WO2022168959A1 - 人工多能性幹細胞由来γδT細胞及びその作製方法 - Google Patents
人工多能性幹細胞由来γδT細胞及びその作製方法 Download PDFInfo
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Definitions
- the present invention relates to ⁇ T cells derived from induced pluripotent stem cells (iPS cells) and methods for producing them. Specifically, the present invention relates to iPS cell-derived ⁇ T cells that act in an MHC-unrestricted manner and a method for producing the same. Furthermore, it relates to a cell population containing the produced iPS cell-derived ⁇ T cells.
- iPS cells induced pluripotent stem cells
- ⁇ T cells Human mature T cells are roughly divided into two groups: ⁇ T cells whose T cell receptors are composed of ⁇ and ⁇ chains, and ⁇ T cells whose T cell receptors are composed of ⁇ and ⁇ chains.
- ⁇ T cells are extremely diverse, and while the types of cells that can be attacked by one type of ⁇ T cells are MHC-restricted and few, one type of ⁇ T cells is MHC-unrestricted and many ⁇ T cells can attack. It is known to attack different types of cancer cells.
- ⁇ T cells are a type of T cell receptor (TCR) that recognizes and directly kills many types of cancer cells.
- ⁇ T cells are usually present in only 1-5% of peripheral blood, even if a small amount of blood is collected to activate and/or proliferate ⁇ T cells, sufficient purity and cell numbers for treatment should be ensured. There is a problem that it is not possible to In addition, there is also the problem that if the amount of blood collected from a patient is increased in order to ensure sufficient purity and cell count for treatment, the patient will suffer a great burden. ⁇ T cells isolated from the patient's peripheral blood have already been treated by in vitro expansion and infusion into the patient. No activation was obtained.
- Patent Document 1 A method for producing iPS cells ( ⁇ TCR-type iPS cells) having a ⁇ TCR rearrangement gene is disclosed (Patent Document 1, Non-Patent Document 1).
- Patent Document 1 and Non-Patent Document 1 further disclose that ⁇ TCR-type iPS cells were induced to differentiate into blood cell progenitor cells. However, it has not been disclosed that the hematopoietic progenitor cells were further induced to differentiate into T cells.
- Patent Document 2 A method for inducing the differentiation of T cell-derived iPS cells into T cells is disclosed (Patent Document 2).
- Patent Document 3 A method for inducing the differentiation of T cell-derived iPS cells into T cells.
- T cells with the same rearrangement as the original cells can be obtained.
- all of them are reports on ⁇ T cells, and ⁇ T cells are not disclosed. Since all of the ⁇ T cells have a specific ⁇ TCR, there are few types of cancers that express the antigen, and they are MHC-restricted, which limits the number of patients who can be treated. .
- Non-Patent Document 4 T cells induced to differentiate from stem cells such as ES cells or iPS cells exhibit a ⁇ T cell-like phenotype.
- T cells shown in the above literature have a gene expression pattern that resembles the phenotype characteristic of ⁇ T, they actually express ⁇ T cell receptors, thereby recognizing antigens and damaging target cells. That is, it cannot be said that they are ⁇ T cells.
- a method for effectively preparing T cells capable of attacking various types of cancer cells in an MHC-unrestricted manner is desired.
- ⁇ T cells normally exist in only 1-5% of peripheral blood, there was the problem of not being able to secure sufficient purity and cell numbers for treatment. In addition, there is also a problem that if a large amount of blood is collected in order to ensure sufficient purity and cell count for treatment, a great burden is placed on the person receiving the blood. In vitro expansion of ⁇ T cells isolated from peripheral blood was difficult to obtain, and sufficient expansion and activation were not achieved due to cell exhaustion.
- An object of the present invention is to effectively produce and provide ⁇ T cells. More specifically, the object is to provide excellent ⁇ T cells that are homogenous ⁇ T cells and are not affected by cell exhaustion.
- the present inventors focused on iPS cells and extensively studied differentiation-inducing treatment methods. We have completed the present invention.
- the present invention consists of the following.
- An iPS cell-derived ⁇ T cell which is an induced pluripotent stem cell (iPS cell)-derived T cell, wherein the T cell has antigen-specific cytotoxic activity in an MHC-unrestricted manner.
- the iPS cell-derived ⁇ T cell according to the preceding item 1 wherein the iPS cell is an iPS cell not derived from ⁇ T cell.
- 3. The iPS cell-derived ⁇ T cell according to the preceding item 1 or 2, wherein the iPS cell is an iPS cell having a ⁇ TCR rearrangement gene.
- Blood cell progenitor cells obtained by differentiation induction treatment of iPS cells with ⁇ TCR reconstruction gene were added to the basal medium with FLT3L (tyrosine kinase 3 ligand), SCF (stem cell factor), IL-2, IL-7, TPO (thrombopoietin ), a method for producing iPS cell-derived ⁇ T cells, comprising the step of culturing using a medium to which one or more selected from L-ascorbic acid is added. 6.
- a ⁇ T cell stimulating agent 6 After the step of culturing using a medium in which one or more selected from FLT3L, SCF, IL-2, IL-7, TPO, and L-ascorbic acid is added to a basal medium, a ⁇ T cell stimulating agent 6.
- the step of culturing using a medium in which one or more selected from FLT3L, SCF, IL-2, IL-7, TPO, and L-ascorbic acid is added to a basal medium is coculturing with feeder cells 7.
- the step of culturing without co-culturing with feeder cells is VCAM1 (vascular cell adhesion molecule-1) and DLL4 (Delta-Like Protein 4) or DLL1 (Delta-Like Protein 1) using a culture substrate coated with 9.
- the method for producing iPS cell-derived ⁇ T cells according to the preceding item 8 comprising the step of culturing. 10.
- the method for producing iPS cell-derived ⁇ T cells according to the preceding item 8 or 9, wherein the step of culturing without coculturing with feeder cells further comprises a step of culturing using a medium containing DKK1 and/or AZA (Azelaic acid). . 11. 11.
- the iPS cell-derived ⁇ T according to any one of 6 to 10 above, wherein the medium containing the ⁇ T cell stimulating agent is a medium containing one or more selected from a ⁇ T cell stimulating agent, IL-2 and IL-15.
- a method for making cells 12. 6 to 11 above, wherein the ⁇ T cell stimulating agent is a phosphoric acid compound or derivative thereof that is an isoprenoid biosynthetic pathway metabolite, or a specific inhibitor of FPP (farnesyl pyrophosphate) synthase that is the rate-limiting enzyme in the isoprenoid biosynthetic pathway. 3.
- the method for producing iPS cell-derived ⁇ T cells according to any one of 1. 13. 13.
- the cell population according to 16 above wherein the cell population containing iPS cell-derived ⁇ T cells has high antigen-specific cytotoxic activity compared to the cell population of ⁇ T cells isolated from peripheral blood. 18.
- a cell population containing ⁇ T cells, characterized in that ⁇ T cells having the same nucleotide sequence in the CDR3 region of the TCR gene comprise 90% or more of the ⁇ T cells constituting the cell population. group. 19.
- 20. A cell population containing ⁇ T cells, wherein 90% or more of the ⁇ T cells constituting the cell population are ⁇ T cells that exhibit higher expression levels of CD7 and CD8a than ⁇ T cells isolated from peripheral blood.
- 21. The cell population according to any one of the preceding items 18 to 20, which is a cell population containing ⁇ T cells, wherein undifferentiated cells account for 10% or less of the ⁇ T cells constituting the cell population.
- 22. An antigen-specific cellular immunotherapeutic agent comprising the iPS cell-derived ⁇ T cells according to any one of 1 to 4 and 15 above as an active ingredient.
- 23. 16 The method for culturing iPS cell-derived ⁇ T cells according to any one of the above items 1 to 4 and 15, wherein the culture is performed using a medium containing a bead-like carrier in a liquid medium. 24.
- a therapeutic agent for diseases such as cancer, infectious diseases, and autoimmune disorders comprising the iPS cell-derived ⁇ T cells according to any one of 1 to 4 and 15 above as an active ingredient.
- a pharmaceutical composition comprising the iPS cell-derived ⁇ T cells according to any one of 1 to 4 and 15 above as an active ingredient.
- An antigen-specific cell-mediated immune cell therapy method comprising administering the iPS cell-derived ⁇ T cells selected from 1 to 4 and 15 above.
- ⁇ T cells can be effectively produced without burdening the blood recipient and without being affected by cell exhaustion. Furthermore, according to the method for producing iPS cell-derived ⁇ T cells of the present invention, excellent ⁇ T cells can be produced even under conditions that do not contain feeder cells and/or serum, or that do not contain animal-derived components.
- the ⁇ T cells of the present invention have excellent functions of non-MHC-restricted antigen-specific cytotoxic activity, and are homogeneous and more effective ⁇ T cell populations than ⁇ T cells isolated from peripheral blood. was made.
- FIG. 1A shows the results of flow cytometry evaluation of CD34/CD43 expression in cells on day 10 of induction of differentiation.
- FIG. 1B shows the results of flow cytometry evaluation of CD3/ ⁇ TCR expression in cells on day 31 of induction of differentiation.
- FIG. 2A shows the results of flow cytometry evaluation of the expression of CD7 (T cell differentiation marker) in cells on day 17 of induction of differentiation.
- FIG. 2B shows the results of evaluating the expression of CD3/ ⁇ TCR/CD45RA in cells on day 54 of differentiation induction by flow cytometry.
- FIG. 3A shows the results of evaluation of CD7 expression by flow cytometry on cells on day 17 of induction of differentiation.
- FIG. 1A shows the results of flow cytometry evaluation of CD34/CD43 expression in cells on day 10 of induction of differentiation.
- FIG. 1B shows the results of flow cytometry evaluation of CD3/ ⁇ TCR expression in cells on day 31 of induction of differentiation.
- FIG. 2A shows the results of flow cytometry evaluation of
- FIG. 3B shows the results of flow cytometry evaluation of CD3/ ⁇ TCR expression in cells on day 55 of induction of differentiation.
- FIG. 3C shows the results of confirming the cytotoxic activity against Jurkat cells of cells on day 55 of induction of differentiation.
- Fig. 2 shows a protocol for induction of differentiation from iPS cells under conditions that do not use feeder cells.
- Example 4) 3 shows the results of evaluation by flow cytometry for the expression of CD3/ ⁇ TCR in cells on days 33, 35, and 37 of induction of differentiation under conditions in which feeder cells were not used.
- Fig. 2 shows a protocol for induction of differentiation from iPS cells under conditions that do not use feeder cells.
- FIG. 5 shows the results of flow cytometry evaluation of CD3/ ⁇ TCR expression in cells on day 55 of induction of differentiation.
- FIG. 7A shows the results of observation of cells on day 37 of differentiation induction using a phase-contrast microscope.
- FIG. 7B shows the results of evaluating CD3/ ⁇ TCR expression by flow cytometry.
- Example 5 shows a protocol for induction of differentiation from iPS cells under conditions that do not use feeder cells.
- Example 6 Shown are the results of phase-contrast microscopy observation of cells on day 32 of induction of differentiation when cultured in each medium without feeder cells.
- Example 3 shows that cells on day 35 of induction of differentiation have cytotoxicity against Jurkat cells when cultured in each medium without feeder cells.
- Example 6 The results of confirming the cytotoxic activity of the cells on day 35 of induction of differentiation with Jurkat cells 1 day and 4 days after the start of mixed culture when cultured in each medium without feeder cells are shown.
- Example 6) 24 shows the results of flow cytometry evaluation of the expression of CD7, a T cell differentiation marker, in cells on the 24th day of induction of differentiation under conditions in which feeder cells were not used.
- Example 7 In the condition that feeder cells are not used, instead of coating the culture dish, magnetic beads coated with VCAM1 and DLL4 are co-cultured to induce differentiation into T cells.
- FIG. 10 shows a protocol for inducing differentiation of ⁇ T cells produced in Example 9 from iPS cells.
- FIG. 16A shows the results of phase-contrast microscopy observation of the shape of cells in the process of differentiation.
- FIG. 16B shows the results of confirmation of cell surface markers by flow cytometry for cells in the process of differentiation.
- Fig. 3 shows the results of confirming the antitumor activity against various tumor cells of ⁇ T cells on day 38 of induction of differentiation.
- FIG. 17A shows the results of confirming the cytotoxic activity against Jurkat cells.
- FIG. 17B shows the results of confirming the cytotoxic activity against Huh-7 cells.
- FIG. 17C shows the results of confirming the cytotoxic activity against SW480 cells.
- FIG. 17D shows the viability of iPS cell-derived ⁇ T cells (E) and Jurkat cells (T) in mixed culture when the E:T ratio was changed stepwise.
- Fig. 3 shows the results of confirming the retention of TCR reconstitution and the cytotoxic mechanism of ⁇ T cells on day 36 of differentiation induction.
- FIG. 18A shows the results of evaluating the expression of ⁇ TCR on the cell surface of unpurified ⁇ T cells (igdT) and peripheral blood mononuclear cells (PB).
- FIG. 18B shows the results of confirming the rearrangement of TCR genes (V ⁇ 9, V ⁇ 2) by genomic PCR.
- FIG. 18C shows the results confirming that ⁇ T cells express GranzymeB and Perforin.
- FIG. 18D shows the results of confirming the cytotoxic activity of purified ⁇ T cells (igdT). There was no significant difference in the dead cell rate between ⁇ T cells with and without purification.
- Fig. 2 shows results of confirming gene expression patterns in iPS cell-derived ⁇ T cells and ⁇ T cells isolated from peripheral blood by single-cell RNA-seq analysis.
- FIG. 1 shows the results of analysis by flow cytometry for CD25 among the cell surface expression markers for ⁇ T cells derived from iPS cells and ⁇ T cells isolated from peripheral blood.
- FIG. 1 shows a protocol for inducing differentiation of ⁇ T cells from iPS cells to confirm the activation method of iPS cell-derived ⁇ T cells.
- Fig. 2 shows the results of investigations on IL-2 and/or IL-15 regarding the method of activating iPS cell-derived ⁇ T cells.
- FIG. 22A shows the results of confirming the number of viable cells
- FIG. 22B shows the results of evaluating CD3 + / ⁇ TCR + cells by flow cytometry.
- ⁇ T cells obtained by inducing differentiation from the ⁇ T cell-derived iPS cell line 121-3 are shown.
- FIG. 23A shows the results of genomic PCR confirming the rearrangement of TCR genes (V ⁇ 9, V ⁇ 2) in undifferentiated iPS cells (121-3 strain) and ⁇ T cells obtained by differentiation induction therefrom.
- FIG. 23B shows the results of confirming the sequences of TCR ⁇ and TCR ⁇ of ⁇ T cells obtained by expanding and culturing ⁇ T cells and peripheral blood mononuclear cells using a next-generation sequencer.
- ⁇ T cells derived from iPS cells on day 39 of differentiation induction or ⁇ T cells obtained by expanding and culturing peripheral blood mononuclear cells were co-cultured with Jurkat cells for 4 hours, and then the expression of IFN ⁇ was evaluated by flow cytometry. show.
- Example 13 A cell population containing ⁇ T cells derived from iPS cells on day 40 of differentiation induction obtained by differentiation induction using a feeder cell and a cell population containing ⁇ T cells obtained by expanding and culturing peripheral blood mononuclear cells (CD3 It shows the results of flow cytometry evaluation of the expression of various surface markers in TCR ⁇ 9-positive or TCR ⁇ 9-positive).
- FIG. 26A shows a protocol in which the step of stimulating ⁇ T cells is performed from day 17.
