WO2016076415A1 - 多能性幹細胞からt細胞への誘導方法 - Google Patents
多能性幹細胞からt細胞への誘導方法 Download PDFInfo
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Definitions
- the present invention relates to a method for producing CD4CD8 both positive T cells using a culture solution to which vitamin C is added in each step of producing CD4CD8 both positive T cells from pluripotent stem cells, and the CD4CD8 both positive T cells.
- the present invention relates to a method for producing CD8-positive T cells comprising a step of culturing in a culture solution containing a corticosteroid.
- T cells play a central role in the immune system against foreign cells such as bacteria and viruses, and abnormal cells such as cancer cells. It is thought to be affected by cancer. If such a disease can be replenished or regenerated with immune cells or the like, it will be an extremely effective means for improving the disease state of the disease and improving the therapeutic effect.
- immune cell replacement therapy functional supplementation and regeneration of T lymphocytes responsible for cellular immunity are strongly demanded, but no effective therapeutic method has been established at present.
- T lymphocyte replacement therapy it has been proposed to introduce an antigen-specific T cell receptor (TCR) gene into various lymphocyte cells to replenish or activate a specific immune reaction ( Non-Patent Document 1 or 2).
- TCR antigen-specific T cell receptor
- CD34 positive cells which are bone marrow progenitor cells, naive T lymphocytes, and the like are used as gene transfer cells, but these have low self-regenerative ability in Ex-vivo and low gene transfer efficiency. It has many problems such as difficulty in controlling differentiation by gene transfer.
- replacement therapy using T lymphocytes derived from pluripotent stem cells such as iPS cells has also been proposed (Non-patent Document 3 or Patent Document 1).
- a step of inducing hematopoietic progenitor cells from pluripotent stem cells (2) a step of inducing both CD4CD8 negative cells from hematopoietic progenitor cells, (3) CD4CD8
- a process of inducing both CD4CD8 positive cells from both negative cells and (4) a process of inducing T lymphocytes from both CD4CD8 positive cells.
- ES-sac net-like structure sac
- Non-Patent Document 5 or 6 a method of culturing in a medium supplemented with IL-7 and Flt-3L on an OP9-DL1 cell layer is known (Non-Patent Document 5 or 6). Furthermore, in the step (4), a method of culturing in a medium supplemented with anti-CD3 antibody (OKT-3) and IL-2 is known. However, the efficiency of producing T lymphocytes from pluripotent stem cells by these methods is not sufficient, and improvements are desired.
- An object of the present invention is to efficiently produce hematopoietic progenitor cells from pluripotent stem cells.
- a further object of the present invention is to efficiently produce CD8-positive T cells from both hematopoietic progenitor cell-derived CD4CD8-positive T cells obtained by the method.
- the present inventors searched for an effective substance for efficiently inducing hematopoietic progenitor cells from pluripotent stem cells. As a result, it was found that hematopoietic progenitor cells are efficiently induced by adding vitamin Cs to the culture medium and culturing in each step of differentiation from pluripotent stem cells to hematopoietic progenitor cells. Furthermore, the present inventors searched for an effective substance for efficiently inducing CD8-positive T cells from both CD4CD8-positive T cells. As a result, it was found that CD8-positive T cells are efficiently induced by adding an adrenocortical hormone agent to the culture medium and culturing, and the present invention has been completed.
- a method for inducing CD8 positive T cells from pluripotent stem cells comprising the following steps; (1) culturing pluripotent stem cells in a culture medium supplemented with vitamin C to induce hematopoietic progenitor cells, (2) a step of culturing the cells obtained in the step (1) in a culture medium supplemented with vitamin C to induce CD4CD8-positive T cells, and (3) obtained in the step (2).
- Inducing CD8-positive T cells by culturing cultured cells in a culture medium supplemented with corticosteroids.
- the culture solution further contains vitamin Cs.
- step of culturing pluripotent stem cells is a step of culturing pluripotent stem cells on C3H10T1 / 2 cells.
- step of culturing the pluripotent stem cells is performed under a hypoxic condition of 5% or less.
- the culture solution further contains VEGF, SCF and FLT-3L.
- CD8 positive T cells can be efficiently produced from pluripotent stem cells by adding vitamin Cs to the culture solution. Furthermore, according to the present invention, it is possible to efficiently produce CD8-positive T cells from both CD4CD8-positive T cells by adding an adrenocortical hormone agent to the culture medium. Therefore, according to the present invention, it is possible to efficiently produce CD8-positive T cells from pluripotent stem cells, and a therapeutic agent that activates an immune function including CD8-positive T cells derived from pluripotent stem cells. Can be provided.
- FIG. 1 shows the results of flow cytometry of cells after culturing for 37 days.
- the left figure shows the figure developed with FSC and SSC, the middle figure shows the figure developed with the staining intensity of CD3 and CD45, and the right figure shows the figure developed with the staining intensity of CD4 and CD8.
- FIG. 2 shows the results of flow cytometry of cells after culturing both CD4CD8 positive T cells for 3 days.
- the left figure shows the figure developed with FSC and SSC, the middle figure shows the figure developed with the staining intensity of CD3 and CD45, and the right figure shows the figure developed with the staining intensity of CD4 and CD8.
- FIG. 1 shows the results of flow cytometry of cells after culturing for 37 days.
- the left figure shows the figure developed with FSC and SSC, the middle figure shows the figure developed with the staining intensity of CD3 and CD45, and the right figure shows the figure developed with the staining intensity of CD4 and CD8.
- FIG. 3 shows the results of flow cytometry of cells after culturing CD4CD8-positive T cells derived from iPS cells (GPC strain) for 3 days.
- FIG. 4 shows the results of flow cytometry after contacting cells after culturing CD4CD8 positive T cells derived from iPS cells (GPC strain) for 3 days with GPC3 Dextramer.
- the left figure shows a diagram developed with the staining intensities of CD4 and CD8 ⁇
- the right figure shows the diagram developed with the staining intensities of GPC3 ⁇ Dextramer and CD8 ⁇ .
- FIG. 5 shows the results of flow cytometry after the cells after culturing CD4CD8 positive T cells derived from iPS cells (GPC strain) for 3 days were contacted with GPC3extDextramer.
