WO2021157685A1 - ヒトＴ細胞由来ｉＰＳ細胞由来の細胞傷害性Ｔ細胞 - Google Patents

ヒトＴ細胞由来ｉＰＳ細胞由来の細胞傷害性Ｔ細胞 Download PDF

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WO2021157685A1
WO2021157685A1 PCT/JP2021/004232 JP2021004232W WO2021157685A1 WO 2021157685 A1 WO2021157685 A1 WO 2021157685A1 JP 2021004232 W JP2021004232 W JP 2021004232W WO 2021157685 A1 WO2021157685 A1 WO 2021157685A1
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cells
hla
cell
derived
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French (fr)
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美樹 安藤
安藤　純
翠 石井
則夫 小松
啓光 中内
素生 渡部
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University of Tokyo NUC
Juntendo Educational Foundation
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University of Tokyo NUC
Juntendo Educational Foundation
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Priority to EP21750181.6A priority Critical patent/EP4101924A4/en
Priority to CA3170361A priority patent/CA3170361A1/en
Priority to CN202180012822.XA priority patent/CN115087732B/zh
Priority to US17/760,245 priority patent/US20230071538A1/en
Priority to JP2021575877A priority patent/JP7743980B2/ja
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Definitions

  • the present invention relates to cytotoxic T cells derived from human T cell-derived iPS cells that can be administered allogeneically and a method for producing the same.
  • Antigen-specific cytotoxic T cells are class 1 major tissue-compatible antigens (MHC class I, HLA class I) of antigen-presenting cells via T cell receptors (TCRs) present on the cell surface. It recognizes the antigen peptide derived from a virus, tumor, etc. presented together with, and specifically exerts cytotoxic activity and attacks the cell presenting the antigen peptide which is a foreign substance. In addition, a part of CTL becomes a long-term viable memory T cell, which is stored in the host while maintaining cytotoxicity against foreign substances, and then can be dealt with when exposed to foreign substances. Therefore, CTL is expected as a cell for immuno-cell therapy in virus-infected patients and cancer diseases.
  • MHC class I major tissue-compatible antigens
  • HLA class I T cell receptors
  • iPS cell-derived rejuvenation T cell therapy which functionally rejuvenates exhausted T cells using iPS technology and administers the obtained rejuvenation T cells to patients, may be an effective means for improving the therapeutic effect on cancer.
  • CTLs T cell-derived iPS cells
  • T-iPS cells T cell-derived iPS cells
  • HLA class I expression is abolished and allogeneic regT that is not rejected by patient CD8 + T cells is prepared, many severe cases are maintained while maintaining the strong antitumor effect of antigen-specific CTL. It can be administered to the patient quickly. Since iPS cells derived from healthy donors can be administered to many patients by validation stock, a method of knocking out B2M by HLA genome editing to eliminate HLA class I antigen is promising. It is necessary to suppress the missing-self response of NK cells. As this means, HLA class I and class II are means for eliminating HLA class I of iPS cells, inducing differentiation of cells expressing only HLA-E, and avoiding attack from NK cells (Non-Patent Document 1).
  • HLA-A and B are eliminated, and HLA-C is a means for expressing PD-L1, HLA-G and "don't-eat me” signal CD47 (Non-Patent Document 2).
  • Non-Patent Document 3 Several editing methods for avoiding the missing-self response of NK cells, such as retention means (Non-Patent Document 3), have already been reported.
  • the subject of the present invention is the cytotoxicity derived from human T cell-derived iPS cells, which can avoid the missing-self response of NK cells while maintaining the strong antitumor effect of antigen-specific CTL, and can be administered allogeneically.
  • the purpose is to provide T cells and a method for producing the same.
  • the present inventor has a class I molecule of HLA-binding of an antigen epitope of CTL against iPS cells derived from human T cells (for example, HLA-A24, HLA-A02 in the case of HLA-A24-binding CTL).
  • HLA-A24 HLA-A02 in the case of HLA-A24-binding CTL.
  • HLA-A02 HLA-A02 in the case of HLA-A24-binding CTL.
  • the present invention provides the following [1] to [4].
  • [1] Cytotoxic T cells derived from human T cell-derived iPS cells that express HLA-binding class I molecules of CTL antigen epitopes and HLA class I of HLA-E.
  • [2] The cytotoxic T cell derived from the human T cell-derived iPS cell according to [1], wherein the HLA-binding class I molecule of the antigen epitope of CTL is HLA-A24 or HLA-A02.
  • HLA-binding class I molecule of CTL antigen epitope and HLA-E gene are added to T cells in which all HLA class I is knocked out.
  • HLA-binding class I molecules and HLA-E of CTL antigen epitopes comprising a step of introduction and a step of redifferentiating the gene-introduced T-iPS cells into CD8 single positive T cells.