- FIG. 26B shows the results of flow cytometry evaluation of CD3/ ⁇ TCR expression in cells on day 17 of induction of differentiation.
- FIG. 26C shows the results of flow cytometry evaluation of CD3/CD7 expression in cells on day 24 of induction of differentiation.
- FIG. 1 shows the results of investigations of IL-2 or IL-15, or IL15 or IL-15+HMBPP regarding the method of activating iPS cell-derived ⁇ T cells.
- FIG. 27A shows the results of flow cytometry evaluation of CD3/ ⁇ TCR expression in cells on day 37 or 33 of induction of differentiation.
- FIG. 27B shows the results of flow cytometry evaluation of CD3/CD7 expression in cells on day 23 of induction of differentiation.
- 24 shows the results of confirming the cytotoxic activity against Jurkat cells after freezing and thawing the cells on the 24th day of induction of differentiation under the condition that no feeder cells were used.
- FIG. 17 shows the results of confirming the cytotoxic activity against Jurkat cells after freezing and thawing the cells on the 24th day of induction of differentiation under the condition that no feeder cells were used.
- FIG. 29A shows the results of flow cytometry evaluation of CD34/CD43 expression in cells on day 10 of induction of differentiation.
- FIG. 29B shows the results of freezing and thawing the cells on day 10 of differentiation induction, and evaluating the expression of CD3/ ⁇ TCR on the cells on day 37 of differentiation induction by flow cytometry.
- FIG. 29C shows the results of confirming the cytotoxic activity against Jurkat cells of cells on day 37 of induction of differentiation. (Example 18)
- FIG. 30A shows a protocol for inducing differentiation under serum-free conditions without using feeder cells after freezing and thawing iPS cell-derived progenitor cells.
- FIG. 30B shows the results of flow cytometry evaluation of CD3/ ⁇ TCR expression in cells on day 17 of induction of differentiation.
- FIG. 31A shows a protocol for inducing the differentiation of blood cell progenitor cells into ⁇ T cells under hypoxic conditions.
- FIG. 31B shows the results of flow cytometry evaluation of CD3/CD7 expression in cells on day 17 of induction of differentiation.
- FIG. 31C shows the results of confirming the cytotoxic activity against Jurkat cells of cells on day 29 of induction of differentiation.
- Fig. 10 shows that iPS cell-derived ⁇ T cells were induced to differentiate under animal-derived component-free conditions.
- FIG. 10 shows that iPS cell-derived ⁇ T cells were induced to differentiate under animal-derived component-free conditions.
- FIG. 32A shows the results of flow cytometry evaluation of CD3/CD7 expression in cells on day 17 of induction of differentiation.
- FIG. 32B shows the results of confirming the cytotoxic activity against Jurkat cells of cells on day 31 of induction of differentiation. (Example 21) It shows the absence of undifferentiated cells in the cell population.
- FIG. 33A shows the results of flow cytometry evaluation of the expression of the undifferentiated marker TRA-1-85 in the cell population on day 35 of induction of differentiation under serum-free conditions without using feeder cells.
- FIG. 33B shows a protocol for confirming the appearance of colonies of undifferentiated cells for a cell population.
- FIG. 33C shows that no colonies of undifferentiated cells appear for the cell population.
- FIG. 34A shows purification of CD3/ ⁇ T-positive cells from a cell population under serum-free conditions without using feeder cells.
- FIG. 34B further shows the results of confirming the cytotoxic activity against Jurkat cells of the purified cells.
- the present invention relates to iPS cell-derived ⁇ T cells, which are iPS cell-derived T cells characterized by having antigen-specific cytotoxic activity in an MHC-unrestricted manner.
- ⁇ -type T cells whose T cell receptor (TCR) is composed of ⁇ and ⁇ chains
- TCR T cell receptor
- ⁇ -type T cells composed of ⁇ and ⁇ chains.
- TCR T cell receptor
- ⁇ T cells refers to ⁇ T cells.
- ⁇ T cells In the blood, ⁇ T cells account for the majority, whereas ⁇ T cells are a minority, accounting for 1-5% of all T cells.
- ⁇ T cells can be regarded as an element of the adaptive immune system because of the rearrangement of the TCR gene to bind to various antigens and the presence of memory cells. It also has the function of attacking, for example, tumor cells by antigen recognition similar to NK cells of innate immune cells.
- ⁇ T cells are considered to have both innate and adaptive immune system functions.
- ⁇ T cell-derived cytotoxic T cells (CTL) against tumor antigens can be said to be an adaptive immune system that requires antigen information from dendritic cells.
- CTL cytotoxic T cells
- iPS cells refer to undifferentiated cells established by reprogramming somatic cells by various methods.
- the iPS cells that are the starting material for the present invention are preferably iPS cells that are not iPS cells having the ⁇ TCR rearrangement gene. Most preferred are iPS cells having a ⁇ TCR rearrangement gene.
- the iPS cells having the ⁇ TCR-rearranged gene are hereinafter simply referred to as “ ⁇ TCR-type iPS cells”.
- ⁇ TCR-rearranged gene refers to a TCR-encoding gene in which both the TCRG region and the TCRD region are rearranged.
- the TCRG region consists of V ⁇ -J ⁇ and the TCRD region consists of V ⁇ -D ⁇ -J ⁇
- the iPS cells in the present specification can be produced by a method known per se or any method that will be developed in the future. For example, it can be produced based on the descriptions of Patent Document 1 and Non-Patent Document 1.
- the iPS cells used for producing the ⁇ T cells of the present invention can be produced by a method known per se or any method that will be developed in the future. Specifically, it can be produced by the method described in Patent Document 1 or Non-Patent Document 1, for example. For example, they can be produced by a method for producing iPS cells, including the following steps 1) to 3).
- IL-2 and bisphosphonates e.g., zoledronic acid, pamidronic acid, alendronic acid, risedronic acid, ibandronic acid, incadronic acid, etidronic acid, minodronic acid, their salts and their hydrates stimulating with one or more, preferably zoledronic acid
- IL-2 and bisphosphonates e.g., zoledronic acid, pamidronic acid, alendronic acid, risedronic acid, ibandronic acid, incadronic acid, etidronic acid, minodronic acid, their salts and their hydrates stimulating with one or more, preferably zoledronic acid
- introducing at least four genes capable of expressing cell reprogramming factors e.g., OCT3/4, SOX2, KLF4 and c-MYC
- SeV Sendai virus
- StemFit (R) AK02N (trade name), StemFit (R) AK03N (trade name), ReproStem (trade name), iPSellon (trade name), and Essential 8 (trade name) are basal media that can be used to maintain and culture iPS cells. ), TeSR-E8 (trade name), and various other stem cell maintenance media can be used. Especially preferred is StemFit (R) AK02N (trade name). Substances added to each medium can be appropriately increased or decreased depending on the purpose. Y27632, which is a Rho-Associated Coil Kinase (ROCK) inhibitor, can be used as an example of the substance to be added.
- ROCK Rho-Associated Coil Kinase
- a laminin 511-E8 fragment can be used in culture substrates such as culture dishes to promote cell attachment and proliferation.
- culture substrates such as culture dishes to promote cell attachment and proliferation.
- iMatrix-511 silk (trade name) and iMatrix-511 (trade name) can be used. Manufacturers and distributors of reagents and the like to be used are not particularly limited as long as they can exhibit equivalent functions.
- a protease such as trypsin can be used to detach the cells from the culture vessel.
- TrypLE Select (trade name) can be used.
- iPS cells are first induced to differentiate into blood cell progenitor cells.
- iPS cell-derived ⁇ T cells are produced by using cells that have been induced to differentiate from iPS cells into ⁇ T cells as starting materials, and performing a differentiation-inducing treatment step from hemocyte progenitor cells to ⁇ T cells. can be a method. Furthermore, it can be a method for producing iPS cell-derived ⁇ T cells that includes a step of converting iPS cells into blood cell progenitor cells.
- iPS cells obtained by freezing and thawing iPS cell-derived progenitor cells can also be used in the method of the present invention.
- the freezing period is not particularly limited, it may be, for example, 2 weeks to 1 year.
- the iPS cells of the present invention are preferably iPS cells other than iPS cells having the ⁇ TCR rearrangement gene. ⁇ TCR-type iPS cells are most preferred.
- the process of inducing differentiation from iPS cells to blood cell progenitor cells is not particularly limited, and any process known per se or any process that will be developed in the future can be adopted.
- the medium contains, for example, FLT3L (tyrosine kinase 3 ligand), SCF (stem cell factor), BMP4 (bone morphogenetic protein-4), bFGF (basic fibroblast growth factor), VEGF (vascular endothelial growth factor), IL-6, IGF-1 (insulin-like growth factors), IL-7, IL-11, EPO (erythropoietin), TPO (thrombopoietin), IL-15, IL-3, etc. It is possible to appropriately select and add one or a plurality of types.
- FBS fetal bovine serum
- FCS fetal calf serum
- the iPS cells of the present invention can be cultured, for example, in the media shown in the following 1-1) to 1-4) and subjected to differentiation induction treatment under conditions that do not use feeder cells.
- a ROCK inhibitor at a final concentration of 0-50 ⁇ M, preferably 1-30 ⁇ M, more preferably 10 ⁇ M
- Laminin-511 E8 fragment such as iMatrix-511 (trade name)
- 0-50 ⁇ l preferably 1 ⁇ 30 ⁇ l , more preferably around 5 ⁇ l
- the frequency of medium exchange, the amount of medium exchange, and the like are not particularly limited, and an appropriate frequency and amount can be determined as appropriate.
- the number of cells to be seeded can be increased or decreased as appropriate.
- the reagents and the like to be used are not particularly limited in terms of manufacturers and distributors, as long as they can exhibit equivalent functions. All cultures can be performed at 37 ⁇ 0.5°C and 5% CO2 conditions.
- a protease such as trypsin, such as TrypLE Select (trade name)
- TrypLE Select trade name
- Day 0 of Differentiation Induction StemFit (R) AK02N (trade name) can be used as a basal medium.
- Further GSK-3 ⁇ / ⁇ inhibitor (CHIR99021, CAS number: 252917-06-9) 0-20 ⁇ M, preferably 0.5-10 ⁇ M, more preferably 4 ⁇ M, BMP4 0-400 ng/ml, preferably 10-200 ng/ ml, more preferably 80 ng/ml, VEGF 0-400 ng/ml, preferably 10-200 ng/ml, more preferably 80 ng/ml.
- Day 2 of Differentiation Induction Advanced DMEM/F12 (trade name) or Essential 6 (trade name) can be used as a basal medium.
- Further selective ALK5, 4, 7 inhibitor (SB431542) 0-20 ⁇ M, preferably 0.5-10 ⁇ M, more preferably 2-4 ⁇ M, bFGF 0-200 ng/ml, preferably 1-100 ng/ml, more preferably 50 ng/ml, SCF 0-200 ng/ml, preferably 1-100 ng/ml, more preferably 50 ng/ml and VEGF 0-400 ng/ml, preferably 10-200 ng/ml, more preferably It can be cultured in a culture system containing 80 ng/ml.
- L-Glutamine, penicillin/streptomycin, differentiation-inducing supplements for iPS/ES cells for example, StemFit (trade name) For Differentiation: hereinafter "AS401"
- AS401 StemFit
- the optimum addition amount can be determined as appropriate.
- Day 4 of Differentiation Induction Advanced DMEM/F12 (trade name) or StemPro-34 SFM (trade name) can be used as a basal medium.
- Days 6 to 8 of Differentiation Induction Advanced DMEM/F12 (trade name) or StemPro-34 SFM (trade name) can be used as a basal medium.
- L-Glutamine 0-50 mM, preferably 1-20 mM, more preferably 2 mM, IL-3 0-200 ng/ml, preferably 1-100 ng/ml, more preferably 50 ng/ml, IL -6 0-200 ng/ml, preferably 1-100 ng/ml, more preferably 50 ng/ml, SCF 0-200 ng/ml, preferably 1-100 ng/ml, more preferably 50 ng/ml and EPO 0-100 IU/ml, preferably 1-50 IU/ml, more preferably 10 IU/ml.
- penicillin/streptomycin, differentiation-inducing supplements for iPS/ES cells for example, AS401
- the optimum addition amount can be determined
- Feeder cells can be co-cultured when iPS cells are cultured or iPS cells are subjected to differentiation induction treatment.
- feeder cells for example, MEF (mouse embryonic fibroblast), OP9, OP9/DLL1, OP9-DL4, and 10T1/2/DL4 cell line selected from one or more cell lines to use can be done.
- MEF mouse embryonic fibroblast
- OP9, OP9/DLL1, OP9-DL4, and 10T1/2/DL4 cell line selected from one or more cell lines to use can be done.
- OP9 embryonic fibroblast
- OP9/DLL1 OP9-DL4 cell line selected from one or more cell lines to use
- 10T1/2/DL4 cell line selected from one or more cell lines to use
- cells obtained by differentiation induction of iPS cells are administered to humans by cell therapy or the like, a stable production method that does not contain animal-derived substances is desired.
- differentiation into the ⁇ T cells of the present invention can be induced without
- induction of differentiation from iPS cell-derived progenitor cells to ⁇ T cells In the process of inducing the differentiation of iPS cell-derived blood cell progenitor cells into ⁇ T cells, co-culturing with feeder cells may be performed, or culture may be performed under conditions in which feeder cells are not used. Furthermore, the cells may be cultured under serum-free conditions or animal-derived components-free conditions. In addition, the process of inducing differentiation from iPS cell-derived blood cell progenitor cells to ⁇ T cells can also be performed by culturing under hypoxic conditions.
- hypoxic conditions mean that the O 2 concentration in the culture conditions during the differentiation induction process from iPS cell-derived blood progenitor cells to ⁇ T cells is lower than the O 2 concentration in normal culture.
- the O 2 concentration for culturing under hypoxic conditions is not particularly limited, but is, for example, less than 20% (v/v), preferably less than 10% (v/v).
- a ⁇ T cell stimulating agent may be added, or may not be added depending on the culture conditions.
- ⁇ T cell stimulators include phosphate compounds that are metabolites of the mevalonate pathway or non-mevalonate pathway of the isoprenoid biosynthetic pathway, or derivatives thereof.
- phosphate compounds that are metabolites of the mevalonate pathway or non-mevalonate pathway of the isoprenoid biosynthetic pathway include, for example, HMBPP ((E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate) and IPP ( isopentenyl diphosphate).
- the derivative include BrHBP (bromohydrin diphosphate).
- ⁇ T cell stimulating agents also include specific inhibitors of FPP (farnesyl pyrophosphate) synthase, which is the rate-limiting enzyme in the biosynthetic pathway.
- FPP farnesyl pyrophosphate
- a specific inhibitor of FPP synthase promotes intracellular accumulation of the phosphate compound.
- FPP synthase-specific inhibitors include nitrogen-containing bisphosphonates (N-BPs), specifically zoledronic acid and pamidronate.
- N-BPs nitrogen-containing bisphosphonates
- IL-15 and 1L-2 also function as ⁇ T cell stimulators.
- ⁇ MEM (trade name) can be used as a basal medium for culturing 10 days after induction of differentiation from iPS cells (blood cell progenitor cells) by the above treatments 1-1) to 1-4).
- penicillin/streptomycin and the like can be appropriately selected and added.
- 0.1% Polyvinyl alcohol + 4% B27 (trade name) supplement may be used instead of FBS. Manufacturers and distributors of reagents and the like to be used are not particularly limited as long as they can exhibit equivalent functions. The optimum addition amount can be determined as appropriate.
