能性幹細胞をin vitroで誘導してT前駆細胞を得ることを特徴とする、免疫細胞療法用T前駆細胞を得る方法。
- T前駆細胞がCD34+CD5+CD4-CD8-細胞、CD34+CD38-CD45RA-CD10-細胞、CD45RA+CD10+CD7-CD5-細胞、CD45RA+CD10+CD7+CD5-細胞、CD45RA+CD10+CD7+CD5+細胞、CD3-CD4+CD8-細胞およびCD4+CD8+細胞からなる群から選択される、請求項1記載の方法。
- 再構成されたT細胞レセプター遺伝子を有する多能性幹細胞が、ヒト細胞傷害性T細胞から誘導されたiPS細胞である、請求項1または2記載の方法。
- ヒト細胞傷害性T細胞が、ヒト末梢血単核球を抗原により刺激して誘導された細胞傷害性T細胞である、請求項3記載の方法。
- 抗原ががん抗原であり、免疫細胞療法ががん患者を治療するために行われる、請求項4記載の方法。
- 再構成されたT細胞レセプター遺伝子を有する多能性幹細胞からT前駆細胞を得、治療を必要とする対象へ当該T前駆細胞を投与することを含む、免疫細胞療法。
- T前駆細胞がCD34+CD5+CD4-CD8-細胞、CD34+CD38-CD45RA-CD10-細胞、CD45RA+CD10+CD7-CD5-細胞、CD45RA+CD10+CD7+CD5-細胞、CD45RA+CD10+CD7+CD5+細胞、CD3-CD4+CD8-細胞およびCD4+CD8+細胞からなる群から選択される、請求項6記載の免疫細胞療法。
- 再構成されたT細胞レセプター遺伝子を有する多能性幹細胞が、ヒト細胞傷害性T細胞から誘導されたiPS細胞である、請求項6または7記載の免疫細胞療法。
- ヒト細胞傷害性T細胞が、ヒト末梢血単核球を抗原により刺激して誘導された細胞傷害性T細胞である、請求項8記載の方法。
- 抗原ががん抗原であり、治療を必要とする対象が、がんを患っている患者である、請求項9記載の方法。
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Cited By (5)
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WO2016010148A1 (ja) * | 2014-07-18 | 2016-01-21 | 国立大学法人京都大学 | 多能性幹細胞から免疫細胞療法用t細胞を誘導する方法 |
US10472610B2 (en) | 2014-05-21 | 2019-11-12 | Kyoto University | Method for generating pancreatic bud cells and therapeutic agent for pancreatic disease containing pancreatic bud cells |
US11426430B2 (en) | 2017-02-13 | 2022-08-30 | Assistance Publique—Hopitaux de Paris | Method for generating T cells progenitors |
RU2797260C2 (ru) * | 2017-02-13 | 2023-06-01 | Ассистанс Публик - Опито Де Пари | Способ получения предшественников т-клеток |
US12071635B2 (en) | 2014-10-06 | 2024-08-27 | Assistance Publique-Hopitaux De Paris | Method for generating T-cell progenitors |
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JP6948072B2 (ja) | 2016-04-15 | 2021-10-13 | 国立大学法人京都大学 | Cd8陽性t細胞を誘導する方法 |
US20230071538A1 (en) * | 2020-02-07 | 2023-03-09 | Juntendo Educational Foundation | Cytotoxic t cells derived from human t cell-derived ips cells |
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2014
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- 2014-12-26 WO PCT/JP2014/084546 patent/WO2015099134A1/ja active Application Filing
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HONG B. ET AL.: "Generating CTLs against the subdominant EBV LMP antigens bytransit expression of an A20 inhibitor with EBV LMP proteins inhuman DCs", GENE THER., vol. 19, no. 8, 2012, pages 818 - 827 * |
NISHIMURA T. ET AL.: "Generation of rejuvenated antigen-specific T cells by reprogramming to pluripotency and redifferentiation.", CELL STEM CELL, vol. 12, no. 1, January 2013 (2013-01-01), pages 114 - 126, XP055218131, DOI: doi:10.1016/j.stem.2012.11.002 * |
VIZCARDO R. ET AL.: "Regeneration of human tumor antigen-specific T cells from iPSCs derived from mature CD 8(+) T cells.", CELL STEM CELL, vol. 12, no. 1, January 2013 (2013-01-01), pages 31 - 36, XP055240588, DOI: doi:10.1016/j.stem.2012.12.006 * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10472610B2 (en) | 2014-05-21 | 2019-11-12 | Kyoto University | Method for generating pancreatic bud cells and therapeutic agent for pancreatic disease containing pancreatic bud cells |
WO2016010148A1 (ja) * | 2014-07-18 | 2016-01-21 | 国立大学法人京都大学 | 多能性幹細胞から免疫細胞療法用t細胞を誘導する方法 |
US12071635B2 (en) | 2014-10-06 | 2024-08-27 | Assistance Publique-Hopitaux De Paris | Method for generating T-cell progenitors |
US11426430B2 (en) | 2017-02-13 | 2022-08-30 | Assistance Publique—Hopitaux de Paris | Method for generating T cells progenitors |
US11638723B2 (en) | 2017-02-13 | 2023-05-02 | Assistance Publique—Hopitaux de Paris | Method for generating T cells progenitors |
US11642376B2 (en) | 2017-02-13 | 2023-05-09 | Assistance Publique—Hopitaux de Paris | Method for generating T cell progenitors |
RU2797260C2 (ru) * | 2017-02-13 | 2023-06-01 | Ассистанс Публик - Опито Де Пари | Способ получения предшественников т-клеток |
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