  • the production method according to [3], wherein the HLA-restricted class I molecule of the antigen epitope of CTL is HLA-A24 or HLA-A02.
  • cytotoxic T cells derived from human T cell-derived iPS cells that can avoid the missing-self response of NK cells and can be administered allogeneically while maintaining the strong antitumor effect of antigen-specific CTL. And its manufacturing method can be stably provided.
  • HPV-regT shows HLA class I expression of HPV-regT after genome editing.
  • Post-edit HPV-regT indicates HPV-regt (upper row) in which only HLA-A24 was knocked in after HLA class I disappeared, and HPV-regt (lower row) expressing both HLA-A24 and HLA-E.
  • HPV-regT was stained with antibodies against each HLA class 1 (ABC, A24, BC, E) and analyzed by flow cytometry.
  • Control isotype indicates a negative control in which HPV-regT was stained with the isotype control.
  • HPV-regT which expressed both HLA-A24 and HLA-E significantly suppresses the damaging activity of NK cells.
  • the K562 cell line is a positive control that is injured by NK cells because it does not express class I.
  • KI-A24 indicates HPV-regT in which only HLA-A24 is knocked in after the disappearance of HLA class I.
  • KI-E indicates HPV-regT in which only HLA-E is knocked in after the disappearance of HLA class I.
  • KI-A24 & E indicates HPV-regT in which both HLA-A24 and HLA-E are knocked in after the disappearance of HLA class I.
  • WT indicates that the HLA has not been edited, that is, the wild type that expresses the HLA.
  • auto CTL shows the donor-derived CTL of NK cells used in this analysis. Since it is an autologous cell, NK cells do not attack auto CTL, so it is used as a negative control. It is a figure which shows that HLA-A24 + HLA-E expression HPV-regT has a strong inhibitory effect on NK cell injury activity even in the 107a assay. Since the K562 cell line does not express class I, NK cells attack K562 by co-culture and highly express 107a. It is used as a positive control. Positive Ctrl was stimulated by adding Cell Stimulation Cocktail to NK cells, so that NK cells highly express 107a.
  • HLA-E KI indicates HPV-regT in which only HLA-E is knocked in after the disappearance of HLA class I.
  • HLA-A24 KI indicates HPV-regT in which only HLA-A24 is knocked in after the disappearance of HLA class I.
  • HL-A24 + E KI indicates HPV-regT in which both HLA-A24 and HLA-E are knocked in after the disappearance of HLA class I.
  • Negative Ctrl shows a negative control cultured only with NK cells. The survival time prolonging effect of HLA-edited HPV-regT on cervical cancer-bearing mice is shown.
  • n For non-treatment group, Occasionallyriginal CTL indicates an original CTL treatment group, WTregT indicates a rejT treatment group, and EXregT indicates an HPV-regT treatment group of the present invention. Shows the in vivo durability of HLA-edited HPV-regT when co-administered with NK cells.
  • the numbers in parentheses in HPV-regT indicate HLA class I editing.
  • the cytotoxic T cells of the present invention express HLA of human T cell-derived iPS cells (T-iPS cells), HLA-binding class I molecules of CTL antigen epitopes, and HLA class I of HLA-E. These are cytotoxic T cells. Further, in addition to the HLA-binding class I molecule and HLA-E of the antigen epitope of CTL, the cytotoxic T cells of the present invention may further express HLA class I such as HLA-G and HLA-C. Often, CD47, PD-L1, iCaspase9 and the like may be further expressed.
  • HLA class I HLA-binding class I molecule of CTL antigen epitope
  • HLA-E HLA-E
  • Human T cell-derived iPS cells used as a raw material can be obtained by inducing human T cells into iPS cells (T-iPS cells).
  • the method for producing T-iPS cells is preferably carried out by the method described in Patent Document 1.
  • the T cells used are preferably human T cells.
  • the human origin of these T cells may be a human suffering from a viral infection, malignant tumor, etc., but after genome editing in producing therapeutic allogeneic antigen-specific cytotoxic T cells. It is preferable to be a healthy person from the viewpoint of forming a bank and administering it to many people.
  • a human preferred as a T cell origin needs to have an HLA type that is completely consistent with the patient to whom the regenerated CTL or CART cell produced using the T-iPS cell produced by the present invention should be administered. There is no.
  • the T cells induced by T-iPS cells are preferably T cells having antigen specificity.
  • T cells expressing CD3 and CD8, specifically CTL which is a CD8 positive cell can be mentioned.
  • T cells expressing CD3 and CD4, specifically, T cells which are CD4 positive cells can be mentioned.
  • the antigen specificity in T cells is provided by the antigen-specific, reconstituted TCR gene. From the viewpoint of production efficiency, the method is not particularly limited to this, but in order to obtain antigen-specific CD8-positive cells, antigen-specific CD8-positive T cells are used as human T cells induced into T-iPS cells.