- Culture can be performed by seeding cells (blood cell progenitor cells) 10 days after induction of differentiation on a culture substrate such as a culture plate seeded with feeder cells.
- the medium can be replaced, for example, every two days, and the supernatant can be recovered by pipetting 12 days, 18 days and 24 days after induction of differentiation, transferred to new feeder cells, and culture continued.
- the frequency of medium exchange, the amount of medium exchange, and the like are not particularly limited, and an appropriate frequency and amount can be determined as appropriate.
- A-2) 30 days or 31 days after differentiation induction Cells cultured in the above medium from day 10 to day 30 or 31 after induction of differentiation can be cultured under conditions in which feeder cells are not used.
- RPMI1640 medium can be used as a basal medium for culture under such conditions.
- it can be cultured in a medium containing 0-30%, preferably 0-20%, more preferably 10-20% FBS. 0.1% Polyvinyl alcohol + 4% B27 (trade name) supplement may be used instead of FBS.
- IL-2 and/or IL-15 0 to 200 ng/ml, preferably 1 to 100 ng/ml, more preferably 10 ng/ml, or Immunace (trade name ) 0-1000 IU/ml, 10-500 IU/ml, preferably 100 IU/ml and 2-Me (2-Mercaptoethanol) 0-100 ⁇ M, 1-50 ⁇ M, preferably 10 ⁇ M. good.
- penicillin/streptomycin and the like can be added as appropriate.
- HMBPP may be added as a ⁇ T cell stimulant.
- concentration to be added is not particularly limited as long as it stimulates ⁇ T cells and does not cause cytotoxicity.
- Day 10 of differentiation induction For example, culture after 10 days (blood cell progenitor cells) after induction of differentiation from iPS cells by the above treatments 1-1) to 1-4), VCAM1 (vascular cell adhesion molecule-1) and DLL4 (Delta-Like Protein 4) or can be cultured using a culture substrate coated with DLL1 (Delta-Like Protein 1). From 10 to 24 days after induction of differentiation, the cells can be cultured, for example, in Lymphoid progenitor Expansion Medium (trade name) included in the StemSpan TM T cell generation kit (trade name). Medium exchange was performed according to the StemSpan TM kit protocol.
- Lymphoid progenitor Expansion Medium (trade name) included in the StemSpan TM T cell generation kit (trade name). Medium exchange was performed according to the StemSpan TM kit protocol.
- an additional medium can be added on day 13 of differentiation induction, and the medium can be replaced on days 17 and 20 of differentiation induction, respectively. It can be replaced with T cell progenitor maturation medium (trade name) included in the above-mentioned kit at around 17 to 24 days of differentiation induction. On the 27th day of differentiation induction, the above medium is additionally added, and thereafter, the medium can be replaced about twice a week, such as on the 31st and 34th days of differentiation induction.
- the frequency of medium exchange, the amount of medium exchange, and the like are not particularly limited, and an appropriate frequency and amount can be determined as appropriate.
- the cells can be continuously cultured by the method described in B-1), they can be cultured in a medium supplemented with a ⁇ T cell stimulating agent from around 17 to 24 days after differentiation induction. From around 17th to 24th day of induction of differentiation, the number of cells tends to decrease, which can be improved by adding a ⁇ T cell stimulant.
- a medium shown in A-2 in which IL-2 and/or IL-15 and ⁇ T cell stimulating agents such as HMBPP and FPP synthase-specific inhibitors are added.
- HMBPP and FPP synthase-specific inhibitors can.
- RPMI1640 medium containing AS401 in which IL-2 and/or IL-15 and HMBPP are added.
- the cells can be cultured in a medium supplemented with a ⁇ T cell stimulating agent.
- the cells can be cultured in the medium shown in A-2, in which HMBPP is added. It is also possible to culture in a medium containing no FBS among the medium shown in A-2 and similarly containing HMBPP.
- RPMI1640 medium containing AS401 which is a medium supplemented with IL-2 and/or IL-15 and a ⁇ T cell stimulant such as HMBPP, can also be used.
- Cells cultured by the differentiation induction method of the present invention can be cultured using beads.
- the size of the beads is not particularly limited, and may be smaller than the cell size or larger than the cell size.
- beads can be mixed in the culture medium and cultured.
- the beads are not particularly limited as long as they are made of a material that can be used for cell culture. Specifically, Dynabeads Protein G (trade name) can be used.
- VCAM1 and DLL4 the cells can be cultured under feeder-free conditions.
- Cells cultured by the differentiation induction method of the present invention can also be cultured under conditions using a medium that does not contain animal-derived components. For example, culture after 10 days (blood cell progenitor cells) after induction of differentiation from iPS cells by the above treatments 1-1) to 1-4), VCAM1 (vascular cell adhesion molecule-1) and DLL4 (Delta-Like Protein 4) or can be cultured using a culture substrate coated with DLL1 (Delta-Like Protein 1).
- RPMI1640 containing AS401 can be used as a basal medium as a medium containing no animal-derived components.
- SCF, IL-7, FLT3L, L-ascorbic adid, IL2, TPO, etc. shown in A-1 may be included.
- the cells can be cultured in a medium supplemented with IL-2, IL-15, and ⁇ T cell stimulating agent shown in A-2.
- a medium can be RPMI1640 containing AS401 as a basal medium.
- cells can be cultured in a medium supplemented with one or more of IL-2, IL-15 and HMBPP.
- ⁇ T cells produced by the method of inducing differentiation of the present invention are T cells that have a unique T cell receptor (TCR) consisting of ⁇ and ⁇ chains on their surface. Such cell surfaces can be checked for expression of cell markers such as CD3, CD7, CD8a, CD45RA and ⁇ TCR.
- the ⁇ T cells of the present invention preferably express one or more selected from CD7, CD8a and CD45RA, while not expressing one or more selected from CD25, IFN ⁇ , CD5 and CD27. is preferred.
- the obtained iPS cell-derived ⁇ T cells are characterized by having non-MHC-restricted antigen-specific cytotoxic activity.
- iPS cell-derived ⁇ T cells tend to express CD7 and CD8a higher
- ⁇ T cells isolated from peripheral blood tend to express IL2RA (CD25), CD5, and IFN ⁇ higher.
- CD45RA tends to be expressed more in iPS cell-derived ⁇ T cells
- CD27 tends to be expressed more in ⁇ T cells isolated from peripheral blood.
- T cells induced to differentiate in this way can be isolated by appropriately selecting known techniques.
- known techniques include, for example, flow cytometry using antibodies against cell surface markers and a cell sorter, as shown in Examples below.
- T cells having desired antigen specificity are isolated from humans, a method of purification using an affinity column or the like on which the desired antigen is immobilized can also be employed.
- a cell population of purified ⁇ T cells is composed of homogeneous cells and is distinguished from a cell population composed of ⁇ T cells isolated from peripheral blood, and the ⁇ T cell population of the present invention is composed of ⁇ T cells isolated from peripheral blood. It has a high antigen-specific cytotoxic activity compared to the husk.
- the cell population containing the ⁇ T cells contains, for example, many cells having the same nucleotide sequence in the complementarity determining regions (CDRs) of the TCR gene.
- CDRs complementarity determining regions
- ⁇ T cells having the same nucleotide sequence in the CDR3 region in particular are included in a large proportion of the ⁇ T cells constituting the cell population, for example, 90% or more.
- a cell population containing ⁇ T cells of the present invention can contain 1 ⁇ 10 5 or more ⁇ T cells.
- ⁇ T cells exhibiting a higher expression level of CD7 and/or CD8a than ⁇ T cells isolated from peripheral blood are ⁇ T cells that constitute the cell population. contained at a rate of 90% or more of Furthermore, 90% of the ⁇ T cells constituting the cell population are ⁇ T cells that exhibit a lower expression level of one or more expression levels selected from CD25, INF ⁇ , and CD5 than the ⁇ T cells isolated from peripheral blood. It is included in the ratio above.
- ⁇ T cells exhibiting a higher expression level of CD45RA than ⁇ T cells isolated from peripheral blood and expanded in vitro and a lower expression level of CD27 than ⁇ T cells isolated from peripheral blood and expanded in vitro are Contained in 70% or more of the ⁇ T cells that make up the cell population.
- the cell population containing ⁇ T cells of the present invention is characterized in that undifferentiated cells account for 10% or less of the ⁇ T cells that make up the cell population, and the undifferentiated cells are present in the ⁇ T cells that make up the cell population. preferably not. Whether a certain cell is an undifferentiated cell can be determined by a marker indicating undifferentiation such as TRA-1-85.
- ⁇ T cells produced by treatment with the differentiation-inducing treatment method of the present invention have excellent immune functions, and therefore are used for the treatment or prevention of diseases such as tumors, infectious diseases (e.g., viral infections), and autoimmune disorders. can be used. Furthermore, it can be used as an antigen-specific cellular immunotherapeutic agent or pharmaceutical composition containing the ⁇ T cell population produced by the method of the present invention as an active ingredient.
- the ⁇ T cells produced by the differentiation-inducing treatment method of the present invention can be used for these formulations even after freezing and thawing. It is expected that the ⁇ T cell population can also be applied to immune cell therapy methods.
- the ⁇ T cell population of the present invention is expected to further enhance the effects of ⁇ T cells when used in combination with an immune checkpoint inhibitor.
- Immune checkpoint inhibitors are not limited to those known per se or those to be developed in the future, but include, for example, drugs targeting immune checkpoints such as PD-1, PD-L1 and CTLA-4.
- Antibody-dependent cellular cytotoxicity (ADCC) is expected to enhance the effects of molecular targeted drugs and antibody preparations (e.g. Herceptin, Rituxan, etc.) that are used to treat various cancers, similar to NK cells.
- a high therapeutic effect can be expected when used in combination with a preparation.
- a pharmaceutical composition containing the ⁇ T cell population of the present invention can be prepared by formulating with a known pharmaceutical method.
- pharmacologically acceptable carriers or media specifically sterile water, physiological saline, vegetable oils, solvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, vehicles, Appropriate combination with preservatives, binders, diluents, tonicity agents, soothing agents, bulking agents, disintegrants, buffers, coating agents, lubricants, coloring agents, solubilizers, or other additives. can be done. In addition, it may be used in combination with known pharmaceutical compositions, immunostimulants, and the like used for treatment or prevention of the aforementioned diseases. When administering the pharmaceutical composition of the present invention, the dosage is appropriately selected according to the subject's age, body weight, symptoms, health condition, type of composition, and the like.
- the present invention also includes an antigen-specific cellular immunotherapy method by administering the iPS cell-derived ⁇ T cells of the present invention. Furthermore, the present invention also includes therapeutic methods for diseases such as cancer, infectious diseases, and autoimmune disorders by administering the iPS cell-derived ⁇ T cells of the present invention.
- the dosage of the active ingredient to the subject varies depending on the body weight, age, symptoms, administration method, etc. of the subject, and can be appropriately selected by those skilled in the art.
- Example 1 Induction of Differentiation from iPS Cells
- Example 2 Induction of Differentiation from iPS Cells
- a method for inducing differentiation of ⁇ T cells produced from ⁇ TCR-type iPS cells produced by the method of Non-Patent Document 1 will be described.
- 0.5 ⁇ TrypLE TM select (manufactured by ThermoFisher) was used for detachment and dispersion of cells during passage, and for subculture, StemFit (R) AK02N with Y27632 (manufactured by Wako Pure Chemical Industries) at a final concentration of 10 ⁇ M and iMatrix-511 A culture medium added to 3.2 ⁇ l was used. The next day, the medium was replaced with StemFit (R) AK02N containing no Y27632 and iMatrix-511, and thereafter the medium was replaced every two days. A medium was added at 1.5 ml/well. All cultures, including the following steps and examples described later, were performed under conditions of 37 ⁇ 0.5° C. and 5% CO 2 .
- Example 2 Differentiation induction from iPS cells
- medium components and differentiation induction after 10 days of differentiation induction The medium components on and after the 31st day are different from those in Example 1.
- medium components after 31 days of induction of differentiation contain HMBPP, which is a ⁇ T cell stimulant.
- Example 3 Differentiation induction from iPS cells under conditions using feeder cells
- ⁇ TCR-type iPS cells prepared by the method of Non-Patent Document 1 were prepared by differentiation induction treatment. ⁇ T cells are shown. Differentiation induction treatment was performed in the same manner as in Example 1, and after the 31st day, half of the ⁇ T cell stimulation medium (containing HMBPP and FBS) was replaced every 2 days in the same manner as in (2-4) of Example 2. A cytotoxicity assay was then performed along with evaluation of marker expression.
- cytotoxicity assay was performed on Jurkat cells. 5 ⁇ 10 4 CFSE-stained Jurkat cells were added per well of a 96-well culture dish, and 1 ⁇ 10 5 iPS cell-derived ⁇ T cells on day 55 of differentiation induction were added. After culturing at 2:1 for 16 hours, 7-AAD staining (dead cell staining) was performed. Many Jurkat cells (CFSE-positive cells) were 7-AAD-positive, and many dead cells were confirmed. That is, it was confirmed that the iPS cell-derived ⁇ T cells have a cytotoxic function against tumor cells (Fig. 3C).
- Example 4 Differentiation induction from iPS cells under conditions without feeder cells
- ⁇ TCR-type iPS cells prepared by the method of Non-Patent Document 1 were prepared by differentiation induction treatment.
- ⁇ T cells a method of inducing differentiation without using feeder cells is shown.
- the differentiation-inducing treatment was performed according to the following procedure according to the protocol shown in FIG.
- Example 5 Induction of differentiation from iPS cells under conditions without feeder cells
- ⁇ T cells prepared from ⁇ TCR-type iPS cells by differentiation induction treatment in the same manner as in Example 4 were treated under conditions without feeder cells.
- the differentiation induction method in is shown.
- the differentiation-inducing treatment was performed according to the following procedure according to the protocol shown in FIG.
- Example 6 Induction of differentiation from iPS cells under conditions without feeder cells
- ⁇ T cells prepared from ⁇ TCR-type iPS cells by differentiation induction treatment in the same manner as in Example 4 were treated under conditions without feeder cells.
- the differentiation induction method in is shown.
- the differentiation-inducing treatment was performed according to the following procedure according to the protocol shown in FIG.
- the cells were treated in the same manner as (4-1)-(4-3) in Example 4, and cultured under the condition that neither feeder cells nor serum was used on days 10 to 24 of induction of differentiation.
- (6-2) Day 24 of differentiation induction (day24) On day 24 of differentiation induction, a. ⁇ T cell stimulation medium (containing HMBPP and FBS) shown in Table 7 of Example 2 (2-4), b. ⁇ T cell stimulation medium shown in Table 7 (10% FBS/RPMI1640) instead of RPMI1640 (including HMBPP) medium containing AS401 and c. Lymphoid progenitor expansion medium included in the StemSpan TM kit. was replaced.
- Example 7 Induction of Differentiation from iPS Cells without Feeder Cells
- ⁇ TCR-type iPS cells were differentiated in the same manner as in Example 4 to produce ⁇ T cells.
- Example 8 Differentiation induction method using magnetic beads
- VCAM1 and DLL4-coated magnetic beads were mixed and cultured under conditions that did not use feeder cells, thereby producing T cells. Differentiation was induced.
- Example 9 ⁇ T cells produced from ⁇ TCR-type iPS cells
- the characteristics of ⁇ T cells produced from ⁇ TCR-type iPS cells were confirmed.
- a method for producing iPS cell-derived ⁇ T cells is shown, and then various characteristics of the cells are shown.