- the figure which developed by the staining intensity of FSC and SSC, the staining intensity of CD3 and CD45, the staining intensity of CD4 and CD8 ⁇ , the staining intensity of CD8 ⁇ and CD8 ⁇ , and the staining intensity of GPC3 ⁇ Dextramer and CD8 ⁇ is shown.
- FIG. 6 shows the results of flow cytometry of cells after culturing CD4CD8-positive T cells produced from iPS cells (TKT3v 1-7 strain and 4GAD 1-8 strain) without using feeder cells for 3 days.
- the present invention provides a method for producing CD8-positive T cells from pluripotent stem cells.
- the production method includes (1) a step of inducing hematopoietic progenitor cells from pluripotent stem cells, (2) a step of inducing CD4CD8 positive T cells from the hematopoietic progenitor cells, and (3) from both CD4CD8 positive positive cells. It can be divided into steps for inducing CD8 positive T cells.
- a pluripotent stem cell is a stem cell having pluripotency that can be differentiated into many cells existing in a living body and also having proliferative ability, and at least used in the present invention. Any cell derived from a hematopoietic progenitor cell is included.
- pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, cloned embryo-derived embryonic stem (ntES) cells obtained by nuclear transfer, sperm stem cells (“GS cells”), embryonic Examples include germ cells (“EG cells”), induced pluripotent stem (iPS) cells, cultured fibroblasts and bone marrow stem cell-derived pluripotent cells (Muse cells).
- ES embryonic stem
- ntES cloned embryo-derived embryonic stem
- GS cells sperm stem cells
- GS cells sperm stem cells
- EG cells germ cells
- iPS induced pluripotent stem
- Muse cells bone marrow stem cell-derived pl
- IPS cell production methods are known in the art and can be produced by introducing reprogramming factors into any somatic cells.
- the reprogramming factor is, for example, 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 or Glis1, etc. genes or gene products are exemplified, and these reprogramming factors may be used alone or in combination. Also good.
- Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells. , Passage cells, and established cell lines.
- somatic cells are, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, (2) tissue progenitor cells, (3) blood cells (peripheral) Blood cells, umbilical cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (pancreatic exocrine cells) Etc.), differentiated cells such as brain cells, lung cells, kidney cells and fat cells.
- tissue stem cells such as neural stem cells, hematopoietic stem cells, mesenchymal stem
- lymphocytes preferably T cells that have undergone gene rearrangement of a T cell receptor (TCR) are used as somatic cells for the purpose of producing CD8 positive T cells. It is preferable to produce iPS cells.
- the lymphocytes when using lymphocytes, prior to the initialization step, the lymphocytes may be activated by stimulation with anti-CD3 antibody and anti-CD28 antibody in the presence of interleukin-2 (IL-2). preferable. Such stimulation can be performed, for example, by adding IL-2, anti-CD3 antibody and anti-CD28 antibody to the medium and culturing the lymphocytes for a certain period.
- IL-2 interleukin-2
- the anti-CD3 antibody and the anti-CD28 antibody may be ones to which magnetic beads or the like are bound, and instead of adding these antibodies to the medium, the anti-CD3 antibody and the anti-CD28 antibody are bound to the surface.
- Stimulation may be given by culturing the T cells for a certain period on a cultured dish. Further, stimulation may be given by adding an antigen peptide recognized by human T cells to the medium together with feeder cells.
- the CD8-positive T cells produced in the present invention preferably have a desired antigen specificity. Therefore, it is desirable that the lymphocytes that are the source of iPS cells have the desired antigen specificity, and the lymphocytes are specifically isolated by purification using an affinity column or the like on which the desired antigen is immobilized. Also good. In this purification, MHC (major histocompatibility complex) to which a desired antigen is bound is tetramerized (so-called “MHC tetramer”) and has a desired antigen specificity from human tissues. A method of purifying lymphocytes can also be employed.
- the mammal individual from which somatic cells are collected is not particularly limited, but is preferably a human.
- somatic cells that are the source of iPS cells are CD8 from the viewpoint that the patient to be transfused easily matches the type of human leukocyte antigen (HLA).
- HLA human leukocyte antigen
- positive T cells are isolated from the subject to be transfused.
- hematopoietic progenitor cells are lymphocytes, eosinophils, neutrophils, basophils, erythrocytes, megakaryocytes, etc.
- hematopoietic progenitor cells and hematopoietic stem cells are not distinguished from each other, and are the same cells unless otherwise specified.
- Hematopoietic stem / progenitor cells can be recognized by, for example, positive surface antigens CD34 and / or CD43.
- hematopoietic progenitor cells can be produced by a method including a step of culturing pluripotent stem cells in a culture solution supplemented with vitamin Cs.
- vitamin C means L-ascorbic acid and derivatives thereof
- L-ascorbic acid derivative means a substance that becomes vitamin C by an enzymatic reaction in vivo.
- L-ascorbic acid derivatives include vitamin C phosphate, ascorbyl glucoside, ascorbyl ethyl, vitamin C ester, ascovir tetrahexyldecanoate, ascovir stearate and ascorbyl 2-phosphate-6 palmitic acid.
- Preferred is vitamin C phosphate and examples thereof include phosphate-L-ascorbate such as sodium phosphate-L-ascorbate or Mg phosphate-L-ascorbate.
- the culture solution used for the production of hematopoietic progenitor cells in the present invention is not particularly limited, but can be prepared by adding vitamin Cs to a basal medium as a medium used for animal cell culture.
- Basal media include, for example, Iscove's'Modified Dulbecco's Medium (IMDM), Medium 199, Eagle's Minimum Essential Medium (EMEM), ⁇ MEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 ', , Neurobasal Medium (Life Technologies) and mixed media thereof. Serum may be contained in the medium, or serum-free may be used.
- the basal medium can be, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low It may also contain one or more substances such as molecular compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines and the like.
- a preferred basal medium in the present invention is an IMDM medium containing serum, insulin, transferrin, serine, thiolglycerol, L-glutamine, and ascorbic acid.