  • an antigen-specific CD4 positive T cell it is preferable to use, and in order to obtain an antigen-specific CD4 positive cell, it is preferable to use an antigen-specific CD4 positive T cell as the human T cell induced into the T-iPS cell. Further, when immunotherapy is performed, it is preferable that the human T cells differentiated from the iPS cells have the same or substantially the same antigen specificity as the human T cells induced into the iPS cells. In addition, T cells that do not have antigen specificity are also included as T cells that induce T-iPS cells. Specific examples thereof include genetically modified T cells such as CART cells and TCR-T cells.
  • T cells can be isolated, for example, from human tissues by known techniques.
  • human tissues include tissues containing the T cells, such as peripheral blood, lymph nodes, bone marrow, thymus, spleen, umbilical cord blood, and lesion tissue.
  • peripheral blood is preferable from the viewpoint of low invasiveness to humans and easy preparation.
  • TIL tumor-infiltrating lymphocytes
  • Known methods for isolating human T cells include, for example, magnetic selection using magnetic beads for cell separation, flow cytometry using an antibody against a cell surface marker such as CD4 or CD8, and a cell sorter, and anti-virus.
  • Examples thereof include an activated T cell induction method using a CD3 antibody and an anti-CD28 antibody. It is also possible to isolate desired T cells using the secretion of cytokines, the expression of functional molecules, or a signal molecule such as PD-1 as an index. In addition, cytotoxic T cells (CTL) can be isolated using the secretion or production of granzyme, perforin, etc. as an index. Further, when isolated from a human tissue containing T cells having antigen specificity, a multimerized MHC (major histocompatibility complex) to which a desired antigen is bound (for example, "MHC"). "Tetramer", “Pro 5 (registered trademark) MHC class I pentamer”) can be used to purify T cells having the desired antigen specificity from human tissues.
  • MHC major histocompatibility complex
  • the genes introduced to convert T cells into iPS cells are (a) Oct3 / 4, (b) c-Myc gene, (c) Sox2 gene, (d) Klf4 gene, and (e).
  • a combination of at least four types of genes such as the NANOG gene and (f) LIN28 gene is preferable.
  • the method for introducing the gene cluster into T cells is not particularly limited, and a known method can be appropriately selected and used.
  • the nucleic acid encoding the gene group for example, cDNA, RNA
  • the expression vector can be inserted into a vector and introduced into cells by an infection, lipofection method, liposome method, electroporation method, calcium phosphate co-precipitation method, DEAE dextran method, microinjection method, or electroporation method.
  • a stealth RNA expression vector is a vector designed to prevent the vector from entering the chromosome and to express the gene continuously and stably in the cytoplasm rather than in the nucleus. It is possible to introduce a large gene of 13,000 base pairs or more and 10 genes at the same time, it does not damage cells, it can be removed when the transgene is unnecessary, and stealth that cells cannot recognize the vector as a foreign substance. Have sex.
  • Such stealth RNA expression vectors include minus single-stranded RNA (A) containing the following RNA sequences (1) to (8), single-stranded RNA-binding protein (B), and RNA-dependent RNA synthase. Examples include complexes that consist of and do not activate the natural immune structure. (1) RNA sequence for the gene cluster, (2) Human mRNA-derived RNA sequences constituting non-coding regions, (3) Transcription initiation signal sequence recognized by the RNA-dependent RNA synthase, (4) Transcription termination signal sequence recognized by the RNA-dependent RNA synthase, (5) An RNA sequence containing an origin of replication recognized by the RNA-dependent RNA synthase.
  • RNA sequence encoding the RNA-dependent RNA synthase (7) An RNA sequence encoding a protein that regulates the activity of the RNA-dependent RNA synthase, (8) An RNA sequence encoding the single-stranded RNA-binding protein.
  • the T cells are interleukin-2 (IL-2) or interleukin-7 (IL-7) and interleukin-15 before the introduction of the gene group. It is preferably activated by stimulating with anti-CD3 antibody and anti-CD28 antibody in the presence of (IL-15), phytohemaglutinin (PHA), interleukin-2 (IL-2), allogeneic antigen-expressing cells, anti. It may be stimulated and activated by at least one substance selected from the group consisting of CD3 antibody and anti-CD28 antibody, CD3 and CD28 agonist.
  • Such stimulation can be performed, for example, by adding PHA, IL-2, anti-CD3 antibody and / or anti-CD28 antibody or the like to a medium and culturing the T cells for a certain period of time.
  • the anti-CD3 antibody and the anti-CD28 antibody may be those to which magnetic beads or the like are bound, and instead of adding these antibodies to the medium, the anti-CD3 antibody and the anti-CD28 antibody are bound to the surface.
  • Stimulation may be given by culturing the T cells on a culture medium for a certain period of time.
  • stimulation may be given by adding an antigen peptide recognized by the T cells (for example, human T cells) to the medium together with the feeder cells.