- ⁇ Day 0 of differentiation induction (Day0): State of ⁇ TCR-type iPS cells (HPC1) Stemfit AK02N (Ajinomoto, Tokyo, Japan, AK02N) CHIR99021 (Tocris, Bristol, UK, 4423) 4 ⁇ M BMP4 (R&D, Minneapolis, MN, 314-BP) 80 ng/ml VEGF (R&D, Minneapolis, MN, 293-VE) 80ng/ml
- ⁇ From day 30 of differentiation induction (Day 30-): Cultivation in ⁇ T active medium
- ⁇ T active medium Accutase-treated cells were suspended in the following ⁇ T active medium and cultured in a feeder cell-free medium. Thereafter, half of the medium was replaced every 2 days. Cells at 7-14 days of active culture were subjected to a cytotoxicity assay.
- ⁇ T activity medium RPMI1640 (Nacalai Tesque, Kyoto, Japan, 30264-56) FBS (Sigma-Aldrich, St. Louis, MO, F7524) 10% HMBPP (Cayman chemical, Ann Arbor, MI, 13580) 1 nM Immunace (Shionogi pharmaceuticals, Osaka, Japan) 100IU/ml 2-Me (Nacalai Tesque, Kyoto, Japan) 10 ⁇ M
- ⁇ T cells 9-3) Antitumor effect Antitumor activity against various tumor cells was confirmed using iPS cell-derived ⁇ T cells on day 38 of differentiation induction (Fig. 17). Unpurified ⁇ T cells were used in these experiments. As a control, conditions were used in which only tumor cells were cultured without addition of ⁇ T cells.
- a cytotoxicity assay was performed on Huh-7 cells (derived from human hepatoma cells).
- E:T (effector:target) ratio 2:1, 5 x 10 4 Huh-7 cells stained with fluorescent dye CFSE were added to one well of a 96-well culture dish, and 1 x 10 5 iPS cells were added to each well.
- the tumor area was measured by observing with a phase-contrast microscope.
- the ⁇ T cells of the present invention clearly had higher cytotoxic activity against Huh-7 cells than the control (Fig. 17B).
- a cytotoxicity assay was performed on SW480 cells (derived from human colon cancer).
- E:T (effector:target) ratio 2:1, 5 ⁇ 10 4 SW480 cells stained with the fluorescent dye CFSE were added to one well of a 96-well culture dish, and 1 ⁇ 10 5 iPS cell-derived ⁇ T cells were added to each well. After adding the cells and culturing for 16 hours, the tumor area was measured by observing with a phase-contrast microscope.
- the ⁇ T cells of the present invention clearly had higher cytotoxic activity against SW480 cells than the control (Fig. 17C).
- iPS cell-derived ⁇ T cells on day 36 of differentiation induction, maintenance of TCR rearrangement and cytotoxic mechanism were confirmed (Fig. 18).
- A. Non-purified iPS cell-derived ⁇ T cells (igdT) and peripheral blood mononuclear cells (PB) were evaluated for cell surface ⁇ TCR expression. Expression of ⁇ TCR was detected in PB, but not in ⁇ T cells (igdT) of the present invention (Fig. 18A).
- igdT Non-purified iPS cell-derived ⁇ T cells
- PB peripheral blood mononuclear cells
- ⁇ TCR was detected in PB, but not in ⁇ T cells (igdT) of the present invention (Fig. 18A).
- B. Genomic PCR for TCR gene rearrangements The rearrangement of TCR genes (Vg9, Vd2) was confirmed by genomic PCR.
- ⁇ T cells sorted by flow cytometry were confirmed to retain the TCR gene rearrangement as in the undifferentiated state (Fig. 18B).
- Peripheral blood mononuclear cells PBMC
- Cytotoxicity assays were performed on ⁇ T cells (igdT) purified by flow cytometry (FACS). The conditions for the cytotoxicity assay are the same as A. of (9-3). was performed by the method shown in .
- As a control (ctrl) in FIG. 18D only Jurkat cells were cultured without adding iPS cell-derived ⁇ T cells.
- iPS cell-derived ⁇ T cells that have not been purified are indicated as bulk, and purified iPS cell-derived ⁇ T cells are indicated as sort.
- iPS cell-derived ⁇ T cells there was no significant difference in cell death rate between the presence and absence of purification (Fig. 18D).
- HLA types of iPS cell lines and tumor cells Table 8 shows the results of confirming the HLA types of the iPS cells used in the iPS cell-derived ⁇ T cells of the present invention and the tumor cells used in Examples 3 and 6 and this example. show. Although the HLA type of iPS cells did not match the HLA type of each tumor cell, an antitumor effect was observed for each tumor cell (this example AC). As a result, it was confirmed that the iPS cell-derived ⁇ T cells of the present invention have antigen-specific cytotoxic activity in an MHC-unrestricted manner.
- Example 10 Comparison of iPS cell-derived ⁇ T cells and ⁇ T cells isolated from peripheral blood
- iPS cell-derived ⁇ T cells prepared by inducing the differentiation of iPS cells and ⁇ T present in peripheral blood Cell surface expression marker genes in cells (PB-gdT) were compared.
- the iPS cell-derived ⁇ T cells of this example were cultured by the method shown in Example 1 and the method shown in Example 9 (9-1), and cells after 36 to 42 days of induction of differentiation were used.
- Cells obtained by culturing mononuclear cells isolated from peripheral blood in the ⁇ T active medium shown in Example 9 (9-1) were used as ⁇ T cells isolated from peripheral blood in this example.
- Example 11 Method for activating iPS cell-derived ⁇ T cells
- a method for activating iPS cell-derived ⁇ T cells was examined. Specifically, in the production method shown in (9-1) of Example 9, for cells on day 30 of differentiation induction, either IL-2 and/or IL-15 is added to the following ⁇ T activity medium. It was examined whether it is possible to produce iPS cell-derived ⁇ T cells more effectively (see FIG. 21).
- activation medium RPMI1640 (Nacalai Tesque, Kyoto, Japan, 30264-56)
- FBS Sigma-Aldrich, St. Louis, MO, F7524
- 10% HMBPP Cayman chemical, Ann Arbor, MI, 13580
- 1 nM 2-Me Nacalai Tesque, Kyoto, Japan
- Example 12 Characteristics of ⁇ T cells produced from ⁇ TCR-type iPS cells (121-3 strain) In this example, characteristics of ⁇ T cells produced from ⁇ TCR-type iPS cells (121-3 strain) were confirmed.
- TCR reconstructionA Retention of TCR reconstructionA.
- the nucleotide sequences and amino acid sequences of the CDR3 regions of each of TCR ⁇ and TCR ⁇ were identified, and the frequencies for each sequence were shown in a pie chart (Fig. 23B). It was confirmed that the PB ⁇ T cell population was composed of cells with diverse sequences, whereas the i ⁇ T cell population was composed of cells with a single type of TCR ⁇ and TCR ⁇ gene rearrangement.
- IFN ⁇ interferon gamma
- i ⁇ T iPS cell-derived ⁇ T cells
- PB ⁇ T ⁇ T cells
- Example 14 Comparison of iPS cell-derived ⁇ T cells and ⁇ T cells obtained by amplifying peripheral blood
- iPS prepared by inducing differentiation of ⁇ TCR-type iPS cells (62B3 strain or 121-3 strain) Cell surface expression markers were compared between cell-derived ⁇ T cells (igdT) and ⁇ T cells obtained by expanding peripheral blood (PB-gdT).
- iPS cell-derived ⁇ T cells (i ⁇ T CD3-positive or TCR ⁇ 9-positive cells) had a higher percentage of cells expressing CD7, a lower percentage of cells expressing CD5 and CD25, and a higher percentage of CD45RA + CD27 ⁇ cells. It was confirmed to have the characteristic of having a high ratio (Fig. 25).
- Example 15 Examination of the process of stimulating ⁇ T cells
- ⁇ T cells prepared by differentiation induction treatment from ⁇ TCR-type iPS cells in the same manner as in Example 5 were treated under conditions in which neither feeder cells nor serum were used, and ⁇ T cells were A method of inducing differentiation under the condition that the step of stimulating is performed on the 17th day instead of the 24th day is shown.
- differentiation was induced by the following procedure according to the protocol shown in FIG. 26A (New protocol).
- Example 5 The same treatment as in Example 5 (5-1) was performed and cultured. However, the step of stimulating ⁇ T cells was performed from day 17 of induction of differentiation.
- Example 16 Method for activating iPS cell-derived ⁇ T cells without feeder cells
- a method for activating iPS cell-derived ⁇ T cells without using feeder cells or serum was examined. .
- Example 15 The same treatments as in Example 15 (15-1) and (15-3) were performed, and the cells were cultured under conditions in which neither feeder cells nor serum were used. However, cells on the 17th day of induction of differentiation were tested under the same conditions as in Example 15 (15-3) and under the conditions in (15-3) in which IL-2 was replaced with IL-15. On day 33 or 37 of differentiation induction, the expression of CD3/ ⁇ TCR was evaluated by flow cytometry to determine whether iPS cell-derived ⁇ T cells could be produced more effectively. CD3 + /TCR + cells were detected, differentiation into TCR cells was confirmed, and they were confirmed to be iPS cell-derived ⁇ T cells (Fig. 27A).
- iPS cell-derived ⁇ T cells can be produced with either IL-2 or IL-15 in the step of ⁇ T cell stimulation. In addition, more iPS cell-derived ⁇ T cells were obtained with the addition of IL-15 than with IL-2.
- the expression of CD3/CD7 was evaluated by flow cytometry in cells on day 23 of induction of differentiation. CD3 + /TCR + cells were detected, differentiation into TCR cells was confirmed, and they were confirmed to be iPS cell-derived ⁇ T cells.
- iPS cell-derived ⁇ T cells were obtained even under the condition that HMBPP, which is a ⁇ TCR stimulant, was not added (Fig. 27B).
- Example 17 Cytotoxic Activity of iPS Cell-Derived ⁇ T Cells After Freezing and Thawing
- iPS cell-derived ⁇ T cells were freeze-thawed under the condition that neither feeder cells nor serum were used, and a cytotoxicity assay was performed.
- Example 15-1) The same treatment as (15-1) and (15-3) of Example 15 was performed, and culture was performed under the condition that neither feeder cells nor serum was used. However, IL-2 in (15-3) of Example 15 was replaced with IL-15. On the 24th day of differentiation induction, the cells were frozen using CS10 (manufactured by Cosmo Bio).
- Example 18 Induction of differentiation after freezing and thawing of iPS cell-derived progenitor blood cells
- differentiation was induced after freezing and thawing of iPS cell-derived progenitor blood cells to prepare ⁇ T cells.
- iPS cell-derived ⁇ T cells were added and cultured for 16 hours. Dead cells were stained with 7-AAD (7-Amino-Actinomycin D) staining. Cell death (7-AAD positive) was confirmed for many Jurkat cells (CFSE positive cells) (Fig. 29C). That is, it was confirmed that the iPS cell-derived ⁇ T cells have a cytotoxic function even after freezing and thawing.
- Example 19 Differentiation induction under conditions in which neither feeder cells nor serum was used after iPS cell-derived progenitor cells were frozen and thawed
- iPS cell-derived progenitor cells were freeze-thawed under conditions in which neither feeder cells nor serum were used. was induced to differentiate into ⁇ T cells.
- differentiation was induced by the following procedure according to the protocol shown in FIG. 30A. The freezing in this example was performed for 18 days.
- Example 20 Differentiation induction from blood cell progenitor cells under hypoxic conditions
- the conditions were carried out without using feeder cells or serum.
- we generated ⁇ T cells by inducing differentiation from blood cell progenitor cells under hypoxic conditions.
- differentiation was induced by the following procedure according to the protocol shown in FIG. 31A.
- Example 21 Induction of differentiation from iPS cells under animal-derived component-free medium
- iPS cell-derived ⁇ T cells were produced under animal-derived component-free medium.
- Example 22 Confirmation of Undifferentiated Cells for iPS Cell-Derived ⁇ T Cells
- the conditions were carried out without using feeder cells or serum.
- undifferentiated cells were confirmed for iPS cell-derived ⁇ T cells.
- Example 23 Cytotoxicity Assay for CD3/ ⁇ T-Positive Cells
- CD3/ ⁇ T-positive cells were purified from the cell population obtained by the same treatment as in Example 22, and a cytotoxicity assay was performed.
- E:T (effector:target) ratio 0.2:1, 5 ⁇ 10 4 Jurkat cells stained with the fluorescent dye CFSE were added to one well of a 96-well culture dish, and 1 ⁇ 10 5 cells on day 35 of induction of differentiation were added. iPS cell-derived ⁇ T cells were added and cultured for 16 hours. Dead cells were stained with 7-AAD (7-Amino-Actinomycin D) staining and graphed (Fig. 34B). It exhibited strong cytotoxic activity in spite of the condition of an E:T ratio of 0.2:1, in which the number of attacking (effector) cells to tumor cells was extremely small. In the cytotoxicity assay, which was previously performed using unpurified cell populations, it was clarified that the target cells, CD3/ ⁇ T-positive cells (i.e., ⁇ T cells), possessed cytotoxic activity. Became.
- ⁇ T cells can be effectively produced without burdening the blood recipient and without being affected by cell exhaustion. Furthermore, according to the production method of the present invention, excellent iPS cell-derived ⁇ T cells can be produced even by a method that does not use feeder cells. Furthermore, according to the production method of the present invention, excellent iPS cell-derived ⁇ T cells can be produced by a method that does not use feeder cells or serum, or even a method that uses a medium that does not contain animal-derived components. Further, according to the production method of the present invention, excellent iPS cell-derived ⁇ T cells can be produced even after freezing and thawing during production.
- the ⁇ T cell population produced by the method of the present invention can be a ⁇ T cell population that is more homogeneous and highly effective than the ⁇ T cell population isolated from peripheral blood, and is more effective in producing MHC-free cells. It has an excellent function of restricted antigen-specific cytotoxic activity. Furthermore, the ⁇ T cell population produced by the method of the present invention can be a ⁇ T cell population with no remaining undifferentiated cells, which is excellent for clinical application.
