[go: up one dir, main page]

WO2024228384A1 - Composition destinée à induire une tolérance immunitaire - Google Patents

Composition destinée à induire une tolérance immunitaire Download PDF

Info

Publication number
WO2024228384A1
WO2024228384A1 PCT/JP2024/016734 JP2024016734W WO2024228384A1 WO 2024228384 A1 WO2024228384 A1 WO 2024228384A1 JP 2024016734 W JP2024016734 W JP 2024016734W WO 2024228384 A1 WO2024228384 A1 WO 2024228384A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
pluripotent stem
stem cells
cba
immune tolerance
Prior art date
Application number
PCT/JP2024/016734
Other languages
English (en)
Japanese (ja)
Inventor
研一郎 清野
智紀 鎌谷
Original Assignee
国立大学法人北海道大学
住友ファーマ株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人北海道大学, 住友ファーマ株式会社 filed Critical 国立大学法人北海道大学
Publication of WO2024228384A1 publication Critical patent/WO2024228384A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a composition for inducing immune tolerance and its use.
  • the main approach to controlling rejection is to suppress the recipient's immune response by administering drugs such as cyclosporine and tacrolimus.
  • drugs such as cyclosporine and tacrolimus.
  • immunosuppressants can cause infections and malignant tumors, and long-term use can lead to complications such as nephrotoxicity, hypertension, hyperlipidemia, and diabetes. Therefore, new methods of rejection control that do not rely on immunosuppressants or that use reduced amounts of immunosuppressants are needed.
  • Immune tolerance induction has attracted attention as a means of controlling the rejection reaction of recipients in allogeneic transplants.
  • an immunomodulator for immune tolerance induction containing regulatory T cells (also called Treg) as an active ingredient (Patent Document 1), an immune tolerance inducer containing hematopoietic stem cells, hematopoietic progenitor cells or a mixture of these as the tolerance-inducing active ingredient (Patent Document 2), and that the tissue retention rate in allogeneic transplants is improved by administering donor-derived B cells or dendritic cells to the recipient before transplantation (Non-Patent Documents 4 and 5).
  • An approach has also been reported that attempts to use cells with immune regulatory ability differentiated from pluripotent stem cells to induce immune tolerance (Non-Patent Document 6).
  • the inventors have found that in allogeneic recipients with MHC compatibility between the transplanted pluripotent stem cells and the pluripotent stem cells, the transplanted pluripotent stem cells form teratomas without being rejected, even without the use of immunosuppressants, and that immune tolerance is induced by the transplantation of pluripotent stem cells.
  • Item 1 A composition for inducing immune tolerance, comprising pluripotent stem cells having teratoma-forming ability, or a teratoma derived from the pluripotent stem cells.
  • Item 2. The composition according to Item 1, wherein the pluripotent stem cells express CD24.
  • Item 3. The composition according to Item 1 or 2, wherein the teratoma expresses CD24.
  • Item 4. The composition according to any one of Items 1 to 3, wherein the teratoma has an ability to produce erythropoietin.
  • Item 7. The composition according to any one of Items 1 to 6, for use in a mammalian individual having MHC compatibility with the pluripotent stem cell.
  • Item 8. The composition according to any one of Items 1 to 7, for use in a recipient receiving a transplant of donor-derived cells, tissue or organs.
  • Item 9. The composition according to Item 8, wherein the pluripotent stem cells are derived from a donor.
  • Item 10A The composition according to any one of Items 1 to 7, for use in a mammalian individual having an autoimmune disease.
  • Item 11 A method for inducing immune tolerance in a mammalian individual, comprising administering an effective amount of a pluripotent stem cell having teratoma-forming ability, or a teratoma derived from the pluripotent stem cell, to the mammalian individual in which immune tolerance is desired.
  • Paragraph 11A The method of paragraph 11, wherein the pluripotent stem cells express CD24.
  • Paragraph 11B The method of paragraph 11 or 11A, wherein the teratoma expresses CD24.
  • Item 11C The method according to Item 11, 11A or 11B, wherein the teratoma has an ability to produce erythropoietin.
  • Item 11D The method according to any one of Items 11 and 11A to 11C, wherein the teratoma has the ability to accumulate or infiltrate regulatory T cells.