- the culture medium used for the production of hematopoietic progenitor cells in the present invention is selected from the group consisting of BMP4 (Bone morphogenetic protein 4), VEGF (vascular endothelial growth factor), SCF (Stem cell factor) and FLT-3L Ligand. Further cytokines may be added. More preferably, it is a culture solution to which VEGF, SCF and FLT-3L are added.
- vitamin Cs are preferably added (supplemented) separately every 4 days, every 3 days, every 2 days, or every day during the culture period, more preferably every day.
- the vitamin C is added in an amount corresponding to 5 ng / ml to 500 ng / ml in the culture solution.
- the amount corresponds to 5 ng / ml, 10 ng / ml, 25 ng / ml, 50 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, or 500 ng / ml.
- the concentration of BMP4 used for the production of hematopoietic progenitor cells in the present invention in the culture solution is 10 ng / ml to 100 ng / ml, such as 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng. / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml.
- it is 20 ng / ml or 40 ng / ml.
- the concentration of VEGF used for the production of hematopoietic progenitor cells in the present invention in the culture solution is 10 ng / ml to 100 ng / ml, such as 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng. / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml.
- it is 20 ng / ml.
- the concentration of SCF used for the production of hematopoietic progenitor cells in the present invention in the culture solution is 10 ng / ml to 100 ng / ml, such as 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng. / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml.
- it is 30 ng / ml.
- the concentration of FLT-3L used for the production of hematopoietic progenitor cells in the present invention in the culture solution is 1 ng / ml to 100 ng / ml, such as 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 20 ng / ml, 50 ng / ml, 100 ng / ml is there.
- it is 10 ng / ml.
- the pluripotent stem cells may be adhesion culture or suspension culture, and in the case of adhesion culture, they may be performed using a culture vessel coated with a coating agent. It may be co-cultured with cells. Other cells to be co-cultured include C3H10T1 / 2 (Takayama N., et al. J Exp Med. 2817-2830, 2010), heterologous stromal cells (Niwa A et al. J Cell Physiol. 2009 Nov; 221 ( 2): 367-77.).
- the coating agent Matrigel (Niwa A, et al.
- PLoS One.6 (7): e22261, 2011 is exemplified.
- suspension culture the method described in Chadwick et al. Blood 2003, 102: 906-15, Vijayaragavan et al. Cell Stem Cell 2009, 4: 248-62, and Saeki et al. Stem Cells 2009, 27: 59-67 Is exemplified.
- hematopoietic progenitor cells can also be prepared from a net-like structure (also referred to as ES-sac or iPS-sac) obtained by culturing pluripotent stem cells.
- a net-like structure also referred to as ES-sac or iPS-sac
- the “net-like structure” is a three-dimensional sac-like structure (with space inside) derived from pluripotent stem cells, which is formed by an endothelial cell population and the like, and contains hematopoietic progenitor cells inside. It is a structure.
- the temperature conditions for culturing for producing hematopoietic progenitor cells are not particularly limited, but for example, about 37 ° C to about 42 ° C, preferably about 37 to about 39 ° C are preferable.
- the culture period can be appropriately determined by those skilled in the art while monitoring the number of hematopoietic progenitor cells.
- the number of days is not particularly limited, for example, at least 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, preferably 14 days.
- the long culture period is not a problem in the production of hematopoietic progenitor cells.
- the culture may be performed under hypoxic conditions.
- hypoxic conditions are exemplified by oxygen concentrations of 15%, 10%, 9%, 8%, 7%, 6%, 5% or less.
- the culture for producing the hematopoietic progenitor cells of the present invention can be performed by appropriately combining the above conditions.
- (i) culturing pluripotent stem cells in basal medium supplemented with vitamin C under low oxygen conditions on C3H10T1 / 2, and (ii) VEGF, SCF and FLT-3L The step of further adding to the culture solution of 1) and culturing under normal oxygen conditions is exemplified.
- the period for performing the step (i) is at least 6 days or more, preferably 7 days or more, and more preferably 7 days.
- the period for performing the step (ii) is at least 6 days or more, preferably 7 days or more, and more preferably 7 days.
- CD4CD8-positive T cells mean cells (CD8 + CD4 + ) that are both positive for surface antigens CD4 and CD8.
- both CD4 and CD8 positive T cells can be identified as cells positive for CD4, CD8, CD3 and CD45. it can. Both CD4CD8 positive T cells can be differentiated into CD4 positive cells or CD8 positive cells by induction.
- both CD4CD8-positive T cells can be produced by a method including a step of culturing hematopoietic progenitor cells in a culture solution to which vitamin Cs are added.
- the culture solution used for the production of both CD4CD8 positive T cells is not particularly limited, but can be prepared by adding vitamin Cs to a basal medium as a medium used for culturing animal cells.
- Basal media include, for example, Iscove's'Modified Dulbecco's Medium (IMDM), Medium 199, Eagle's Minimum Essential Medium (EMEM), ⁇ MEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 ', , Neurobasal Medium (Life Technologies) and mixed media thereof. Serum may be contained in the medium, or serum-free may be used.
- the basal medium can be, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low It may also contain one or more substances such as molecular compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines and the like.
- a preferred basal medium used for the production of both CD4CD8 positive T cells of the present invention is an ⁇ MEM medium containing serum, transferrin, serine, and L-glutamine. Vitamin C added to the basal medium is the same as in the case of induction of hematopoietic progenitor cells described above.
- the culture medium used for the production of both CD4CD8 positive T cells of the present invention may further contain a cytokine selected from the group consisting of FLT-3L and IL-7. More preferably, it is a culture solution to which FLT-3L and IL-7 are added.
- the concentration of IL-7 used in the production of both CD4CD8 positive T cells in the culture solution is 1 ng / ml to 50 ng / ml, such as 1 ng / ml, 2 ng / ml, 3 ng / ml. ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, or 50 ng / ml.
- it is 5 ng / ml.
- FLT-3L used for the production of both CD4CD8 positive T cells can be used under the same conditions as described above.
- hematopoietic progenitor cells may be adhered or suspended, and in the case of adhesion culture, the culture vessel may be coated and used or co-cultured with feeder cells and the like. May be.