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Abstract
Description
1.人工多能性幹細胞(iPS細胞)由来のT細胞であって、前記T細胞がMHC非拘束性に抗原特異的細胞傷害活性を有することを特徴とするiPS細胞由来γδT細胞。
2.前記iPS細胞が、αβT細胞由来ではないiPS細胞である、前項1に記載のiPS細胞由来γδT細胞。
3.前記iPS細胞が、γδTCR再構成遺伝子を有するiPS細胞である、前項1又は2に記載のiPS細胞由来γδT細胞。
4.γδTCR再構成遺伝子を有するiPS細胞を分化誘導処理して作製されたiPS細胞由来γδT細胞。
5.γδTCR再構成遺伝子を有するiPS細胞を分化誘導処理して得た血球前駆細胞を、基本培地にFLT3L(tyrosine kinase 3 ligand)、SCF(stem cell factor)、IL-2、IL-7、TPO(thrombopoietin)、L-アスコルビン酸から選択される1種又は複数種を選択して加えた培地を用いて培養する工程を含む、iPS細胞由来γδT細胞の作製方法。
6.基本培地にFLT3L、SCF、IL-2、IL-7、TPO、L-アスコルビン酸から選択される1種又は複数種を選択して加えた培地を用いて培養する工程の後、γδT細胞刺激剤を含む培地を用いて培養する工程を含む、前項5に記載のiPS細胞由来γδT細胞の作製方法。
7.基本培地にFLT3L、SCF、IL-2、IL-7、TPO、L-アスコルビン酸から選択される1種又は複数種を選択して加えた培地を用いて培養する工程が、フィーダー細胞と共培養して培養する工程である、前項5又は6に記載のiPS細胞由来γδT細胞の作製方法。
8.基本培地にFLT3L、SCF、IL-2、IL-7、TPO、L-アスコルビン酸から選択される1種又は複数種を選択して加えた培地を用いて培養する工程が、フィーダー細胞と共培養せずに培養する工程である、前項5又は6に記載のiPS細胞由来γδT細胞の作製方法。
9.フィーダー細胞と共培養せずに培養する工程が、VCAM1(vascular cell adhesion molecule-1)、並びにDLL4(Delta-Like Protein 4)若しくはDLL1(Delta-Like Protein 1)でコートした培養基材を用いて培養する工程を含む、前項8に記載のiPS細胞由来γδT細胞の作製方法。
10.フィーダー細胞と共培養せずに培養する工程が、更にDKK1及び/又はAZA(Azelaic acid)を含む培地を用いて培養する工程を含む、前項8又は9に記載のiPS細胞由来γδT細胞の作製方法。
11.γδT細胞刺激剤を含む培地が、γδT細胞刺激剤、IL-2及びIL-15から選択される1種又は複数種を含む培地である、前項6~10のいずれかに記載のiPS細胞由来γδT細胞の作製方法。
12.γδT細胞刺激剤が、イソプレノイド生合成経路代謝物であるリン酸化合物又はその誘導体、あるいはイソプレノイド生合成経路の律速酵素であるFPP(farnesyl pyrophosphate)合成酵素の特異的阻害物質である、前項6~11のいずれかに記載のiPS細胞由来γδT細胞の作製方法。
13.血清を含まない条件で培養することを特徴とする前項6~12のいずれかに記載のiPS細胞由来γδT細胞の作製方法。
14.低酸素条件下で培養することを含む、前項6~13のいずれかに記載のiPS細胞由来γδT細胞の作製方法。
15.前項5~14のいずれかに記載のiPS細胞由来γδT細胞の作製方法により作製されたiPS細胞由来γδT細胞。
16.前項1~4及び前項15より選択されるいずれかに記載のiPS細胞由来γδT細胞を含む細胞集団。
17.末梢血から分離されたγδT細胞の細胞集団と比較して、iPS細胞由来γδT細胞を含む細胞集団が抗原特異的に高い細胞傷害活性を有することを特徴とする、前項16に記載の細胞集団。
18.γδT細胞を含む細胞集団であって、TCR遺伝子のCDR3領域が同一の塩基配列を有するγδT細胞が当該細胞集団を構成するγδT細胞の90%以上の割合で含むことを特徴とするγδT細胞の細胞集団。
19.γδT細胞が1×105個以上含まれることを特徴とする、前項18に記載の細胞集団。
20.γδT細胞を含む細胞集団であって、CD7及びCD8aの発現量について、末梢血から分離されたγδT細胞に比べて高い発現量を示すγδT細胞が、当該細胞集団を構成するγδT細胞の90%以上の割合で含むことを特徴とするγδT細胞の細胞集団。
21.γδT細胞を含む細胞集団であって、未分化細胞が当該細胞集団を構成するγδT細胞の10%以下であることを特徴とする、前項18~20のいずれかに記載の細胞集団。
22.前項1~4及び前項15より選択されるいずれかに記載のiPS細胞由来γδT細胞を有効成分とする、抗原特異的細胞性免疫治療剤。
23.液体培地中でビーズ状担体を含む培地を用いて培養することを特徴とする、前項1~4及び15より選択されるいずれかに記載のiPS細胞由来γδT細胞の培養方法。
24.前項1~4及び前項15より選択されるいずれかに記載のiPS細胞由来γδT細胞を有効成分とする、がん、感染症、自己免疫不全等の疾患の治療剤。
25.前項1~4及び前項15より選択されるいずれかに記載のiPS細胞由来γδT細胞を有効成分とする、医薬組成物。
26.前項1~4及び前項15より選択されるいずれかに記載のiPS細胞由来γδT細胞を投与することによる、抗原特異的細胞性免疫細胞治療方法。
27.前項1~4及び前項15より選択されるいずれかに記載のiPS細胞由来γδT細胞を投与することによる、がん、感染症、自己免疫不全等の疾患に対する治療方法。
本発明のγδT細胞作製のために使用するiPS細胞は、自体公知の方法あるいは今後開発されるあらゆる方法で作製することができる。具体的には例えば特許文献1や非特許文献1に記載の方法で作製することができる。例えば、以下の1)~3)の工程を含む、iPS細胞の作製方法により作製することができる。
1)採取した血液細胞を、IL-2及びビスホスホネート(例えば、ゾレドロン酸、パミドロン酸、アレンドロン酸、リセドロン酸、イバンドロン酸、インカドロン酸、エチドロン酸、ミノドロン酸、それらの塩及びそれらの水和物から選択される1種若しくは複数種であり、好ましくはゾレドロン酸)で刺激する工程;
2)センダイウイルス(SeV)ベクターを用いて、前記血液細胞に細胞初期化因子(例えば、OCT3/4、SOX2、KLF4及びc-MYC)を発現しうる遺伝子を少なくとも4種類導入する工程;
3)遺伝子が導入された細胞を培養する工程。
iPS細胞の維持培養に使用可能な基本培地として、StemFit(R)AK02N(商品名)、StemFit(R)AK03N(商品名)、ReproStem(商品名)、iPSellon(商品名)、Essential 8(商品名)、TeSR-E8(商品名)などの各種幹細胞維持培地を使用することができる。特に好ましくはStemFit(R)AK02N(商品名)である。各培地に添加する物質は、目的に応じて、適宜増減することができる。添加する物質の例として、Rho-Associated Coil Kinase (ROCK) inhibitorであるY27632を用いることができる。細胞接着や増殖を促進するために、培養皿等の培養基材に例えばラミニン511-E8フラグメントを使用することができる。具体的にはiMatrix-511 silk(商品名)やiMatrix-511(商品名)を使用することができる。使用する試薬等は同等の機能を発揮しうるものであれば、製造・販売元は特に限定されない。iPS細胞の継代時、培養容器から細胞を剥がす際にはトリプシンなどのプロテアーゼを使用することができ、例えばTrypLE Select(商品名)を使用することができる。
iPS細胞からγδT細胞の分化誘導処理の工程で、まずiPS細胞から血球前駆細胞へ分化誘導される。本発明のiPS細胞由来γδT細胞の作製方法では、iPS細胞から血球前駆細胞へ分化誘導された細胞を出発原料とし、血球前駆細胞からγδT細胞への分化誘導処理工程をiPS細胞由来γδT細胞の作製方法とすることができる。さらには、iPS細胞から血球前駆細胞への工程を含むiPS細胞由来γδT細胞の作製方法とすることができる。またiPS細胞由来血球前駆細胞を凍結して融解した細胞を、本発明の方法に用いることもできる。凍結期間は特に限定されないが、例えば2週間~1年等が挙げられる。いずれの場合であっても、本発明のiPS細胞は、αβTCR再構成遺伝子を有するiPS細胞ではないiPS細胞が好適である。最も好適にはγδTCR型iPS細胞である。
StemFit(R)AK02N(商品名)を基本培地とすることができる。さらにGSK-3α/β阻害剤(CHIR99021、CAS番号:252917-06-9)0~20μM、好ましくは0.5~10μM、より好ましくは4μM、BMP4 0~400 ng/ml、好ましくは10~200 ng/ml、より好ましくは80 ng/ml、VEGF 0~400 ng/ml、好ましくは10~200 ng/ml、より好ましくは80 ng/mlを含む培養系で培養することができる。
Advanced DMEM/F12(商品名)又はEssential6(商品名)を基本培地とすることができる。さらに選択的ALK5, 4, 7阻害剤(SB431542)0~20μM、好ましくは0.5~10μM、より好ましくは2~4μM、bFGF 0~200 ng/ml、好ましくは 1~100 ng/ml、より好ましくは50 ng/ml、SCF 0~200 ng/ml、好ましくは1~100 ng/ml、より好ましくは50 ng/ml及びVEGF 0~400 ng/ml、好ましくは10~200 ng/ml、より好ましくは80 ng/mlを含む培養系で培養することができる。上記のほか、さらにL-Glutamine、ペニシリン/ストレプトマイシン、iPS/ES細胞用の分化誘導サプリメント(例えばStemFit(商品名) For Differentiation:以下「AS401」)等を適宜選択して加えることができる。至適添加量は適宜決定することができる。
Advanced DMEM/F12(商品名)又はStemPro-34 SFM(商品名)を基本培地とすることができる。さらにL-Glutamine 0~20 mM、好ましくは0.5~10 mM、より好ましくは2 mM、IL-3 0~200 ng/ml、好ましくは1~100 ng/ml、より好ましくは50 ng/ml、IL-6 0~200 ng/ml、好ましくは1~100 ng/ml、より好ましくは50 ng/ml、FLT3L 0~200 ng/ml、好ましくは1~100 ng/ml、より好ましくは50 ng/ml、SCF 0~200 ng/ml、好ましくは1~100 ng/ml、より好ましくは50 ng/ml、VEGF 0~200 ng/ml、好ましくは1~100 ng/ml、より好ましくは20 ng/ml及びEPO 0~100 IU/ml、好ましくは1~50 IU/ml、より好ましくは10 IU/mlを含む培養系で培養することができる。上記のほか、さらにペニシリン/ストレプトマイシン、iPS/ES細胞用の分化誘導サプリメント(例えばAS401)等を適宜選択して加えることができる。至適添加量は適宜決定することができる。
Advanced DMEM/F12(商品名)又はStemPro-34 SFM(商品名)を基本培地とすることができる。さらにL-Glutamine 0~50 mM、好ましくは1~20 mM、より好ましくは2 mM、IL-3 0~200 ng/ml、好ましくは1~100 ng/ml、より好ましくは50 ng/ml、IL-6 0~200 ng/ml、好ましくは1~100 ng/ml、より好ましくは50 ng/ml、SCF 0~200 ng/ml、好ましくは1~100 ng/ml、より好ましくは50 ng/ml及びEPO 0~100 IU/ml、好ましくは1~50 IU/ml、より好ましくは10 IU/mlを含む培養系で培養することができる。上記のほか、さらにペニシリン/ストレプトマイシン、iPS/ES細胞用の分化誘導サプリメント(例えばAS401)等を適宜選択して加えることができる。至適添加量は適宜決定することができる。
iPS細胞の培養やiPS細胞を分化誘導処理する際に、フィーダー細胞を共培養することができる。フィーダー細胞としては、例えばMEF(マウス胎児線維芽細胞)や、OP9, OP9/DLL1、OP9-DL4、及び10T1/2/DL4細胞等から選択される1種又は複数種の細胞株を使用することができる。一方、iPS細胞を分化誘導して得た細胞を、細胞療法等でヒトに投与する場合には、動物由来の物質を含まず、安定して生産方法が望まれている。本発明では、上述のラミニン-511 E8断片や培地成分を工夫することでフィーダー細胞を使わずに本発明のγδT細胞へと分化誘導することもできる。
iPS細胞由来血球前駆細胞からγδT細胞への分化誘導過程は、フィーダー細胞との共培養を行ってもよいし、フィーダー細胞を用いない条件で培養してもよい。さらに血清を含まない条件で培養してもよいし、動物由来成分を含まない条件で培養してもよい。またiPS細胞由来血球前駆細胞からγδT細胞への分化誘導過程は低酸素条件下で培養することもできる。低酸素条件下とは、iPS細胞由来血球前駆細胞からγδT細胞への分化誘導過程における培養条件でのO2濃度が、通常培養されるO2濃度より低いことを意味する。低酸素条件下で培養するO2濃度は、特に限定されるものではないが、例えば20%(v/v)未満であり、好ましくは10%(v/v)未満である。
A-1)分化誘導10日目~
例えば上記の処理1-1)~1-4)によりiPS細胞から分化誘導後10日目(血球前駆細胞)以降の培養は、αMEM(商品名)を基本培地とすることができる。さらにFBS 0~30%、好ましくは 0~20%、より好ましくは10~20%、SCF 0~100 ng/ml、好ましくは1~50 ng/ml、より好ましくは10 ng/ml、IL-7 0.1~20 ng/ml、好ましくは0.5~10 ng/ml、より好ましくは5 ng/ml、FLT3L 0.1~50 ng/ml、好ましくは1~20 ng/ml、より好ましくは5 ng/ml、L-ascorbic acid 1~1000μg/ml、好ましくは10~500μg/ml、より好ましくは100μg/mlを含む培養系で培養することができる。さらにIL-2 0~200 ng/ml、好ましくは 1~100 ng/ml、より好ましくは10 ng/mlを含んでいてもよいし、又はTPO 0~200 ng/ml、好ましくは1~100 ng/ml、より好ましくは10 ng/mlを含んでいてもよい。上記のほか、さらにペニシリン/ストレプトマイシン等を適宜選択して加えることができる。また、FBSの代わりに0.1%Polyvinyl alcohol+4% B27(商品名)サプリメント等を用いてもよい。使用する試薬等は同等の機能を発揮しうるものであれば、製造・販売元は特に限定されない。至適添加量は適宜決定することができる。培養はフィーダー細胞を播種した培養皿等の培養基材に、分化誘導後10日目の細胞(血球前駆細胞)を播種して培養することができる。培地は例えば2日ごとに交換し、分化誘導後12日目、18日目及び24日目にピペッティングして上清を回収し新しいフィーダー細胞へ移して培養を継続することができる。培地交換の頻度、培地交換量等は特に限定されず適宜適切な頻度、量を決定することができる。
上記培地を用いて分化誘導後10日目から30日目又は31日目まで培養した細胞は、フィーダー細胞を用いない条件で培養することができる。係る条件での培養は、RPMI1640培地を基本培地とすることができる。さらにFBS 0~30%、好ましくは 0~20%、より好ましくは10~20%含む培地で培養することができる。FBSの代わりに0.1%Polyvinyl alcohol+4% B27(商品名)サプリメント等を用いてもよい。さらにIL-2及び/又はIL-15 0~200 ng/ml、好ましくは1~100 ng/ml、より好ましくは10 ng/mlを含む培養系で培養してもよいし、又はImmunace(商品名)0~1000 IU/ml、10~500 IU/ml、好ましくは100 IU/mlと2-Me(2-Mercaptoethanol)0~100μM、1~50μM、好ましくは10μMを含む培養系で培養してもよい。上記のほか、さらにペニシリン/ストレプトマイシン等を適宜加えることができる。