  • the concentration of PHA added into the medium in order to give such stimulation to the T cells is not particularly limited, but is preferably 1 to 100 ⁇ g / mL.
  • the concentration of IL-2 added to the medium is not particularly limited, but is preferably 1 to 200 ng / mL.
  • the concentrations of the anti-CD3 antibody and the anti-CD28 antibody added to the medium are not particularly limited, but are preferably 1 to 10 times the amount of the T cell culture.
  • the concentrations of the anti-CD3 antibody and the anti-CD28 antibody bound on the surface of the culture dish in order to give such stimulation to the T cells are not particularly limited, but the concentration at the time of coating is 0 for the anti-CD3 antibody. .1 to 100 ⁇ g / mL, preferably 1 to 100 ⁇ g / mL, preferably 0.1 to 10 ⁇ g / mL for anti-CD28 antibody.
  • the culture period for performing such stimulation is a period sufficient to give such stimulation to the T cells, and is a period during which T cells can be proliferated to the number of cells required for the introduction of the four genes. If there is no particular limitation, it is usually 2 to 7 days, but from the viewpoint of gene transfer efficiency, it is preferably 3 to 5 days. From the viewpoint of infecting by mixing T cells and a vector in a 15 mL tube or increasing the efficiency of gene transfer, it is preferable to culture on a culture dish coated with retronectin.
  • a medium for culturing the T cells and adding PHA, IL-2, anti-CD3 antibody and / or anti-CD28 antibody for example, a known medium suitable for culturing the T cells (more specifically, more specifically, The Roswell Park Memorial Institute (RPMI) 1640 medium, AIM VTM medium, NS-A2, which contains other cytokines, human serum, can be used.
  • the medium is PHA, IL-2, anti-CD3 antibody and /.
  • amino acids necessary for culture for example, L-glutamine
  • antibiotics for example, streptomycin, penicillin
  • IL-2 may be added to the medium instead of IL-2. It is also preferable to add -7 and IL-15.
  • the concentration of IL-7 and IL-15 added is not particularly limited, but is preferably 1 to 100 ng / mL, respectively.
  • the conditions for introducing the 4 genes into the T cells or thereafter are not particularly limited, but the T cells into which the 4 genes have been introduced are preferably cultured under feeder-free conditions.
  • laminin 511E8 fragment iMatrix-511 solution or vitronectin coated wells can be mentioned. It can also be established by culturing under feeder cell conditions, and examples of feeder cells include mouse embryonic fibroblasts (MEFs), STO cells, and SNL cells whose cell division has been stopped by irradiation with radiation or treatment with antibiotics. ..
  • the iPS cell medium from the next day. After that, it is preferable to replace the medium by half every other day and gradually replace the T cell medium with the iPS medium.
  • a known medium suitable for culturing the T cells with a medium suitable for culturing the iPS cells in accordance with the transition from the T cells to the iPS cells.
  • a medium suitable for culturing such iPS cells a known medium can be appropriately selected and used.
  • StemFit AK03N in the case of iMatrix coating
  • Essential 8 Medium in the case of coating with Vitronectin, MEF cells, etc.
  • a modified Dalveco eagle medium / F12 medium (human iPS cell medium) containing knockout serum substitute, L-glutamine, non-essential amino acids, 2-mercaptoethanol, b-FGF, etc. is desirable on the feeder cells of.
  • T-iPS cells can be performed by appropriately selecting a known method.
  • a known method for example, a method of observing and selecting the morphology of ES cells / iPS cell-like colonies under a microscope can be mentioned.
  • the properties are often similar, so a method in which all the established colonies are subcultured as they are without selecting each colony of T-iPS cells. There is also.
  • T-iPS cells can be determined, for example, by undifferentiated cell-specific markers (ALP, SSEA-4, Tra-1-60, and Tra-) in the selected cells.
  • ALP undifferentiated cell-specific markers
  • SSEA-4 Tra-1-60
  • Tra- Tra-
  • the expression of (1-81, etc.) can be detected by immunostaining, RT-PCR, or the like, or by transplanting selected cells into mice and observing their teratoma formation. Further, confirmation that the cells selected in this manner are derived from the T cells can be performed by detecting the state of TCR gene rearrangement by genomic PCR.
  • the culture environment is preferably 5% CO 2 , 35 to 38 ° C, more preferably 5% CO 2 , 37 ° C.
  • T cells expressing the HLA-binding class I gene expression of the CTL antigen epitope and the HLA class I of HLA-E for example.
  • the step (a) of knocking out all HLA class I of human T cell-derived iPS cells, and the HLA-binding class I gene and HLA-E of the antigen epitope of CTL were added to the T-iPS cells in which all HLA class I was knocked out.
  • a method including the step (b) of introducing the gene of.