- feeder cells to be co-cultured are bone marrow stromal cell line OP9 cells (available from RIKEN BioResource Center).
- the OP9 cells may preferably be OP-DL1 cells that constantly express Dll1 (HolmesolR1 and Zuniga-Pflucker JC. Cold Spring Harb Protoc. 2009 (2)).
- Dll1 when OP9 cells are used as feeder cells, Dll1 prepared separately or a fusion protein such as Dll1 and Fc can be added to the culture medium as appropriate.
- Dll1 has a protein encoded by the gene having the nucleotide sequence described in NCBI accession number NM # 005618 for humans and NM # 007865 for mice, as well as higher sequences thereof. Naturally occurring variants having identity (eg, 90% or more) and equivalent functions are included.
- the exchange of the feeder cells can be performed by transferring the target cells in culture onto the feeder cells previously seeded. The exchange can occur every 5 days, every 4 days, every 3 days, or every 2 days.
- the culture temperature conditions for culturing hematopoietic progenitor cells to produce both CD4CD8-positive T cells are not particularly limited, but for example, about 37 ° C to about 42 ° C, about 37 ° C to about 39 ° C Is preferred.
- the culture period can be appropriately determined by those skilled in the art while monitoring the number of CD4CD8 positive T cells.
- the number of days is not particularly limited as long as hematopoietic progenitor cells can be obtained.For example, at least 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 20 days, 22 days, 23 days or more Yes, preferably 23 days.
- the obtained CD4CD8 both positive T cells may be isolated and used, or a cell population containing other cell types.
- it can be isolated using any one indicator consisting of CD4, CD8, CD3 and CD45, and the isolation method can be a method well known to those skilled in the art, for example, Examples thereof include a method of labeling with CD4, CD8, CD3 and CD45 antibodies and isolating using a flow cytometer, or a method of purification using an affinity column to which a desired antigen is immobilized.
- the CD8-positive T cell means a cell (CD8 + CD4 ⁇ ) that is positive for the surface antigen CD8 among T cells. Also called damaging T cells. Because T cells can be recognized by the positive surface antigens CD3 and CD45, CD8 positive T cells should be identified as cells positive for CD8, CD3 and CD45 and negative for CD4 Can do.
- CD8-positive T cells can be produced by a method including a step of culturing both CD4 and CD8-positive T cells in a culture solution to which a corticosteroid is added.
- the corticosteroid agent used in the present invention is a glucocorticoid or a derivative thereof, and examples thereof include cortisone acetate, hydrocortisone, fludrocortisone acetate, prednisolone, triamcinolone, methylprednisolone, dexamethasone, betamethasone, and beclomethasone propionate. Preferable is dexamethasone.
- the concentration in the culture is 1 nM to 100 nM, for example, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM or 100 nM It is. Preferably, it is 10 nM.
- the culture medium used for the production of CD8 positive T cells in the present invention is not particularly limited, but can be prepared by adding a corticosteroid to a basal medium as a medium used for culturing animal cells.
- Basal media include, for example, Iscove's'Modified Dulbecco's Medium (IMDM), Medium 199, Eagle's Minimum Essential Medium (EMEM), ⁇ MEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 ', , Neurobasal Medium (Life Technologies) and mixed media thereof. Serum may be contained in the medium, or serum-free may be used.
- the basal medium can be, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low It may also contain one or more substances such as molecular compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines and the like.
- a preferred basal medium in the present invention is an ⁇ MEM medium containing serum, transferrin, serine, L-glutamine, and ascorbic acid.
- the culture solution used for the production of CD8-positive T cells preferably further contains a CD3 antibody, vitamin Cs, and cytokines.
- the cytokine include IL-2 and IL-7.
- a more preferred cytokine for use in the production of CD8 positive T cells is a combination of IL-2 and IL-7.
- the CD3 antibody is not particularly limited as long as it is an antibody that specifically recognizes CD3, and examples thereof include an antibody produced from an OKT3 clone.
- the concentration of CD3 antibody in the culture solution is 10 ng / ml to 1000 ng / ml, for example, 10 ng / ml, 50 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml 500 ng / ml, 600 ng / ml, 700 ng / ml, 800 ng / ml, 900 ng / ml or 1000 ng / ml.
- it is 500 ng / ml.
- vitamin Cs used for producing CD8-positive T cells can be used under the same conditions as described above.
- the concentration of IL-2 used in the production of CD8-positive T cells in the present invention in the culture solution is 10 ⁇ U / ml to 1000 ⁇ U / ml, for example, 10 ⁇ U / ml, 20 ⁇ U / ml, 30 ⁇ U / ml. 40 U / ml, 50 U / ml, 60 U / ml, 70 U / ml, 80 U / ml, 90 U / ml, 100 U / ml, 500 U / ml, or 1000 U / ml. 100 U / ml is preferable.
- the concentration of IL-7 used in the production of CD8-positive T cells in the present invention in the culture solution is 1 ng / ml to 100 ng / ml, for example, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml or 100 ng / ml.
- it is 10 ng / ml.
- the temperature conditions for culturing both CD4CD8 positive T cells to produce CD8 positive T cells are not particularly limited, but for example, about 37 ° C to about 42 ° C, about 37 ° C to about 39 ° C Is preferred.
- the culture period can be appropriately determined by those skilled in the art while monitoring the number of CD8-positive T cells.
- the number of days is not particularly limited as long as hematopoietic progenitor cells can be obtained. For example, it is at least 1 day, 2 days, 3 days, 4 days, 5 days or more, preferably 3 days.
- Cell iPS cells (TKT3v 1-7 strain) were isolated after consent using the method described in Nishimura T, et al., Cell Stem Cell. 12 (1): 114-126, 2013. It was established from human CD3-positive T cells. C3H10T1 / 2 cells and OP9-DL1 cells were obtained from RIKEN / RIKEN BioResource Center and used.
- Hematopoietic cells (CD34 + hematopoietic stem / progenitor cells) contained in the obtained net-like structure (also referred to as iPS-SAC) were collected and seeded on OP9-DL1 cells.