B-1)分化誘導10日目~
例えば上記の処理1-1)~1-4)によりiPS細胞から分化誘導後10日目(血球前駆細胞)以降の培養は、VCAM1(vascular cell adhesion molecule-1)、並びにDLL4(Delta-Like Protein 4)若しくはDLL1(Delta-Like Protein 1)でコートした培養基材を用いて培養することができる。分化誘導10~24日目までは、例えばStemSpanTM T cell generation kit(商品名)に含まれるLymphoid progenitor Expansion Medium(商品名)で培養することができる。培地交換はStemSpanTM キットのプロトコール通り行った。具体的には分化誘導13日目に培地を追加で加え、分化誘導17日目及び20日目にそれぞれ前記培地を交換することができる。分化誘導17日~24日目ごろで上記キットに含まれるT cell progenitor Maturation Medium(商品名)に交換することができる。分化誘導27日目で前記培地を追加で加え、以降は分化誘導31日目及び34日目等、週2回程度培地を交換することができる。培地交換の頻度、培地交換量等は特に限定されず適宜適切な頻度、量を決定することができる。
B-1)に記載の方法で継続して培養することもできるが、分化誘導17日~24日ごろからγδT細胞刺激剤を加えた培地で培養することができる。分化誘導17日~24日ごろから細胞数の減少傾向が見られる場合があり、γδT細胞刺激剤を加えることで改善される。具体的には、A-2に示す培地であって、IL-2及び/又はIL-15と、HMBPP及びFPP合成酵素特異的阻害物質等のγδT細胞刺激剤を加えた培地で培養することができる。A-2に示す培地のうちFBSを含まない培地であって、同様にHMBPPを加えた培地で培養することもできる。A-2に示す培地の代わりに、AS401を含むRPMI1640培地で培養であって、IL-2及び/又はIL-15と、HMBPPを加えた培地で培養することもできる。
B-1)に記載の培地条件にさらにDKK1(Dickkopf-1)及び/又はAZA(Azelaic acid)を含む方法で継続して培養することもできる。さらに分化誘導17日~24日ごろからγδT細胞刺激剤を加えた培地で培養することができる。分化誘導17日~24日ごろから具体的には、A-2に示す培地であってHMBPPを加えた培地で培養することができる。A-2に示す培地のうちFBSを含まない培地であって、同様にHMBPPを加えた培地で培養することもできる。A-2に示す培地の代わりに、AS401を含むRPMI1640培地であって、IL-2及び/又はIL-15と、HMBPP等のγδT細胞刺激剤を加えた培地で培養することもできる。
本発明の分化誘導方法で培養した細胞は、ビーズを用いて培養することができる。ビーズの大きさは特に限定されず、細胞の大きさより小さいものであってもよいし、細胞の大きさ以上の大きさであってもよい。例えば分化誘導10日目の細胞を上記各種条件で培養する際に、培地にビーズを混入して培養することができる。ビーズとしては、細胞培養に使用可能な材質のビーズであればよく、特に限定されないが、具体的にはDynabeads ProteinG(商品名)を使用することができる。ビーズを例えばVCAM1及びDLL4でコートすることで、フィーダー細胞を用いない条件で培養することもできる。
D-1)分化誘導10日目~
本発明の分化誘導方法で培養した細胞は、動物由来成分を含まない培地を用いた条件で培養することもできる。例えば上記の処理1-1)~1-4)によりiPS細胞から分化誘導後10日目(血球前駆細胞)以降の培養は、VCAM1(vascular cell adhesion molecule-1)、並びにDLL4(Delta-Like Protein 4)若しくはDLL1(Delta-Like Protein 1)でコートした培養基材を用いて培養することができる。分化誘導10日~24日ごろは動物由来成分を含まない培地として例えばAS401を含むRPMI1640を基本培地とすることができる。さらにA-1に示すSCF, IL-7, FLT3L, L-ascorbic adid, IL2, TPO等を含んでもよい。
分化誘導17日目~24日ごろからA-2に示すIL-2、IL-15、γδT細胞刺激剤を加えた培地で培養することができる。係る培地はAS401を含むRPMI1640を基本培地とすることができる。分化誘導17日目~24日ごろから具体的には、IL-2、IL-15及びHMBPPのうち1つまたは複数を加えた培地で培養することができる。
本発明の分化誘導方法により作製されるγδT細胞は、表面にγ鎖とδ鎖からなる特有のT細胞受容体(TCR)を有するT細胞である。係る細胞表面について、例えばCD3、CD7、CD8a、CD45RA及びγδTCRなどの細胞マーカーの発現を確認することができる。本発明のγδT細胞として特にCD7、CD8a及びCD45RAから選択される1つまたは複数を発現していることが好ましく、一方CD25、IFNγ、CD5及びCD27から選択される1つまたは複数を発現していないことが好ましい。得られたiPS細胞由来のγδT細胞はMHC非拘束性に抗原特異的細胞傷害活性を有することを特徴とする。さらに本発明のiPS細胞を分化誘導して作製したγδT細胞と末梢血から分離されたγδT細胞での細胞表面マーカーのパターンには違いが認められる。例えばCD7、CD8aについてはiPS細胞由来γδT細胞のほうが高い発現傾向を示し、IL2RA(CD25)、CD5、IFNγについては末梢血から分離されたγδT細胞のほうが高い発現傾向を示す。また例えばCD45RAについてはiPS細胞由来γδT細胞のほうが高い発現傾向を示し、及びCD27については末梢血から分離されたγδT細胞のほうが高い発現傾向を示す。
本実施例では、非特許文献1の方法で作製したγδTCR型iPS細胞から作製したγδT細胞の分化誘導処理方法について示す。
フィーダー細胞を用いない条件で培養したγδTCR型iPS細胞(62B3株)2×103/wellを6ウェルプレートに継代し、維持培養した。維持培養は、1.6μg/wellのiMatrix-511(ニッピ製)を含むStemFit(R)AK02N(味の素製)を用いた。継代時の細胞の剥離・分散は0.5×TrypLETM select(ThermoFisher製)を使用し、継代培養にはStemFit(R)AK02NにY27632(和光純薬工業製)を終濃度10μMとiMatrix-511 3.2μlとなるように添加した培地を用いた。翌日にY27632及びiMatrix-511を含まないStemFit(R)AK02Nに交換し、以降2日ごとに培地を交換した。培地は1.5 ml/well添加した。培養は以下の工程及び後述の実施例も含め、すべて37±0.5℃、5% CO2条件で行った。
上記(1-5)の2日後に、表4に示す培地(Step4)と同じ培地2 ml/wellを交換した。
CD34/CD43の発現をフローサイトメトリーで評価した。CD34+/CD43+細胞及びCD34-/CD43+細胞が多数検出された。すなわち、血球前駆細胞へと分化していた(図1A)。
上記(1-7)でフローサイトメトリーに供した以外の細胞を、フィーダー細胞であるOP9/N-DLL1細胞を播種した12ウェル培養皿に撒いた。表5に示す組成の培地で培地量は1 ml/wellとし、2日ごとに培地を半量交換した。
CD3/γδTCRの発現をフローサイトメトリーで評価した結果、CD3+/TCR+細胞が多数検出され、TCR細胞への分化が確認された(図1B)。得られた細胞を、以降本実施例では「iPS細胞由来γδT細胞」という。
Jurkat細胞(ヒト白血病T細胞由来)に対する細胞傷害アッセイを行った。E:T(effector:target)比=2:1、蛍光色素CFSEで染色したJurkat細胞(T)を5×104個96ウェル培養皿の1ウェルに加え、ここに1×105個のiPS細胞由来γδT細胞(E)を加えて16時間培養した。7-AAD(7-Amino-Actinomycin D)染色により死細胞を染色した。多くのJurkat細胞(CFSE陽性細胞)について細胞死(7-AAD陽性)が確認された。すなわち、iPS細胞由来γδT細胞は細胞傷害機能を有していることが確認された。本実施例ではγδT細胞の活性化刺激培養を行っていないにも関わらず細胞傷害活性が確認された。
本実施例では非特許文献1の方法で作製したγδTCR型iPS細胞から分化誘導処理により作製したγδT細胞に関し、分化誘導10日以降の培地成分と分化誘導31日目以降の培地成分が実施例1と相違する。特に分化誘導31日目以降の培地成分にはγδT細胞刺激剤であるHMBPPを含む。
(2-2)分化誘導10日目~(Day10~)
上記実施例1の(1-6)で作製した細胞を、フィーダー細胞であるOP9/N-DLL1細胞を播種した12ウェル培養皿に撒いた。表6に示す培地(Step5)の組成の培地1 ml/wellを7日ごとに全量交換した。
CD7(T細胞分化マーカー)の発現をフローサイトメトリーで評価した。CD7陽性細胞を認め、T細胞へと分化が進んでいることが明らかとなった(図2A)。
(2-5)分化誘導54日目(Day54)の細胞の評価
CD3/γδTCRの発現をフローサイトメトリーで評価した。CD3+/TCR+細胞が多数検出され、TCR細胞への分化が確認された。すなわち得られた細胞はiPS細胞由来γδT細胞であることが確認された。また、一般にT細胞の成熟の指標として用いられるCD45RAの発現についても評価したところ、CD3+細胞はCDRA+細胞とCDRA-細胞の両方を含むことが明らかになった(図2B)。
本実施例では実施例1と同様に、非特許文献1の方法で作製したγδTCR型iPS細胞から分化誘導処理により作製したγδT細胞について示す。実施例1と同様に分化誘導処理を行い、31日目以降、実施例2の(2-4)と同様にγδT細胞刺激培地(HMBPP及びFBSを含む)を2日ごとに半量交換した。そのうえで、マーカー発現の評価とともに、細胞傷害アッセイを行った。
(3-2)分化誘導17日目(Day17)の細胞の評価
CD7(T細胞分化マーカー)の発現をフローサイトメトリーで評価した。CD7陽性細胞を検出し、T細胞へと分化が進んでいることが明らかとなった(図3A)。
(3-3)分化誘導55日目(Day55)の細胞の評価
CD3/γδTCRの発現をフローサイトメトリーで評価した。CD3+/TCR+細胞を多数検出し、γδT細胞への分化が確認された(図3B)。得られた細胞を、以降本実施例では「iPS細胞由来γδT細胞」という。
(3-4)分化誘導55日目(Day55)の細胞の評価
Jurkat細胞に対する細胞傷害アッセイを行った。96ウェル培養皿の1ウェルあたりに、CFSEで染色したJurkat細胞5×104個を加え、さらに分化誘導55日目の1×105個のiPS細胞由来γδT細胞を加え、E:T比=2:1にて16時間培養後、7-AAD染色(死細胞染色)を行った。多くのJurkat細胞(CFSE陽性細胞)が7-AAD陽性であり、死細胞が多く確認された。すなわち、iPS細胞由来γδT細胞は、腫瘍細胞に対して細胞傷害機能を有していることが確認された(図3C)。
本実施例では実施例1と同様に、非特許文献1の方法で作製したγδTCR型iPS細胞から分化誘導処理により作製したγδT細胞に関し、フィーダー細胞を用いない条件での分化誘導方法について示す。本実施例では図4に示すプロトコールに従い、以下の手順で分化誘導処理を行った。
(4-2)分化誘導10日目(Day10)
VCAM1とDLL4でコートした48ウェル培養皿を用い、1ウェルあたりStemSpanTM T cell generation kit(Stem Cell Technologies)に含まれるLymphoid progenitor Expansion Medium 250μlに分化誘導10日目の細胞を1.2×104個懸濁したものを播種した。VCAM1 5μg/ml及びDLL4 10μg/mlを溶解したPBS(-)を、細胞接着のための親水化処理をしていない市販の48ウェル培養皿(cell culture-non-treated)に1ウェルあたり100μl加え、4℃で一晩静置して溶液を除去し、PBS(-)で一回洗浄したものをVCAM1とDLL4でコートした培養皿として使用した。Lymphoid progenitor Expansion Mediumを用いる工程ではフィーダー細胞も血清も使用しない条件で培養した。
CD3/γδTCRの発現をフローサイトメトリーで評価した。CD3+/TCR+細胞を多数検出し、TCR細胞への分化が確認され、iPS細胞由来γδT細胞であることが確認された(図5)。本結果は3回の独立した分化誘導実験の結果を示した。評価した日(分化誘導開始をday0)は図中に示した。
本実施例では実施例4と同様にγδTCR型iPS細胞から分化誘導処理により作製したγδT細胞に関し、フィーダー細胞を用いない条件での分化誘導方法について示す。本実施例では図6に示すプロトコールに従い、以下の手順で分化誘導処理を行った。
(5-2)分化誘導24日目(day24)
実施例2(2-4)の表7に示すγδT細胞刺培地(HMBPP及びFBSを含む)に交換し、以降は3日ごとに培地を半量交換した。
(5-3)分化誘導37日目(day37)の細胞の評価
上記細胞について位相差顕微鏡を用いて細胞数を観察した。実施例4でγδT細胞刺激剤(HMBPP)を含まない培地で培養し作製した細胞についても同様に観察した。その結果、γδT細胞刺激培地で培養した方が、明らかに細胞数が多く観察された(図7A)。さらにCD3/γδTCRの発現をフローサイトメトリーで評価したところ、CD3+/TCR+細胞が多数検出され、TCR細胞への分化が確認された(図7B)。得られた細胞はiPS細胞由来γδT細胞であることが確認された。
本実施例では実施例4と同様にγδTCR型iPS細胞から分化誘導処理により作製したγδT細胞に関し、フィーダー細胞を用いない条件での分化誘導方法について示す。本実施例では図8に示すプロトコールに従い、以下の手順で分化誘導処理を行った。
(6-2)分化誘導24日目(day24)
分化誘導24日目に、a.実施例2(2-4)の表7に示すγδT細胞刺激培地(HMBPP及びFBSを含む)、b.表7に示すγδT細胞刺激培地の基礎培地(10%FBS/RPMI1640)の代わりにAS401を含むRPMI1640(HMBPP含む)培地、及びc.StemSpanTM キットに含まれるLymphoid progenitor Expansion Mediumの各培地に交換し、実施例5(5-2)と同手法により培地を交換した。
(6-3)分化誘導32日目(day32)の細胞の評価2
分化誘導32日目の細胞について、位相差顕微鏡を用いて細胞数を観察した。c.のStemSpanTMキットに含まれる培地では明らかに細胞数が少ないのに対して、b.の無血清培地ではa.の血清培地と同等の細胞密度が観察された(図9)。さらに上記細胞についてCD3/γδTCRの発現をフローサイトメトリーで評価した。いずれの条件でもCD3+/TCR+細胞が多数検出され、iPS細胞由来γδT細胞であることが確認された(図10)。得られた細胞を、以降本実施例では「iPS細胞由来γδT細胞」という。
(6-4)分化誘導35日目(day35)の細胞の評価1
分化誘導35日目の細胞について、a.及びb.の培地条件で得られた細胞を用いて、実施例1の(1-10)と同手法によりJurkat細胞に対する細胞傷害アッセイを行った。96ウェル培養皿の1ウェルあたりに、CFSEで染色したJurkat細胞5×104個を加え、さらに分化誘導35日目の1×105個のiPS細胞由来γδT細胞を加え、E:T比=2:1にて混合培養開始1日後(d1)と4日後(d4)に評価したところ、T細胞の活性化を示す細胞集塊が見られた。エフェクター細胞(iPS細胞由来γδT細胞)を添加しないコントロール(ctrl)と比較して、a.及びb.の培地条件で得られた細胞は明らかに少ない様子が観察された(図11)。混合培養開始1日後、a.の培地条件と比べれば少ないものの、b.の血清を含まない培地条件でも細胞傷害が明らかであり、4日後にでは、さらに著明な細胞傷害活性が確認された(図12)。