  • the above knockout step (a) and gene transfer step (b) can be carried out by various methods, but the CRISPR-Cas9 genome editing method is one option.
  • ⁇ 2 microglobin (B2M) is first knocked out.
  • electroporation knockout plasmid for the guide RNA in T-iPSC of about 2 ⁇ 10 5 after cell detachment by 5 [mu] g.
  • the seeds are sown in 3 wells on a 6 well plate and cultured.
  • the knockout plasmid contains the target sequence of the guide RNA used for the second editing and selection markers such as GFP, CD8, and CD19. Approximately 10 days later, positive selection of selection markers is performed at the timing when the cells seeded in 3 wells become confluent. After selection, iPS cells are single-cell cloned by thinly seeding T-iPSC. GFP strongly positive cells are picked up, cultured, and genotyped. After identifying the clone containing the marker in biological by PCR, the clone is expanded and cultured, and the process proceeds to the next step.
  • the guide RNA used for the second editing and selection markers such as GFP, CD8, and CD19.
  • positive selection of selection markers is performed at the timing when the cells seeded in 3 wells become confluent.
  • iPS cells are single-cell cloned by thinly seeding T-iPSC. GFP strongly positive cells are picked up, cultured, and genotyped. After identifying the clone containing the
  • the step (b) of introducing the HLA-binding class 1 gene and the HLA-E gene into the T-iPS cells in which all HLA class I was knocked out by the CRISPR-Cas9 genome editing method is a knock-in plasmid 2.
  • 2.5 ⁇ g of each type and 5 ⁇ g of guide RNA are electroporated after cell detachment of T-iPSC in which B2M was knocked out in the first edit. After electroporation, the cells are sown in 3 wells on a 6 well plate and cultured. Negative selection of selection markers is performed at the timing when the cells seeded in 3 wells become confluent after about 7 days.
  • the iPS cells are single-cell cloned by seeding the T-iPS cells thinly. After picking up GFP-negative cells, they are cultured and genotyped. After identifying the clone containing the marker in biological by PCR, it is expanded and cultured. Further, in addition to the HLA-binding class I molecule and HLA-E of the antigen epitope of CTL, HLA class I such as HLA-G and HLA-C may be further expressed by the same means as described above, and CD47 may be further expressed. , PD-L1, iCaspase9 and the like may be expressed.
  • CTL cells are induced to differentiate from the genome-edited T-iPS cells.
  • a method for inducing redifferentiation a method of differentiating T-iPS cells into CD8 + single positive T cells is preferable, and T-iPS cells are differentiated into CD4 / CD8 double negative T cells, and then the CD4 / CD8 double negative.
  • a method of differentiating T cells into CD8 + single positive T cells is more preferred.
  • T-iPS cells are differentiated into CD4 / CD8 double negative cells, and a substance that stimulates the T cell receptor is added to stimulate the CD4 / CD8 double negative cells. Then, it is preferable to obtain the CD4 / CD8 double negative cells stimulated at the T cell receptor by differentiating them into CD8 single positive T cells in the presence of IL-7 and IL-15 cytokines.
  • T-iPS cells are placed on feeder cells (preferably mouse stromal cells) with cytokines, serum (eg, fetal bovine serum (FBS)), insulin, and It is preferable to culture in a medium containing transtransferase, sodium selenate, L-glutamine, ⁇ -monothioglycerol, ascorbic acid and the like.
  • the stromal cells to be used are preferably OP9 cells and 10T1 / 2 cells (C3H10T1 / 2 cells) that have been subjected to a treatment such as irradiation.
  • the cytokine added to the medium is preferably at least one cytokine selected from the VEGF, SCF, TPO, SCF and FLT3L groups, and is VEGF, SCF and TPO, or VEGF, SCF and FLT3L. Is more preferable.
  • the medium include X-VIVO medium, Iskoff-modified Dulbecco medium (IMDM medium), ⁇ -MEM, and DMEM, and T-iPS sack (bag-shaped structure containing hematopoietic progenitor cells) is used.
  • IMDM medium is preferable from the viewpoint of facilitating formation.
  • the culture period of the T-iPS cells is preferably 8 to 14 days, more preferably 10 to 14 days after the start of the culture of the T-iPS cells.
  • the culture environment is not particularly limited, but is preferably 5% CO 2 , 35 to 38 ° C, more preferably 5% CO 2 , 37 ° C. Further, it is more preferable to culture under a low oxygen concentration condition (oxygen concentration: for example, 5 to 20%) for about one week.
  • the cells contained in the T-iPS sack are further subjected to a medium containing cytokines, serum (for example, FBS), or the like. It is preferable to culture on feeder cells (preferably stromal cells, more preferably human stromal cells).
  • feeder cells preferably stromal cells, more preferably human stromal cells.
  • the cells present inside the T-iPS sack can be separated, for example, by passing them through a sterilized sieve device (eg, cell strainer).