- OP9 medium supplemented with 10 ng / mL FLT-3L and 5 ng / mL IL-7 (15% FBS, 2 mM L-glutamine, 100 U / ml penicillin, 100 ng / ml streptomycin, 5.5 ⁇ g / mL human transferrin and 5 ng / mL
- the cells were cultured for 23 days under normal pressure oxygen conditions ( ⁇ MEM to which sodium selenate was added) (Day 37). Cells were seeded on new OP9-DL1 cells every 3-4 days.
- L-Ascorbic acid 2-phosphate-sesquimagnesium salt was added every day to a final concentration of 50 ng / ml.
- CD3 (+) CD45 (+) CD4 (+) CD8 (+) fractionated cells were isolated using FACS to obtain CD4CD8 double-positive cells (referred to as DP cells) (FIG. 1). ).
- DP cells obtained by the above method are seeded in a 24-well plate, 500 ng / ml anti-CD3 antibody (OKT3), 10 nM Dexamethasone (Dexate R Fuji Pharma), 15% FBS, 2 mM L-glutamine , 100 U / ml penicillin, 100 ng / ml streptomycin, 5.5 ⁇ g / mL human transferrin, 5 ng / mL sodium selenite, 50 ng / ml L-Ascorbic acid 2-phosphate, Non essential Amino Acid, 100 U / ml IL-2, 10 ng
- the cells were cultured in ⁇ MEM supplemented with / ml IL-7 for 3 days. When the obtained cells were examined by FACS, it was confirmed that they were CD3 (+) CD45 (+) CD4 ( ⁇ ) CD8 (+) fractionated cells (FIG. 2).
- Cellular iPS cells (GPC3 strain) were isolated using human CD3 isolated after consent using the method described in Nishimura T, et al., Cell Stem Cell. 12 (1): 114-126, 2013. Established from positive T cells.
- the human CD3-positive T cells used are CD8-positive killer T cells, which have a T cell receptor specific for the GPC3 antigen, which is known to be expressed in liver cells.
- CD8-positive cells When the expression of CD3, CD45, CD4, CD8 ⁇ , CD8 ⁇ , CD5, and CD7 was confirmed with a flow cytometer for the cell group derived from the GPC3 strain by the above method, both CD8 ⁇ -positive CD8-positive cells were found. It was confirmed that it was induced (FIG. 3).
- Cells iPS cells (strain 4GAD 1-8) were isolated after consent using the method described in Nishimura T, et al., Cell Stem Cell. 12 (1): 114-126, 2013. It was established from human CD3-positive T cells.
- the human CD3-positive T cells used are CD4-positive helper T cells and have a T cell receptor specific for the GAD65 antigen known to be expressed in the pancreas.
- OP9 medium (15% ng FBS, 2 mM L-glutamine, 100 U / ml penicillin, 100 ng / ml streptomycin, 5.5 ⁇ g / mL human transferrin and 5 ng / mL supplemented with 10 ng / mL FLT-3L and 5 ng / mL IL-7
- the cells were cultured under normal pressure oxygen conditions for 23 days in ⁇ MEM supplemented with mL sodium sodium selenite (Day 37).
- L-Ascorbic acid 2-phosphate-sesquimagnesium salt was added every day to a final concentration of 50 ng / ml.
- CD3 (+) CD45 (+) CD4 (+) CD8 (+) fractionated cells were isolated using FACS to obtain CD4CD8 double-positive cells (referred to as DP cells) (FIG. 1). ).
- the obtained DP cells were seeded in a 24 well plate, 500 ng / ml anti-CD3 antibody (OKT3), 10 nM Dexamethasone (Dexate R Fuji Pharma), 15% FBS, 2 mM L-glutamine, 100 U / ml penicillin, 100 ng / ml streptomycin, In ⁇ MEM supplemented with 5.5 ⁇ g / mL human transferrin, 5 ng / mL ⁇ ⁇ ⁇ ⁇ sodium selenite, 50 ng / ml L-Ascorbic acid 2-phosphate, Non essential Amino Acid, 100 U / ml IL-2, 10 ng / ml IL-7 Incubated for 3 days.
- CD8-positive cells When the expression of CD3, CD45, CD4, CD8 ⁇ , CD8 ⁇ , CD5, and CD7 was confirmed with a flow cytometer for the cell group derived from the GPC3 strain by the above method, both CD8 ⁇ -positive CD8-positive cells were found. It was confirmed that it was induced (FIG. 6).