本実施例では実施例4と同様にγδTCR型iPS細胞を分化誘導処理し、γδT細胞を作製した。
(7-2)ただし分化誘導10日目において、(4-2)と同条件(i)の他、ここに(ii)DKK1(Dickkopf-1)を終濃度30 ng/ml添加した条件、(iii)AZA(Azelaic acid)を終濃度5 mM添加した条件、(iv)DKK1(Dickkopf-1)とAZAの両方をそれぞれ(ii)と(iii)と同じ濃度で加えた条件で行った。
(7-3)以降の培地交換はStemSpanTM キットのプロトコール通りに従い行った。すなわち、分化誘導13日目(Day13)に(7-2)に示す各培地を250μl追加し、分化誘導17日目(day17)及び20日目(day20)に各培地を半量交換した。
(7-4)分化誘導24日目(Day24)の細胞の評価
分化誘導24日目の細胞についてT細胞分化マーカーであるCD7の発現をフローサイトメトリーで評価した。その結果、DKK1及びAZAについて各々分化誘導効率に正の効果を有し、併用して処理することで、より効果が高いことが分かった(図13)。
本実施例では、フィーダー細胞を用いない条件で、培養皿をコートする代わりにVCAM1とDLL4をコートした磁気ビーズを混合培養することでT細胞へ分化誘導した。
(8-2)VCAM1とDLL4をコートした磁気ビーズ溶液の調製
磁気ビーズ(DynabeadsTM ProteinG(Invitrogen製))をボルテックスにて攪拌し、これを5μlとPBS 1 mlをチューブにいれ、磁気ビーズ捕捉用マグネティックスタンドで1分間静置した。PBSを除去してスタンドから外し、PBS 200μl、VCAM1(100μg/ml溶液)4.26μl、DLL4(100μg/ml溶液)4.26μlを加え、室温に15分静置した。マグネティックスタンドで1分間静置し、上記溶液を除きスタンドから外した。StemSpanTM キットに含まれるLymphoid progenitor Expansion Medium 500μlを加え、ピペッティングにて懸濁した。
(8-3)分化誘導10日目(Day10)
前記(8-1)で作製した細胞4.75×105個を(8-2)で調製した磁気ビーズ溶液500μlで懸濁し、24ウェルの低接着培養皿(PrimeSurfaceTM)に播種し、培養した。
(8-4)分化誘導13日目(Day13)に前記培地500μlを追加し、分化誘導17日目及び20日目にそれぞれ培地を半量交換した。
(8-5)分化誘導24日目(Day24)の細胞の評価
分化誘導24日目の細胞についてT細胞分化マーカーであるCD7の発現をフローサイトメトリーで評価した。その結果、0.3%と割合は低いものの、コントロール(isotype control)と比較すると明らかにCD7陽性細胞が存在していることが確認された。すなわち、磁気ビーズを混合培養する本方法によってもT細胞への分化が可能であることが明らかとなった(図14)。
本実施例では、γδTCR型iPS細胞から作製したγδT細胞(iPS細胞由来γδT細胞)の特性を確認した。まず初めにiPS細胞由来γδT細胞の作製方法を示し、続いて細胞の各種特性について示す。
図15に示す方法でγδT細胞を作製した。
・iPS細胞の樹立
非特許文献1の方法で作製したγδTCR型iPS細胞を用いた。iPS細胞の維持培養にはStemfit(R)AK02N(味の素)を用いた。継代には0.5×TrypLETM select(ThermoFisher製)を用いた。血球前駆細胞への分化誘導処理は、各工程で6ウェルの培養プレートを使用し、2×103cells/wellで播種した。1日おきに培地を吸引し、全培地2.0 ml/wellを交換した。
Stemfit AK02N(Ajinomoto, Tokyo, Japan, AK02N)
CHIR99021(Tocris, Bristol, UK, 4423) 4μM
BMP4 (R&D, Minneapolis, MN, 314-BP) 80 ng/ml
VEGF (R&D, Minneapolis, MN, 293-VE) 80 ng/ml
Essential6 (Thermofisher, Waltham, MA, A1516501)
SB431542 (WAKO, Osaka, Japan, 033-24631)2μM
bFGF(WAKO, Osaka, Japan,060-04543) 50 ng/ml
SCF (R&D, Minneapolis, MN, 255-SC) 50 ng/ml
VEGF (R&D, Minneapolis, MN, 293-VE) 80 ng/ml
StemPRO34SFM (Thermofisher, Waltham, MA,10639-011)
L-Glutamine(Life technologies, 25036-081) 2 mM
IL-3(Peprotech, Cranbury, NJ, AF-200-03) 50 ng/ml
IL-6(R&D, Minneapolis, MN, 206-IL) 50 ng/ml
FLT3L(R&D, Minneapolis, MN, 308-FK) 50 ng/ml
SCF (R&D, Minneapolis, MN, 255-SC) 50 ng/ml
VEGF (R&D, Minneapolis, MN, 293-VE) 20 ng/ml
EPO(Kyowa Kirin, Tokyo, Japan) 10IU/ml
StemPRO34SFM (Thermofisher, Waltham, MA,10639-011)
L-Glutamine(Life technologies, 25036-081) 2 mM
IL-6(R&D, Minneapolis, MN, 206-IL) 50 ng/ml
SCF (R&D, Minneapolis, MN, 255-SC) 50 ng/ml
EPO(Kyowa Kirin, Tokyo, Japan) 10IU/ml
細胞の継代はAccutase(Nacalai Tesque, Kyoto, Japan, 12679-54)を用いた。以後2日ごとに培地を半量交換した。また、12日目、18日目及び24日目にピペッティングして上清を回収し新しいフィーダー細胞(OP9/N-DLL1)上へ播種した。
(Tcell 分化培地)
αMEM(Gibco, 11900-016)
FBS(Sigma-Aldrich, St. Louis, MO, F7524) 20%
SCF (R&D, Minneapolis, MN, 255-SC) 10 ng/ml
TPO(R&D, Minneapolis, MN, ) 10 ng/ml
IL-7 (R&D, Minneapolis, MN, 207-IL) 5 ng/ml
FLT3L(R&D, Minneapolis, MN, 308-FK) 5 ng/ml
L-ascorbic acid(Nacalai Tesque, Kyoto, Japan, 30264-56)100μg/ml
Accutaseで処理した細胞を下記γδT活性培地に懸濁し、フィーダー細胞を含まない培地で培養した。以後2日ごとに培地を半量交換した。活性培養7~14日の細胞を細胞傷害アッセイに供した。
RPMI1640 (Nacalai Tesque, Kyoto, Japan, 30264-56)
FBS(Sigma-Aldrich, St. Louis, MO, F7524) 10%
HMBPP(Cayman chemical, Ann Arbor, MI, 13580) 1 nM
Immunace(Shionogi pharmaceuticals, Osaka, Japan) 100IU/ml
2-Me(Nacalai Tesque, Kyoto, Japan) 10μM
分化の過程での細胞の形状を位相差顕微鏡で観察し(図16A)、及びフローサイトメトリーにて細胞表面マーカーについて確認した(図16B)。
d0 :分化誘導 0日目:γδTCR型iPS細胞
d10:分化誘導10日目:血球前駆細胞へ分化した細胞
d30:分化誘導30日目:γδT細胞の活性化刺激前のγδT細胞
d51:分化誘導51日目:γδT細胞の活性化刺激後のγδT細胞
分化誘導38日目のiPS細胞由来γδT細胞を用いて各種腫瘍細胞に対する抗腫瘍活性を確認した(図17)。これらの実験では純化していないγδT細胞を用いた。コントロールとして、γδT細胞を加えず腫瘍細胞のみを培養した条件を用いた。
A.Jurkat細胞(ヒト白血病T細胞由来)に対する細胞傷害アッセイを行った。E:T(effector:target)比=2:1、蛍光色素CFSEで染色したJurkat細胞5×104個を96ウェル培養皿の1ウェルに加え、ここに1×105個のiPS細胞由来γδT細胞を加えて16時間培養したのち、7-AAD染色により死細胞を染色した。コントロールに比べ、明らかに本発明のγδT細胞はJurkat細胞に対して細胞傷害活性が高かった(図17A)。
分化誘導36日目のiPS細胞由来γδT細胞を用いてTCR再構成の保持及び細胞傷害機構について確認した(図18)。
A.純化していないiPS細胞由来γδT細胞(igdT)と末梢血単核細胞(PB)について細胞表面のαβTCRの発現を評価した。PBではαβTCRの発現が検出されたが、本発明のγδT細胞(igdT)ではαβTCRの発現は検出されなかった(図18A)。
B.TCR遺伝子再構成をゲノムPCR
TCR遺伝子(Vg9、Vd2)の再構成をゲノムPCRで確認した。γδT細胞(igdT)をフローサイトメトリーでソート(sort)したものは未分化(undiff)の状態と同じくTCR遺伝子再構成を保持していることが確認された(図18B)。陽性コントロールとして末梢血単核細胞(PBMC)を用いた。
C.事前にCD3を標識したiPS細胞由来γδT細胞(igdT)とJurkat細胞をBrefeldin A 3μg/ml下で共培養した。iPS細胞由来γδT細胞はGranzymeB, Perforinを発現していることが確認された(図18C)。GranzymeBやPerforinはT細胞による細胞傷害機能の分子実態であり、本発明のiPS細胞由来γδT細胞が細胞傷害性を有することが確認された。
D.γδT細胞(igdT)をフローサイトメトリー(FACS)で純化したものについて細胞傷害アッセイを行った。細胞傷害アッセイの条件は(9-3)のA.に示す方法で行った。図18Dにおいてコントロール(ctrl)としてiPS細胞由来γδT細胞を加えずJurkat細胞のみを培養したものを用いた。また純化していないiPS細胞由来γδT細胞はbulkとし、純化したiPS細胞由来γδT細胞はsortと明記した。iPS細胞由来γδT細胞について純化の有無で死細胞率に大きな違いはなかった(図18D)。
本発明のiPS細胞由来γδT細胞に使用したiPS細胞、並びに実施例3、6及び本実施例で使用した各腫瘍細胞のHLAタイプについて確認した結果を表8に示す。iPS細胞のHLAタイプは各腫瘍細胞のHLAタイプとは一致していないが、各腫瘍細胞に対して抗腫瘍作用が認められた(本実施例A.~C.)。これにより、本発明のiPS細胞由来γδT細胞がMHC非拘束性に抗原特異的細胞傷害活性を有することが確認された。
本実施例では、iPS細胞を分化誘導して作製したiPS細胞由来γδT細胞(igdT)と末梢血に存在するγδT細胞(PB-gdT)での細胞表面発現マーカー遺伝子について比較した。本実施例のiPS細胞由来γδT細胞は、実施例1に示す方法及び実施例9(9-1)に示す方法で培養し、分化誘導36~42日目の細胞を用いた。末梢血から分離された単核球を実施例9(9-1)に示すγδT活性培地で培養して得た細胞を本実施例の末梢血から分離されたγδT細胞として用いた。
iPS細胞由来γδT細胞と末梢血から分離されたγδT細胞及び末梢血中のγδT細胞ではない細胞について、マーカー遺伝子発現の違いをシングルセルRNA-seq解析により解析した。その結果、CD7、CD8a、IL18R1、IL2RA(CD25)、IL2RB及びIFNγについてそれぞれ異なる発現パターンを示した(図19、表9)。
iPS細胞由来γδT細胞と末梢血から分離されたγδT細胞におけるCD25の発現をフローサイトメトリーで比較した。iPS細胞由来TCR-Vγ9陽性細胞ではほとんどがCD25陰性細胞であるのに対し、末梢血から分離されたTCR-Vγ9陽性細胞ではほとんどがCD25陽性細胞であった(図20)。
上記により、iPS細胞由来γδT細胞と末梢血から分離されたγδT細胞では細胞表面マーカーのパターンが相違することが確認された。
本実施例では、iPS細胞由来γδT細胞の活性化方法について検討した。具体的には実施例9の(9-1)に示す作製方法のうち分化誘導30日目の細胞について、以下のγδT活性培地に、さらにIL-2及び/又はIL-15のいずれかを添加するのがより効果的にiPS細胞由来γδT細胞を作製し得るかについて検討した(図21参照)。
(活性化培地)
RPMI1640 (Nacalai Tesque, Kyoto, Japan, 30264-56)
FBS(Sigma-Aldrich, St. Louis, MO, F7524) 10%
HMBPP(Cayman chemical, Ann Arbor, MI, 13580) 1 nM
2-Me(Nacalai Tesque, Kyoto, Japan) 10μM
本実施例では、γδTCR型iPS細胞(121-3株)から作製したγδT細胞の特性を確認した。
A.γδTCR型iPS細胞(121-3株)から分化誘導して得られたγδT細胞(iγδT)のTCR遺伝子(Vγ9、Vγ2)の再構成をゲノムPCRで確認した。iγδTをフローサイトメトリーでソート(sort)したものは未分化(undiff)の状態と同じくTCR遺伝子再構成を保持していることが確認された(図23A)。
B.γδTCR型iPS細胞(121-3株)から分化誘導して得られたγδT細胞(iγδT)と末梢血単核球を増幅培養して得られたγδT細胞(PBγδT)のTCRγとTCRδの配列を次世代シーケンサーによって解析した。各々のTCRγとTCRδのCDR3領域の塩基配列およびアミノ酸配列を特定し、配列ごとの頻度を円グラフで示した(図23B)。PBγδT細胞集団は多様な配列を有する細胞から構成されるのに対し、iγδT細胞集団は全て1種類のTCRγ及びTCRδ遺伝子再構成を有する細胞から構成されることが確認された。
本実施例では、実施例9に示す作製方法により作製したiPS由来γδT細胞に関し、分化誘導39日目の細胞をJurkat細胞と4時間共培養した細胞について、フローサイトメトリーによるIFNγの発現を評価した。
本実施例では、γδTCR型iPS細胞(62B3株又は121-3株)を分化誘導して作製したiPS細胞由来γδT細胞(igdT)と末梢血を増幅して得られたγδT細胞(PB-gdT)での細胞表面発現マーカーについて比較した。
(14-2)分化誘導40日目の細胞についてのγδT細胞を含む細胞集団(iγδT)と末梢血単核球を増幅培養して得られたγδT細胞(PBγδT)を含む細胞集団(CD3陽性またはTCRγ9陽性)における種々の細胞表面マーカー(CD25、CD7、CD5、CD45RA及びCD27)の発現をフローサイトメーターで評価した。PBγδTと比べてiPS細胞由来γδT細胞(iγδTのうちCD3陽性またはTCRγ9陽性細胞)では、CD7を発現する細胞の割合が高く、CD5及びCD25を発現する細胞の割合が低く、さらにCD45RA+CD27-の割合が高いという特徴を有することが確認された(図25)。
本実施例では実施例5と同様にγδTCR型iPS細胞からの分化誘導処理により作製したγδT細胞に関し、フィーダー細胞も血清も用いない条件及びγδT細胞を刺激する工程を24日目からではなく17日目で行う条件での分化誘導方法について示す。本実施例では図26A(New protocol)に示すプロトコールに従い、以下の手順で分化誘導を行った。
分化誘導17日目の細胞についてCD3/γδTCR(gdTCR)の発現をフローサイトメトリーで評価した。CD3+/TCR+細胞を検出し、TCR細胞への分化が確認され、iPS細胞由来γδT細胞であることが確認された(図26B)。得られた細胞を以降本実施例では「iPS細胞由来γδT細胞」という。