  • the stromal cells used in this culture include OP9-DL1 cells, OP9-DL4 cells, and 10T1 / 2 / that have been subjected to treatment such as irradiation from the viewpoint of inducing differentiation into T lymphocytes via notch signaling.
  • Cytokines added to the medium include, for example, IL-7, FLT3L, VEGF, SCF, TPO, IL-2, and IL-15.
  • Examples of the medium include ⁇ -MEM medium, DMEM medium, and IMDM medium, and ⁇ -MEM medium is preferable.
  • amino acids eg, L-glutamine
  • antibiotics eg, streptomycin, penicillin
  • the culture period of the cells contained in this T-iPS sack is until the T cell receptor (TCR) is expressed on the cell surface of the CD4 / CD8 double negative cells thus differentiated.
  • the period is preferably 14 to 28 days after the start of culturing the cells contained in the T-iPS sack.
  • the culture environment is not particularly limited, but is preferably 5% CO 2 , 35 to 38 ° C, more preferably 5% CO 2 , 37 ° C.
  • T cell receptor TCR
  • Whether or not the T cell receptor (TCR) is expressed on the cell surface of the CD4 / CD8 double negative cells is determined by flow cytometry using an anti-TCR ⁇ antibody, an anti-CD3 antibody, an anti-CD4 antibody, and an anti-CD8 antibody. Can be evaluated by.
  • the TCRA gene is stimulated by stimulating CD4 / CD8 double negative cells derived from T-iPS cells via the TCR expressed on the cell surface.
  • CD8 single positive cells obtained by redifferentiation the frequency of appearance of T cells having the same TCR gene rearrangement pattern as the original human T cells is extremely high. can do.
  • T- A method of contacting iPS cell-derived CD4 / CD8 double negative cells is preferable, and a method of contacting specific peptide / HLA complex-expressing cells is more preferable from the viewpoint of giving physiological stimulation. Further, from the viewpoint of emphasizing the uniformity of stimulation, a method of contacting an antibody or a reagent is more preferable.
  • the method of contacting can be carried out, for example, by adding PHA or the like to a medium and culturing the T cells for a certain period of time.
  • the anti-CD3 antibody and the anti-CD28 antibody may be those to which magnetic beads or the like are bound, and instead of adding these antibodies to the medium, the anti-CD3 antibody and the anti-CD28 antibody are bound to the surface.
  • Stimulation may be given by culturing the T cells on a culture medium for a certain period of time.
  • stimulation may be given by adding the antigen peptide together with the feeder cells into the medium.
  • the concentration of PHA added to the medium in order to stimulate the TCR of CD4 / CD8 double negative cells is preferably 1 to 100 ⁇ g / ml.
  • the concentration of the anti-CD3 antibody and the anti-CD28 antibody added to the medium is preferably 1 to 10 times the amount of the T cell culture.
  • the concentration at the time of coating is 0.1 for the anti-CD3 antibody. It is preferably ⁇ 100 ⁇ g / ml, preferably 0.1-10 ⁇ g / ml for anti-CD28 antibody.
  • the T cell receptor As for the culture period of the cells contained in this T-iPS sack, the T cell receptor (TCR) is expressed on the cell surface of the CD4 / CD8 double negative cells thus obtained by differentiation. It is preferable to include a necessary period, and it is preferable that the period is 7 to 29 days after the start of culturing the cells contained in the T-iPS sack.
  • the culture environment is preferably 5% CO 2 , 35 to 38 ° C, more preferably 5% CO 2 , 37 ° C.
  • the CD4 / CD8 double negative cells contain cytokines, serum (for example, human serum) and the like. It is preferable to cultivate in the medium to be used.
  • the cytokine to be added to the medium may be any cytokine capable of differentiating CD4 / CD8 double negative cells into CD8 single positive cells, and examples thereof include IL-7 and IL-15. Among these, IL-7 and IL-15 can be added in combination from the viewpoint of selecting the CD8 lineage and facilitating the generation of memory-type CD8 + T cells in the differentiation into CD8 single positive cells. preferable.
  • the addition concentration of IL-7 and IL-15 is preferably 1 to 20 ng / ml.
  • the medium include RPMI-1640 medium, X-VIVO medium, DMEM medium, and ⁇ -MEM medium, and RPMI-1640 medium or X-VIVO medium is preferable.
  • the medium contains amino acids (eg, L-glutamine), antibiotics (eg, streptomycin, penicillin), IL-7, IL-15, etc., which are necessary for culturing. Cytokine may be added.
  • CD4 / CD8 double negative cells may be co-cultured with feeder cells.
  • the feeder cells are preferably peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • peripheral blood monogenesis presenting an antigen peptide to which human T cells, which are the source of CD4 / CD8 double negative cells, specifically bind. It is more preferable to use nuclear cells.