  • Paragraph 11E The method of any one of paragraphs 11, 11A to 11D, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.
  • Paragraph 11F The method according to any one of paragraphs 11, 11A to 11E, for use in a mammalian individual having MHC compatibility with the pluripotent stem cells.
  • Item 11G The method according to any one of Items 11, 11A to 11F, for use in a recipient receiving a transplant of a donor-derived cell, tissue or organ.
  • Paragraph 11H The method according to any one of Items 11, 11A to 11F, for use in a recipient receiving a transplant of a donor-derived cell, tissue or organ.
  • Paragraph 11G wherein the pluripotent stem cells are derived from a donor.
  • Paragraph 11I The method of paragraph 11G or 11H, wherein the recipient is MHC compatible with the donor.
  • Item 12 A method for improving the engraftment rate of donor-derived cells, tissues or organs in a recipient, comprising administering to a recipient an effective amount of pluripotent stem cells having teratoma-forming ability, or teratomas derived from the pluripotent stem cells, and transplanting the donor-derived cells, tissues or organs into the recipient.
  • Paragraph 12A The method of paragraph 12, wherein the pluripotent stem cells express CD24.
  • Paragraph 12B The method of paragraph 12 or 12A, wherein the teratoma expresses CD24.
  • Item 12C The method according to Item 12, 12A or 12B, wherein the teratoma has an ability to produce erythropoietin.
  • Item 12D The method according to any one of Items 12 and 12A to 12C, wherein the teratoma has the ability to accumulate or infiltrate regulatory T cells.
  • Paragraph 12E The method of any one of paragraphs 12, 12A-12D, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.
  • Paragraph 12F The method of any one of paragraphs 12, 12A-12E, wherein the recipient is MHC compatible with the pluripotent stem cells.
  • Paragraph 12G The method according to Item 12, 12A or 12B, wherein the teratoma has an ability to produce erythropoietin.
  • Item 12D The method according to any one of Items 12 and 12A to 12C, wherein the teratoma has the ability to accumulate or infiltrate regulatory T cells.
  • Paragraph 12E The
  • Item 13 A method for treating an autoimmune disease, comprising administering an effective amount of pluripotent stem cells having teratoma-forming ability, or a teratoma derived from said pluripotent stem cells, to a mammalian individual having the autoimmune disease.
  • Paragraph 13A The method of paragraph 13, wherein the pluripotent stem cells express CD24.
  • Paragraph 13B The method of paragraph 13 or 13A, wherein the teratoma expresses CD24.
  • Item 13C The method according to Item 13, 13A or 13B, wherein the teratoma has an ability to produce erythropoietin.
  • Item 13D The method according to any one of Items 13 and 13A to 13C, wherein the teratoma has the ability to accumulate or infiltrate regulatory T cells.
  • Paragraph 13E The method of any one of paragraphs 13, 13A-13D, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.
  • Paragraph 13F The method according to any one of paragraphs 13, 13A to 13E, wherein the mammalian individual having an autoimmune disease is MHC compatible with the pluripotent stem cells.
  • Item 14 A kit for medical transplantation comprising an effective amount of pluripotent stem cells capable of forming teratomas or teratomas derived from the pluripotent stem cells, and cells, tissues or organs derived from a donor.
  • the present invention makes it possible to induce immune tolerance in mammalian individuals in which induction of immune tolerance is desired.
  • the graph shows the mean value ⁇ standard error.
  • the horizontal axis of the graph represents the period from the date of skin grafting to the date of rejection (this is also true for all graphs showing the survival period of skin fragments).
  • 1 is a graph showing the engraftment period of skin fragments from donor mice (B6C3F1, BALB/c, 129X1, CBA/N) transplanted into recipient mice (B6C3F1) subcutaneously inoculated with CBA/N mouse-derived ES cells (CBA-ES).
  • FIG. 5 is a graph showing the survival period of skin pieces from donor mice (B6C3F1, BALB/c, CBA/N) transplanted onto recipient mice (B6C3F1).
  • A shows the results for untreated recipient mice
  • B and C show the results for recipient mice subcutaneously inoculated with CBA-iPS cells on the same day and 10 days before skin transplantation, respectively.
  • 6 is a graph showing the survival period of skin pieces from donor mice (B6C3F1, BALB/c, CBA/N) transplanted onto recipient mice (B6C3F1).