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Abstract
Description
このようなTリンパ球の補充療法として、抗原特異的なT細胞受容体(TCR)遺伝子を各種リンパ球系細胞に遺伝子導入し、特異的免疫反応を補充や賦活させることが提案されている(非特許文献1または2)。これらの試みでは遺伝子導入細胞として骨髄前駆細胞であるCD34陽性細胞や、ナイーブTリンパ球などが用いられているが、これらは、Ex-vivoでの自己再生能が低い、遺伝子導入の効率が低い、遺伝子導入による分化の調節が困難である、など多くの課題を有している。
また、iPS細胞などの多能性幹細胞から誘導したTリンパ球を用いた補充療法も提案されている(非特許文献3または特許文献1)。多能性幹細胞からTリンパ球の誘導方法においては、(1)多能性幹細胞から造血前駆細胞を誘導する工程、(2)造血前駆細胞からCD4CD8両陰性細胞を誘導する工程、(3)CD4CD8両陰性細胞からCD4CD8両陽性細胞を誘導する工程、および(4)CD4CD8両陽性細胞からTリンパ球を誘導する工程が提案されている。
(1)の工程では、多能性幹細胞からネット様構造物サック(ES-sac)を形成させ造血前駆細胞を製造する方法が知られている(非特許文献4)。また、(2)および(3)の工程は、OP9-DL1細胞層上でIL-7およびFlt-3Lを添加した培地で培養する方法が知られている(非特許文献5または6)。さらに、(4)の工程は、抗CD3抗体(OKT-3)およびIL-2を添加した培地で培養する方法が知られている。
しかし、これらの方法によって多能性幹細胞からTリンパ球を製造する効率は十分ではなく、改良が望まれている。
[1]以下の工程を含む、多能性幹細胞からCD8陽性T細胞を誘導する方法;
(1)多能性幹細胞を、ビタミンC類を添加した培養液中で培養し、造血前駆細胞を誘導する工程、
(2)前記工程(1)で得られた細胞を、ビタミンC類を添加した培養液中で培養し、CD4CD8両陽性T細胞を誘導する工程、および
(3)前記工程(2)で得られた細胞を、副腎皮質ホルモン剤を添加した培養液中で培養し、CD8陽性T細胞を誘導する工程。
[2]前記工程(3)において、培養液はさらにビタミンC類を含む、[1]に記載の方法。
[3]前記ビタミンC類が、リン酸ビタミンCである、[1]または[2]に記載の方法。[4]各工程において、ビタミンC類は1日毎に培養液へ補充される、[1]から[3]のいずれかに記載の方法。
[5]前記工程(1)において、多能性幹細胞はC3H10T1/2細胞上で培養される、[1]から[4]のいずれかに記載の方法。
[6]前記工程(1)が、5%以下の低酸素条件で行われる、[1]から[5]のいずれかに記載の方法。
[7]前記工程(1)において、培養液はさらにVEGF、SCFおよびFLT-3Lを含む、[1]から[6]のいずれかに記載の方法。
[8]前記工程(2)において、工程(1)で得られた細胞はOP9-DL1細胞上で培養される、[1]から[7]のいずれかに記載の方法。
[9]前記工程(2)において、培養液はさらにFLT-3L、およびIL-7を含む、[1]から[8]のいずれかに記載の方法。
[10]副腎皮質ホルモン剤を添加した培養液中でCD4CD8両陽性T細胞を培養する工程を含む、CD8陽性T細胞を誘導する方法。
[11]前記副腎皮質ホルモン剤がデキサメタゾンである、[10]に記載の方法。
[12]前記培養液は、さらに抗CD3抗体、ビタミンC類、IL-2およびIL-7を含む、[10]または[11]に記載の方法。
[13]前記ビタミンC類が、リン酸ビタミンCである、[12]に記載の方法。
[14]ビタミンC類を添加した培養液中で多能性幹細胞を培養する工程を含む、造血前駆細胞を誘導する方法。
[15]前記ビタミンC類が、リン酸ビタミンCである、[14]に記載の方法。
[16]ビタミンC類は1日毎に培養液に補充される、[14]または[15]に記載の方法。
[17]前記多能性幹細胞を培養する工程が、C3H10T1/2細胞上で多能性幹細胞を培養する工程である、[14]から[16]のいずれかに記載の方法。
[18]前記多能性幹細胞を培養する工程が、5%以下の低酸素条件で行われる、[14]から[17]のいずれかに記載の方法。
[19]前記培養液は、さらにVEGF、SCFおよびFLT-3Lを含む、[14]から[18]のいずれかに記載の方法。
本発明において多能性幹細胞とは、生体に存在する多くの細胞に分化可能である多能性を有し、かつ、増殖能をも併せもつ幹細胞であり、少なくとも本発明で使用される造血前駆細胞に誘導される任意の細胞が包含される。多能性幹細胞には、特に限定されないが、例えば、胚性幹(ES)細胞、核移植により得られるクローン胚由来の胚性幹(ntES)細胞、精子幹細胞(「GS細胞」)、胚性生殖細胞(「EG細胞」)、人工多能性幹(iPS)細胞、培養線維芽細胞や骨髄幹細胞由来の多能性細胞(Muse細胞)などが含まれる。好ましい多能性幹細胞は、製造工程において胚、卵子等の破壊をしないで入手可能であるという観点から、iPS細胞であり、より好ましくはヒトiPS細胞である。
本発明において、造血前駆細胞(Hematopoietic Progenitor Cells(HPC))とは、リンパ球、好酸球、好中球、好塩基球、赤血球、巨核球等の血球系細胞に分化可能な細胞である、本発明において、造血前駆細胞と造血幹細胞は、区別されるものではなく、特に断りがなければ同一の細胞を示す。造血幹細胞/前駆細胞は、例えば、表面抗原であるCD34および/またはCD43が陽性であることによって認識できる。
本発明において、CD4CD8両陽性T細胞とは、T細胞のうち、表面抗原のCD4およびCD8が共に陽性である細胞(CD8+CD4+)を意味し、T細胞は、表面抗原であるCD3およびCD45が陽性であることによって認識することができることから、CD4CD8両陽性T細胞は、CD4、CD8、CD3およびCD45が陽性である細胞として同定することができる。CD4CD8両陽性T細胞は、誘導によってCD4陽性細胞またはCD8陽性細胞へと分化させることができる。
本発明において、CD8陽性T細胞とは、T細胞のうち、表面抗原のCD8が陽性である細胞(CD8+CD4-)を意味し、細胞傷害性T細胞とも呼ばれる。T細胞は、表面抗原であるCD3およびCD45が陽性であることによって認識することができることから、CD8陽性T細胞は、CD8、CD3およびCD45が陽性であり、CD4が陰性である細胞として同定することができる。
iPS細胞(TKT3v 1-7株)は、Nishimura T, et al., Cell Stem Cell. 12(1):114-126, 2013に記載の方法を用いて、告知後に同意を得て単離されたヒトCD3陽性T細胞より樹立した。
C3H10T1/2細胞およびOP9-DL1細胞は、理化学研究所・理研 BioResource Center より入手して用いた。
10cm dishにおいてコンフルエントなC3H10T1/2細胞上にTKT3v 1-7株の小塊を播種し(Day0)、EB培地(15%ウシ胎児血清(FBS)、10μg/mL ヒトインスリン、5.5μg/mL ヒトトランスフェリン、5ng/mL 亜セレン酸ナトリウム、2mM L-グルタミンと、0.45mM α-モノチオグリセロール、および50μg/mL アスコルビン酸を添加したIMDM)中で、低酸素条件下(5% O2)にて7日間培養した(Day7)。
上記の方法で得られたDP細胞を24wellプレートに播種し、500ng/ml抗CD3抗体(OKT3)、10nM Dexamethasone(デキサートR Fuji Pharma)、15% FBS、2mM L-グルタミン、100U/ml ペニシリン、100ng/ml ストレプトマイシン、5.5μg/mL ヒトトランスフェリン、5ng/mL 亜セレン酸ナトリウム、50ng/ml L-Ascorbic acid 2-phosphate、Non essential Amino Acid、100U/ml IL-2、10ng/ml IL-7を添加したαMEM中で、3日間培養した。得られた細胞をFACSにて調べたところ、CD3(+) CD45(+) CD4(-) CD8(+) 分画細胞であることが確認された(図2)。