  • the culture period for differentiating the CD4 / CD8 double negative cells into CD8 single positive cells is preferably 2 to 4 weeks.
  • the culture environment is preferably 5% CO 2 , 35 to 38 ° C, more preferably 5% CO 2 , 37 ° C.
  • CD8 single positive cells thus induced to differentiate are derived from T-iPS cells and derived from the T cells that are the source of the T-iPS cells can be confirmed, for example, in the state of TCR gene rearrangement. Can be done by detecting by genomic PCR.
  • the CD8 single positive cells thus obtained can be isolated by appropriately selecting a known method.
  • a known method for example, flow cytometry using an antibody against a cell surface marker of CD8 and a cell sorter can be mentioned.
  • a method of purifying using an affinity column or the like in which an antigen recognized by the T cell that is the source of the CD8 single positive cells is immobilized, or an MHC multimer to which the antigen is bound can also be used for purification.
  • the CD8 single positive cells obtained by the present invention do not express PD-1, whereas CCR7 is expressed together with CD27 and CD28, which represent the phenotype of central memory T cells, and telomea is also present. It is longer than the original T cell and has a high self-renewal ability. Therefore, according to the present invention, a CD8 single positive T cell having the same TCR gene rearrangement pattern as the original T cell, which does not express PD-1, but expresses CD27, CD28 and CCR7. Can be manufactured.
  • the T cells collected from humans are different from the obtained T cells in that they express PD-1 and the proportion of immature memory phenotypes is small.
  • the cells may be stimulated every 1 to 2 weeks.
  • Such stimuli include anti-CD3 antibody, anti-CD28 antibody, IL-2, IL-7, IL-15, an antigen recognized by the CD8SP cell, an MHC multimer to which the antigen is bound, the CD8 single positive cell and allo.
  • the resulting cytotoxic T cells derived from human T cell-derived iPS cells expressing HLA-A24 and HLA-E HLA class I of the present invention are antigen-specific cytotoxic cells of human T cells used as raw materials. It is useful as a cytotoxic T cell derived from human T cell-derived iPS cells that can be administered allogeneically because it can avoid the missing-self response of NK cells while maintaining sex.
  • the NK cell responsiveness of the cytotoxic T cells of the present invention is significantly lower than that of T cells expressing only HLA-A24 or HLA-E.
  • Example 1 Establishment of T-iPS cells using Sendai virus vector from human papillomavirus (HPV) -specific CTL clone.
  • Peripheral blood mononuclear cells were isolated from the peripheral blood of healthy subjects, and then dendritic cells were induced for the purpose of antigen presentation. After 7 days, HPV antigen peptide (HPV16-E6, A2402) was added to the induced dendritic cells, and co-culture with peripheral blood mononuclear cells was started. After about 8 to 10 days, the CTL was stained with MHC tetramer for HPV-specific CTL detection, and then the tetramer positive rate was confirmed by flow cytometry.
  • HPV antigen peptide HPV16-E6, A2402
  • Example 2 Knockout of all HLA class I cells of HPV antigen-specific CTL-derived iPS cells.
  • B2M knock out ⁇ 2 microglobin
  • Electroporation was performed on the T-iPSC after cell detachment by 5 ⁇ g each of the knockout plasmid and the guide RNA using LONZA 4-D Nucleofector. After that, the seeds are sown in 3 wells on a 6 well plate and cultured. Since the knockout plasmid contains the target sequence of the guide RNA used for the second editing and the selection markers of GFP and CD8, the MACS bead positive selection of CD8 is performed at the timing when the cells seeded in 3 well become confident about 10 days later. went.
  • iPS cells were single-cell cloned by thinly seeding T-iPSC. GFP strongly positive cells were picked up, cultured, genotyped, and a clone containing a marker in biological was identified by PCR and then expanded and cultured, and the process proceeded to the next step.
  • HLA-A24 and HLA-E genes were knocked into the T-iPSC after B2M knockout.
  • a knock-in plasmid having an HLA-E trimmer structure was also prepared and HLA-E was knocked in.
  • a plasmid in which HLA-A2402 and HLA-E were knocked in by half at the same time was also prepared.
  • the T-iPSC in which B2M was knocked out in the first edit was electroporated using a LONZA 4-D Nucleofector after cell detachment.
  • iPS cells were single-cell cloned by thinly seeding T-iPSC. GFP-negative cells were picked up and cultured, genotyping was performed, clones were identified, and then expanded culture was performed.
  • Example 4 Redifferentiation of T-iPS cells after genome editing into CD8 single positive T cells.
  • the genome-edited small mass of T-iPS cells obtained in Example 3 was transferred onto C3H10T1 / 2 cells and placed in EB medium in the presence of 20 ng / mL VEGF, 50 ng / mL SCF and 50 ng / mL FLT-3L. Co-cultured. On the 14th day of culture, hematopoietic cells contained in the iPS sack were collected, transferred onto cells on DL1 / 4 expressing C3H10T1 / 2 cells, and 10 ng / mL FLT-3L and 1 ng / mL IL-7. In the presence, hematopoietic cells were differentiated into T lineage cells in OP9 medium.