20% AS401を含むRPMI1640を基本培地として、ここにHMBPP(Cayman chemical, Ann Arbor, MI, 13580) 1 nM及びIL2(Reprotech,200-02)100ng/mlを加えた培地に交換し、以降3日ごとに培地を半量交換した。
さらに分化誘導24日目の細胞についてCD3/CD7の発現をフローサイトメトリーで評価した(図26C)。γδT細胞を刺激する工程を短縮した条件でもiPS細胞由来γδT細胞が得られた。
本実施例でフィーダー細胞も血清も用いない条件でのiPS細胞由来γδT細胞γδT細胞の活性化方法について検討した。
B.上記AのうちγδT細胞刺激の工程でIL-15を用いて分化誘導処理を行い、HMBPPの添加の有無によってiPS細胞由来γδT細胞を作製し得るかについて検討した。分化誘導23日目の細胞について、CD3/CD7の発現をフローサイトメトリーで評価した。CD3+/TCR+細胞を検出し、TCR細胞への分化が確認され、iPS細胞由来γδT細胞であることが確認された。γδTCR刺激剤であるHMBPPを添加しない条件であってもiPS細胞由来γδT細胞が得られた(図27B)。
本実施例ではフィーダー細胞も血清も用いない条件でのiPS細胞由来γδT細胞を凍結融解し細胞傷害アッセイを行った。
上記凍結させた細胞を2週間後に解凍しJurkat細胞に対する細胞傷害アッセイを行った。E:T(effector:target)比=2:1、蛍光色素CFSEで染色したJurkat細胞を5×104個96ウェル培養皿の1ウェルに加え、ここに分化誘導24日目の1×105個のiPS細胞由来γδT細胞を加えて16時間培養した。7-AAD(7-Amino-Actinomycin D)染色により死細胞を染色した。多くのJurkat細胞(CFSE陽性細胞)について細胞死(7-AAD陽性)が確認された(図28)。すなわち、iPS細胞由来γδT細胞は凍結融解後でも細胞傷害機能を有していることが確認された。
本実施例ではiPS細胞由来血球前駆細胞凍結融解後に分化誘導を行い、γδT細胞を作製した。
実施例1に示す(1-1)-(1-6)と同処理を行い、分化誘導10日目の細胞をフローサイトメトリーで評価し、血球前駆細胞の段階であることを確認した(図29A)。
上記細胞を、CS10(コスモバイオ製)を用いて凍結させ約1年後に解凍した。解凍後、実施例9の(9-1)と同処理で行いフィーダー細胞を用いる方法で分化誘導させた。
分化誘導37日目(分化誘導培養期間が凍結前後あわせて37日目)の細胞について、CD3/γδTCRの発現をフローサイトメトリーで評価した。CD3+/TCR+細胞が検出されたため、iPS細胞由来γδT細胞であることが確認された(図29B)。さらに、分化誘導37日目の細胞についてJurkat細胞に対する細胞傷害アッセイを行った。E:T(effector:target)比=2:1、蛍光色素CFSEで染色したJurkat細胞を5×104個96ウェル培養皿の1ウェルに加え、ここに分化誘導24日目の1×105個のiPS細胞由来γδT細胞を加えて16時間培養した。7-AAD(7-Amino-Actinomycin D)染色により死細胞を染色した。多くのJurkat細胞(CFSE陽性細胞)について細胞死(7-AAD陽性)が確認された(図29C)。すなわち、iPS細胞由来γδT細胞は凍結融解後でも細胞傷害機能を有していることが確認された。
本実施例ではiPS細胞由来血球前駆細胞凍結融解後に、フィーダー細胞も血清も使用しない条件での分化誘導を行い、γδT細胞を作製した。本実施例では図30Aに示すプロトコールに従い、以下の手順で分化誘導を行った。なお本実施例での凍結は18日間行った。
上記解凍後の細胞に関し、VCAM1とDLL4でコートした48ウェル培養皿を用い、1ウェルあたりStemSpanTM T cell generation kit(Stem Cell Technologies)に含まれるLymphoid progenitor Expansion MediumにDKK1 終濃度 30ng/ml及びアゼライン酸(AZA)終濃度5 mMを加えた培地に分化誘導10日目の細胞を1.2×104個懸濁したものを播種した。VCAM1 5μg/ml及びDLL4 10μg/mlを溶解したPBS(-)を、細胞接着のための親水化処理をしていない市販の48ウェル培養皿(cell culture-non-treated)に1ウェルあたり100μl加え、4℃で一晩静置して溶液を除去し、PBS(-)で一回洗浄したものをVCAM1とDLL4でコートした培養皿として使用した。
分化誘導17日目では実施例15の(15-3)のうちIL-2をIL-15に交換し実施例15の(15-3)と同手法を行い、フィーダー細胞も血清も使用しない条件で分化誘導を行い、γδT細胞を作製した。
分化誘導17日目(分化誘導培養期間が凍結前後あわせて17日目)の細胞について、CD3/γδTCRの発現をフローサイトメトリーで評価した。CD3+/TCR+細胞が検出されたため、iPS細胞由来γδT細胞であることが確認された(図30B)。
分化誘導24日目(分化誘導培養期間が凍結前後あわせて24日目)の細胞についてJurkat細胞に対する細胞傷害アッセイを行った。E:T(effector:target)比=2:1、蛍光色素CFSEで染色したJurkat細胞を5×104個96ウェル培養皿の1ウェルに加え、ここに分化誘導24日目の1×105個のiPS細胞由来γδT細胞を加えて16時間培養した。7-AAD(7-Amino-Actinomycin D)染色により死細胞を染色した。多くのJurkat細胞(CFSE陽性細胞)について細胞死(7-AAD陽性)が確認された(図30C)。
本実施例ではフィーダー細胞も血清も使用しない条件で行った。ただし低酸素条件下で血球前駆細胞から分化誘導を行い、γδT細胞を作製した。本実施例では図31Aに示すプロトコールに従い、以下の手順で分化誘導を行った。
(20-2)ただし分化誘導10日目の細胞において、実施例4の(4-2)に示すStemSpanTM T cell generation kit(Stem Cell Technologies)に含まれるLymphoid progenitor Expansion MediumにDKK1 終濃度 30ng/ml及びアゼライン酸(AZA)終濃度5 mMを加えた培地でフィーダー細胞も血清も使用しない条件で行い、O2濃度を20%から5%に変えて培養した。
(20-3)以降の培地交換はStemSpanTMキットのプロトコールに従い行った。具体的には分化誘導13日目に(20-2)に示す培地250μlを追加で加えた。
分化誘導17日目の細胞について、CD3/D7の発現をフローサイトメトリーで評価した(図31B)。CD3+/CD7+細胞が検出されたため、iPS細胞由来γδT細胞であることが確認された。20%O2に比べ、低酸素(5%O2)の条件ではiPS細胞由来γδT細胞の割合および絶対数ともに多いことが確認できた。
実施例19の(19-4)と同処理のうちO2濃度を20%から5%に変えて培養した。
分化誘導29日目の細胞についてJurkat細胞に対する細胞傷害アッセイを行った。E:T(effector:target)比=2:1、蛍光色素CFSEで染色したJurkat細胞を5×104個96ウェル培養皿の1ウェルに加え、ここに分化誘導29日目の1×105個のiPS細胞由来γδT細胞を加えて16時間培養した。7-AAD(7-Amino-Actinomycin D)染色により死細胞を染色した。多くのJurkat細胞(CFSE陽性細胞)について細胞死(7-AAD陽性)が確認された(図31C)。すなわち細胞傷害活性は通常酸素条件下で誘導したものより低酸素条件下のほうが効果的であった。
本実施例では動物由来成分を含まない培地条件下でiPS細胞由来γδT細胞を作製した。
(21-2)ただし、分化誘導10日目では、実施例2の表6に示すうち、フィーダー細胞を用いず基礎培地20%FBS/αMEMを20%AS401/RPMI1640に交換したもの(動物由来成分を含まない培地条件となる)を実施例4の(4-2)に示すLymphoid progenitor Expansion Mediumの代わりに用いて、実施例4の(4-2)と同手法により分化誘導を行い、γδT細胞を作製した。分化誘導13日目に培地250μlを追加した。
分化誘導17日目の細胞について、CD3/CD7の発現をフローサイトメトリーで評価した。CD3+/CD7+細胞が検出されたため、iPS細胞由来γδT細胞であることが確認された(図32A)。
分化誘導17日目では、実施例2の(2-4)の表7のうち基礎培地20%FBS/αMEMを20%AS401/RPMI1640に、IL-2をIL-15に交換し実施例2の(2-4)と同手法により分化誘導し培養した。
分化誘導31日目の細胞についてJurkat細胞に対する細胞傷害アッセイを行った。E:T(effector:target)比=2:1、蛍光色素CFSEで染色したJurkat細胞を5×104個96ウェル培養皿の1ウェルに加え、ここに分化誘導31日目の1×105個のiPS細胞由来γδT細胞を加えて16時間培養した。7-AAD(7-Amino-Actinomycin D)染色により死細胞を染色した。多くのJurkat細胞(CFSE陽性細胞)について細胞死(7-AAD陽性)が確認された(図32B)。顕著な細胞傷害活性が確認された。
実施例ではフィーダー細胞も血清も使用しない条件で行った。本実施例ではiPS細胞由来γδT細胞に対する未分化細胞の確認をした。
(22-2)ただし分化誘導10日目の細胞において、実施例4の(4-2)に示すStemSpanTM T cell generation kit(Stem Cell Technologies)に含まれるLymphoid progenitor Expansion MediumにDKK1 終濃度 30ng/ml及びアゼライン酸(AZA)終濃度5 mMを加えた培地でフィーダー細胞も血清も使用しない条件で行った。
フィーダー細胞も血清も使用しない条件で分化させた35日目の細胞集団における未分化マーカーTRA-1-85の発現をフローサイトメトリーで評価した。35日目の細胞集団についてTRA-1-85陽性細胞を全く含まないことが確認された。(図33A)
フィーダー細胞も血清も使用しない条件で分化させた35日目の細胞集団を使用し、未分化細胞のコロニーが出現を確認するプロトコールを示す(図33B)。35日目の細胞の1×104個のiPS由来細胞γδT細胞集団を、未分化iPS細胞の維持培養条件(実施例1の(1-1))に播種し、未分化細胞のコロニーが出現するかを調べた。陽性対照として1×102個の未分化iPS細胞を混合した。11日後にアルカリフォスファターゼ染色(AP染色)を行った。未分化細胞のコロニーはAP染色で赤く染まる。陽性対照であるiPS細胞を添加した条件ではAP染色陽性のコロニーが多数確認されるのに対し、iPS細胞を添加していない分化誘導後の細胞集団ではAP染色陽性のコロニーは一つも確認されなかった(図33C)。
本実施例では、実施例22と同処理により得られた細胞集団中からCD3/γδT陽性細胞を純化して、細胞傷害アッセイを行った。
Claims (25)
- 人工多能性幹細胞(iPS細胞)由来のT細胞であって、前記T細胞がMHC非拘束性に抗原特異的細胞傷害活性を有することを特徴とするiPS細胞由来γδT細胞。
- 前記iPS細胞が、αβT細胞由来ではないiPS細胞である、請求項1に記載のiPS細胞由来γδT細胞。
- 前記iPS細胞が、γδTCR再構成遺伝子を有するiPS細胞である、請求項1又は2に記載のiPS細胞由来γδT細胞。
- γδTCR再構成遺伝子を有するiPS細胞を分化誘導処理して作製されたiPS細胞由来γδT細胞。
- γδTCR再構成遺伝子を有するiPS細胞を分化誘導処理して得た血球前駆細胞を、基本培地にFLT3L(tyrosine kinase 3 ligand)、SCF(stem cell factor)、IL-2、IL-7、TPO(thrombopoietin)、L-アスコルビン酸から選択される1種又は複数種を選択して加えた培地を用いて培養する工程を含む、iPS細胞由来γδT細胞の作製方法。
- 基本培地にFLT3L、SCF、IL-2、IL-7、TPO、L-アスコルビン酸から選択される1種又は複数種を選択して加えた培地を用いて培養する工程の後、γδT細胞刺激剤を含む培地を用いて培養する工程を含む、請求項5に記載のiPS細胞由来γδT細胞の作製方法。
- 基本培地にFLT3L、SCF、IL-2、IL-7、TPO、L-アスコルビン酸から選択される1種又は複数種を選択して加えた培地を用いて培養する工程が、フィーダー細胞と共培養して培養する工程である、請求項5又は6に記載のiPS細胞由来γδT細胞の作製方法。
- 基本培地にFLT3L、SCF、IL-2、IL-7、TPO、L-アスコルビン酸から選択される1種又は複数種を選択して加えた培地を用いて培養する工程が、フィーダー細胞と共培養せずに培養する工程である、請求項5又は6に記載のiPS細胞由来γδT細胞の作製方法。
- フィーダー細胞と共培養せずに培養する工程が、VCAM1(vascular cell adhesion molecule-1)、並びにDLL4(Delta-Like Protein 4)若しくはDLL1(Delta-Like Protein 1)でコートした培養基材を用いて培養する工程を含む、請求項8に記載のiPS細胞由来γδT細胞の作製方法。
- フィーダー細胞と共培養せずに培養する工程が、更にDKK1及び/又はAZA(Azelaic acid)を含む培地を用いて培養する工程を含む、請求項8又は9に記載のiPS細胞由来γδT細胞の作製方法。
- γδT細胞刺激剤を含む培地が、γδT細胞刺激剤、IL-2及びIL-15から選択される1種又は複数種を含む培地である、請求項6~10のいずれかに記載のiPS細胞由来γδT細胞の作製方法。
- γδT細胞刺激剤が、イソプレノイド生合成経路代謝物であるリン酸化合物又はその誘導体、あるいはイソプレノイド生合成経路の律速酵素であるFPP(farnesyl pyrophosphate)合成酵素の特異的阻害物質である、請求項6~11のいずれかに記載のiPS細胞由来γδT細胞の作製方法。
- 血清を含まない条件で培養することを特徴とする請求項6~12のいずれかに記載のiPS細胞由来γδT細胞の作製方法。
- 低酸素条件下で培養することを含む、請求項6~13のいずれかに記載のiPS細胞由来γδT細胞の作製方法。
- 請求項5~14のいずれかに記載のiPS細胞由来γδT細胞の作製方法により作製されたiPS細胞由来γδT細胞。
- 請求項1~4及び請求項15より選択されるいずれかに記載のiPS細胞由来γδT細胞を含む細胞集団。
- 末梢血から分離されたγδT細胞の細胞集団と比較して、iPS細胞由来γδT細胞を含む細胞集団が抗原特異的に高い細胞傷害活性を有することを特徴とする、請求項16に記載の細胞集団。
- γδT細胞を含む細胞集団であって、TCR遺伝子のCDR3領域が同一の塩基配列を有するγδT細胞が当該細胞集団を構成するγδT細胞の90%以上の割合で含むことを特徴とするγδT細胞の細胞集団。
- γδT細胞が1×105個以上含まれることを特徴とする、請求項18に記載の細胞集団。
- γδT細胞を含む細胞集団であって、CD7及び/又はCD8aの発現量について、末梢血から分離されたγδT細胞に比べて高い発現量を示すγδT細胞が、当該細胞集団を構成するγδT細胞の90%以上の割合で含むことを特徴とするγδT細胞の細胞集団。
- γδT細胞を含む細胞集団であって、未分化細胞が当該細胞集団を構成するγδT細胞の10%以下であることを特徴とする、請求項18~20のいずれかに記載の細胞集団。
- 請求項1~4及び請求項15より選択されるいずれかに記載のiPS細胞由来γδT細胞を有効成分とする、抗原特異的細胞性免疫治療剤。
- 液体培地中でビーズ状担体を含む培地を用いて培養することを特徴とする、請求項1~4及び15より選択されるいずれかに記載のiPS細胞由来γδT細胞の培養方法。
- 請求項1~4及び請求項15より選択されるいずれかに記載のiPS細胞由来γδT細胞を有効成分とする、がん、感染症、自己免疫不全等の疾患の治療剤。
- 請求項1~4及び請求項15より選択されるいずれかに記載のiPS細胞由来γδT細胞を有効成分とする、医薬組成物。
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