iPS細胞(GPC3株)は、Nishimura T, et al., Cell Stem Cell. 12(1):114-126, 2013に記載の方法を用いて、告知後に同意を得て単離されたヒトCD3陽性T細胞より樹立した。用いたヒトCD3陽性T細胞はCD8陽性のキラーT細胞であり、肝細胞がんでの発現が知られるGPC3抗原に特異的なT細胞受容体を持つ。
C3H10T1/2細胞上にGPC3株の小塊を播種し、実施例1と同様の方法にて培養し、CD4CD8両陽性を得た。さらに、得られたCD4CD8両陽性を実施例1と同様の方法にて培養し、CD8陽性細胞を誘導した。
GPC3株から上述の方法で誘導した細胞群をフローサイトメーターにてCD3、CD45、CD4、CD8α、CD8β、CD5およびCD7の発現を確認したところ、CD8αβ両陽性のCD8陽性細胞が誘導されることが確認された(図3)。
iPS細胞(4GAD 1-8株)は、Nishimura T, et al., Cell Stem Cell. 12(1):114-126, 2013に記載の方法を用いて、告知後に同意を得て単離されたヒトCD3陽性T細胞より樹立した。用いたヒトCD3陽性T細胞はCD4陽性のヘルパーT細胞であり、膵臓での発現が知られるGAD65抗原に特異的なT細胞受容体を持つ。
10cm dishにTKT3v 1-7株または4GAD 1-8株の小塊を播種し(Day0)、EB培地(15%ウシ胎児血清(FBS)、10μg/mL ヒトインスリン、5.5μg/mL ヒトトランスフェリン、5ng/mL 亜セレン酸ナトリウム、2mM L-グルタミン、0.45mM α-モノチオグリセロール、および50μg/mL アスコルビン酸を添加したIMDM)中で、低酸素条件下(5% O2)にて7日間培養した(Day7)。
GPC3株から上述の方法で誘導した細胞群をフローサイトメーターにてCD3、CD45、CD4、CD8α、CD8β、CD5およびCD7の発現を確認したところ、CD8αβ両陽性のCD8陽性細胞が誘導されることが確認された(図6)。
Claims (19)
- 以下の工程を含む、多能性幹細胞からCD8陽性T細胞を誘導する方法;
(1)多能性幹細胞を、ビタミンC類を添加した培養液中で培養し、造血前駆細胞を誘導する工程、
(2)前記工程(1)で得られた細胞を、ビタミンC類を添加した培養液中で培養し、CD4CD8両陽性T細胞を誘導する工程、および
(3)前記工程(2)で得られた細胞を、副腎皮質ホルモン剤を添加した培養液中で培養し、CD8陽性T細胞を誘導する工程。 - 前記工程(3)において、培養液はさらにビタミンC類を含む、請求項1に記載の方法。
- 前記ビタミンC類が、リン酸ビタミンCである、請求項1または2に記載の方法。
- 各工程において、ビタミンC類は1日毎に培養液へ補充される、請求項1から3のいずれか1項に記載の方法。
- 前記工程(1)において、多能性幹細胞はC3H10T1/2細胞上で培養される、請求項1から4のいずれか1項に記載の方法。
- 前記工程(1)が、5%以下の低酸素条件で行われる、請求項1から5のいずれか1項に記載の方法。
- 前記工程(1)において、培養液はさらにvascular endothelial growth factor (VEGF)、Stem cell factor (SCF)およびFlt3 Ligand (FLT-3L)を含む、請求項1から6のいずれか1項に記載の方法。
- 前記工程(2)において、工程(1)で得られた細胞はOP9-DL1細胞上で培養される、請求項1から7のいずれか1項に記載の方法。
- 前記工程(2)において、培養液はさらにFLT-3L、およびinterleukin (IL)-7を含む、請求項1から8のいずれか1項に記載の方法。
- 副腎皮質ホルモン剤を添加した培養液中でCD4CD8両陽性T細胞を培養する工程を含む、CD8陽性T細胞を誘導する方法。
- 前記副腎皮質ホルモン剤がデキサメタゾンである、請求項10に記載の方法。
- 前記培養液は、さらに抗CD3抗体、ビタミンC類、IL-2およびIL-7を含む、請求項10または11に記載の方法。
- 前記ビタミンC類が、リン酸ビタミンCである、請求項12に記載の方法。
- ビタミンC類を添加した培養液中で多能性幹細胞を培養する工程を含む、造血前駆細胞を誘導する方法。
- 前記ビタミンC類が、リン酸ビタミンCである、請求項14に記載の方法。
- ビタミンC類は1日毎に培養液へ補充される、請求項14または15に記載の方法。
- 前記多能性幹細胞を培養する工程が、C3H10T1/2細胞上で多能性幹細胞を培養する工程である、請求項14から16のいずれか1項に記載の方法。
- 前記多能性幹細胞を培養する工程が、5%以下の低酸素条件で行われる、請求項14から17のいずれか1項に記載の方法。
- 前記培養液は、さらにVEGF、SCFおよびFLT-3Lを含む、請求項14から18のいずれか1項に記載の方法。
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Cited By (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017179720A1 (ja) * | 2016-04-15 | 2017-10-19 | 国立大学法人京都大学 | Cd8陽性t細胞を誘導する方法 |
WO2017221975A1 (ja) * | 2016-06-23 | 2017-12-28 | 国立大学法人京都大学 | Cd4cd8両陽性t細胞の製造方法 |
WO2018135646A1 (ja) | 2017-01-20 | 2018-07-26 | 国立大学法人京都大学 | CD8α+β+細胞傷害性T細胞の製造方法 |
WO2018143454A1 (ja) | 2017-02-06 | 2018-08-09 | 国立研究開発法人国立がん研究センター | 新規t細胞受容体 |
WO2018199186A1 (ja) | 2017-04-26 | 2018-11-01 | 国立大学法人京都大学 | 造血前駆細胞マーカー |
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WO2020032179A1 (ja) | 2018-08-10 | 2020-02-13 | 国立大学法人京都大学 | Cd3陽性細胞の製造方法 |
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WO2021106832A1 (ja) | 2019-11-25 | 2021-06-03 | 国立大学法人京都大学 | T細胞マスターセルバンク |
WO2021241658A1 (ja) | 2020-05-26 | 2021-12-02 | 株式会社ヘリオス | 低免疫原性細胞 |
WO2022255489A1 (ja) | 2021-06-04 | 2022-12-08 | キリンホールディングス株式会社 | 細胞組成物、細胞組成物の製造方法及び細胞組成物を含む医薬組成物 |
WO2023012584A2 (en) | 2021-08-03 | 2023-02-09 | Genicity Limited | Engineered tcr complex and methods of using same |