  • ⁇ -CD3 / CD28 beads or 5 ⁇ g / ml PHA was stimulated by adding to the OP9 medium.
  • T lineage cells were collected and cultured in CTL medium together with irradiated PMBCs in the presence of 10 ng / mL IL-7 and 10 ng / mL IL-15.
  • Example 5 Antigen specificity of CD8 single positive cells of the present invention. It was examined whether the redifferentiated cells obtained in Example 4 had the same antigen specificity as the original T cells even after genome editing. As a result, it was revealed that the obtained redifferentiated CD8 single positive cells were consistent with the antigen specificity of the original HPV-T cell clone (Fig. 1). After successful induction of differentiation of rejuvenated CTL (rejuvenated CTL; rejT) that expresses only HLA-A24, only HLA-E, or both, a chromium assay is used to investigate whether it can be avoided from the missing self-reaction of NK cells. A CD107a assay was performed.
  • HPV-regT which expressed only HLA-A24 and only HLA-E, suppressed NK activity but was insufficient, but HPV-regT, which expressed both, was able to significantly suppress the damaging activity of NK cells. .. Expression of both HLA-restricted HLA class I molecules and HLA-E of CTL antigen epitopes has been demonstrated to be important for suppressing NK activity (FIGS. 2 and 3).
  • Example 6 HLA-edited HPV-regT survival-prolonging effect on cervical cancer-bearing mice.
  • Method After intraperitoneal transplantation of the cervical cancer cell line SiHa into immunodeficient mice (NOG mice), a no-treatment control group and three treatment groups (original HPV-CTL clone, Wild type (WT) HPV-regT, or After HLA editing, HPV-regT) was divided into 2.5x10 6 T cells once a week and administered 3 times by intraperitoneal injection. The survival time was compared for the purpose of confirming the therapeutic effect of the mice in the non-treatment control group and each treatment group. (result) The survival time of cervical cancer-bearing mice is shown in FIG. As shown in FIG.
  • the survival time of cervical cancer-bearing mice was significantly prolonged in the WT regT-administered group and the HLA-edited HPV-regT (EXregT) -administered group as compared with the original CTL-administered group.
  • pathological findings of treated mice that survived for 6 months or longer showed no residual tumor in the lungs, liver, intestinal tract, spleen, and uterus.
  • Example 7 In vivo durability of HLA-edited HPV-rejT when co-administered with NK cells.
  • Methods After intraperitoneal transplantation of the cervical cancer cell line SiHa into immunodeficient mice (NOG mice) into Day-4, they were divided into three groups. 1. 1. HLA edited FFluc-regT + NK cells (HLA-A24 +) 2. HLA edited FFluc-regT + NK cells (HLA-A24-) 3. 3. HLA editing FFluc-regT In addition, Day 0 was administered with 2.5x10 6 rejT cells + 2.5x10 6 NK cells in groups 1 and 2, and group 3 was administered with 2.5x10 6 rejT cells by intraperitoneal injection.

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HAN XWANG MDUAN SFRANCO PJKENTY JHHEDRICK P ET AL.: "Generation of hypoimmunogenic human pluripotent stem cells", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 116, no. 21, 2019, pages 10441 - 6, XP055640699, DOI: 10.1073/pnas.1902566116
See also references of EP4101924A4
XU HUAIGENG, WANG BO, ONO MIYUKI, KAGITA AKIHIRO, FUJII KAHO, SASAKAWA NORIKO, UEDA TATSUKI, GEE PETER, NISHIKAWA MISATO, NOMURA M: "Targeted disruption of HLA genes via CRISPR-Cas9 generates iPSCs with enhanced immune compatibility", CELL STEM CELL, vol. 24, no. 4, 2019, pages 566 - 578, XP085654753 *
XU HWANG BONO MKAGITA AFUJII KSASAKAWA N ET AL.: "Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility", CELL STEM CELL, vol. 24, no. 4, 2019, pages 566 - 78

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023037986A1 (ja) * 2021-09-13 2023-03-16 公益財団法人京都大学ｉＰＳ細胞研究財団 多能性幹細胞の製造方法
JPWO2023037986A1 (https=) * 2021-09-13 2023-03-16
WO2023037544A1 (ja) * 2021-09-13 2023-03-16 公益財団法人京都大学ｉＰＳ細胞研究財団 多能性幹細胞の製造方法

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CA3170361A1 (en) 2021-08-12
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JPWO2021157685A1 (https=) 2021-08-12
US20230071538A1 (en) 2023-03-09
EP4101924A4 (en) 2024-03-13
JP7743980B2 (ja) 2025-09-25

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