TWI860972B (zh) | 2017-04-26 | 2024-11-11 | 國立大學法人京都大學 | 造血前驅細胞標記 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013176197A1 (ja) * | 2012-05-22 | 2013-11-28 | 国立大学法人 東京大学 | 抗原特異的t細胞の製造方法 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2192908A4 (en) * | 2007-07-25 | 2010-09-01 | Bioe Inc | DIFFERENTIATION OF PRECURSOR CELLS OF MULTIPLE ABSTRACTS AGAINST CHONDROCYTES |
US9206394B2 (en) | 2010-02-03 | 2015-12-08 | The University Of Tokyo | Method for reconstructing immune function using pluripotent stem cells |
JPWO2011096482A1 (ja) | 2010-02-03 | 2013-06-13 | 国立大学法人 東京大学 | 多能性幹細胞を用いた免疫機能再建法 |
-
2015
- 2015-11-13 JP JP2016559115A patent/JP6736003B2/ja active Active
- 2015-11-13 US US15/526,294 patent/US10660915B2/en active Active
- 2015-11-13 WO PCT/JP2015/081959 patent/WO2016076415A1/ja active Application Filing
- 2015-11-13 EP EP15859306.1A patent/EP3219791A4/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013176197A1 (ja) * | 2012-05-22 | 2013-11-28 | 国立大学法人 東京大学 | 抗原特異的t細胞の製造方法 |
Non-Patent Citations (4)
Title |
---|
BERKI T. ET AL.: "Glucocorticoid (GC) sensitivity and GC receptor expression differ in thymocyte subpopulations.", INT IMMUNOL., vol. 14, no. 5, May 2002 (2002-05-01), pages 463 - 469, XP003034341 * |
HUIJSKENS MJ. ET AL.: "Technical advance: ascorbic acid induces development of double- positive T cells from human hematopoietic stem cells in the absence of stromal cells.", J LEUKOC BIOL., vol. 96, no. 6, pages 1165 - 1175, XP055441080 * |
SCREPANTI I. ET AL.: "Steroid sensitivity of thymocyte subpopulations during intrathymic differentiation. Effects of 17 beta-estradiol and dexamethasone on subsets expressing T cell antigen receptor or IL -2 receptor.", J IMMUNOL., vol. 142, no. 10, 15 May 1989 (1989-05-15), pages 3378 - 3383, XP055441082 * |
See also references of EP3219791A4 * |
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WO2020013315A1 (ja) | 2018-07-13 | 2020-01-16 | 国立大学法人京都大学 | γδT細胞の製造方法 |
WO2020027094A1 (ja) * | 2018-07-31 | 2020-02-06 | サイアス株式会社 | iPS細胞を介して再生T細胞集団を製造する方法 |
CN112513255A (zh) * | 2018-07-31 | 2021-03-16 | 赛雅思株式会社 | 介由iPS细胞制造再生T细胞群体的方法 |
JPWO2020027094A1 (ja) * | 2018-07-31 | 2021-09-16 | サイアス株式会社 | iPS細胞を介して再生T細胞集団を製造する方法 |
EP4455166A2 (en) | 2018-08-10 | 2024-10-30 | Kyoto University | Method for producing cd3-positive cell |
WO2020032179A1 (ja) | 2018-08-10 | 2020-02-13 | 国立大学法人京都大学 | Cd3陽性細胞の製造方法 |
WO2020116606A1 (ja) | 2018-12-06 | 2020-06-11 | キリンホールディングス株式会社 | T細胞又はnk細胞の製造方法、t細胞又はnk細胞の培養用培地、t細胞又はnk細胞の培養方法、未分化t細胞の未分化状態を維持する方法及びt細胞又はnk細胞の増殖促進剤 |
WO2020138371A1 (ja) | 2018-12-26 | 2020-07-02 | キリンホールディングス株式会社 | 改変tcr及びその製造方法 |
WO2020138256A1 (ja) | 2018-12-27 | 2020-07-02 | 国立大学法人京都大学 | T細胞受容体の改変体 |
WO2020204149A1 (ja) | 2019-03-29 | 2020-10-08 | 公立大学法人横浜市立大学 | スクリーニング方法および毒性評価法 |
WO2021106832A1 (ja) | 2019-11-25 | 2021-06-03 | 国立大学法人京都大学 | T細胞マスターセルバンク |
EP4067490A1 (en) | 2019-11-25 | 2022-10-05 | Kyoto University | T-cell master cell bank |
WO2021241658A1 (ja) | 2020-05-26 | 2021-12-02 | 株式会社ヘリオス | 低免疫原性細胞 |
WO2022255489A1 (ja) | 2021-06-04 | 2022-12-08 | キリンホールディングス株式会社 | 細胞組成物、細胞組成物の製造方法及び細胞組成物を含む医薬組成物 |
WO2023012584A2 (en) | 2021-08-03 | 2023-02-09 | Genicity Limited | Engineered tcr complex and methods of using same |
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US10660915B2 (en) | 2020-05-26 |
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JP6736003B2 (ja) | 2020-08-05 |
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US20170326175A1 (en) | 2017-11-16 |
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