  • Figure 6, A to C show the results of recipient mice subcutaneously inoculated with CBA-iPS cells 20, 30, and 40 days before skin transplantation, respectively.
  • FIG. 7 is a graph showing the survival period of skin fragments from donor mice (B6C3F1, BALB/c, CBA/N) transplanted onto recipient mice (B6C3F1).
  • Figure 7A shows the results of recipient mice inoculated with CBA-iPS subcutaneously 40 days before skin grafting and excised with a teratoma formed 20 days before skin grafting
  • Figure 7B shows the results of recipient mice inoculated with CBA-iPS subcutaneously 40 days before skin grafting and excised with a teratoma formed 20 days after skin grafting.
  • the images show luciferase luminescence in teratomas, spleens, teratoma-draining lymph nodes, and teratoma-distal lymph nodes taken from B6C3F1 mice subcutaneously inoculated with Luc-CBA-iPS.
  • Dot plots showing the results of flow cytometry analysis of CBA-iPS (Fig. 9, right) and teratoma cell populations formed in B6C3F1 mice subcutaneously inoculated with CBA-iPS (Fig. 9, left).
  • the numbers in the upper right corner of each plot indicate the percentage of CD24 positive cells (the percentage of cells within the square in the CD45 negative live cell population).
  • FIG. 10 is a graph showing the survival period of skin pieces from donor mice (BALB/c, CBA/N, B6C3F1) transplanted onto recipient mice (B6C3F1).
  • Fig. 10A shows the results for untreated recipient mice
  • Fig. 10B shows the results for recipient mice subcutaneously inoculated with CBA-iPS before skin transplantation
  • Fig. 10C shows the results for recipient mice subcutaneously inoculated with Cd24a knockout CBA-iPS before skin transplantation.
  • the upper images are immunohistochemical staining images using an anti-erythropoietin antibody and optical microscope images (lower images) of the kidney of a B6C3F1 mouse and teratomas formed in a B6C3F1 mouse subcutaneously inoculated with CBA-iPS (20 and 40 days after subcutaneous inoculation).
  • FIG. 1 is a graph showing the engraftment period of a skin fragment from a donor mouse (CBA/N) grafted onto a recipient mouse (B6C3F1) under administration of erythropoietin.
  • 14A and 14B are graphs showing the anti-dsDNA-IgG concentration in the blood and the lymph node weights of C3H/lpr mice subcutaneously inoculated with CBA-iPS. The plots show the actual measured values for each individual, and the graphs show the mean ⁇ standard error.
  • Statistical analysis was performed using Tukey's HSD test. * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01.
  • Figure 15 shows the results of single-cell RNA analysis of regulatory T cells (Treg) infiltrating teratomas formed in B6C3F1 mice subcutaneously inoculated with CBA-iPS.
  • Figure 15A shows a plot of Treg clustering based on gene expression information.
  • Figure 15B shows a heat map showing the marker genes of each Treg cluster.
  • Figure 15C shows gene expression of immunosuppressive factors (Il10, Il2ra, Clta4, Entpd1, Nt5e) in Treg.
  • Figure 15D is a bar graph showing the abundance ratio of each Treg cluster in teratomas over time.
  • This graph shows the period of survival of skin fragments from a donor mouse (CBA/N) transplanted onto a recipient mouse (B6C3F1) after administration of anti-CD25 antibody or control IgG.
  • 17 is a graph showing the engraftment period of donor mouse (CBA/N) skin pieces transplanted onto recipient mice (B6C3F1). In all trials shown in Fig. 17, subcutaneous inoculation of CBA-iPS cells and skin transplantation were performed on the same day.
  • the present disclosure provides a composition for inducing immune tolerance that contains pluripotent stem cells capable of forming teratomas or teratomas derived from the pluripotent stem cells.
  • the present disclosure also provides an immune tolerance inducer that contains pluripotent stem cells capable of forming teratomas or teratomas derived from the pluripotent stem cells.
  • pluripotent stem cells refer to cells that have the ability to self-replicate and pluripotency, which is the ability to differentiate into all cell lineages belonging to the three germ layers (ectoderm, mesoderm, and endoderm).
  • pluripotent stem cells include iPS cells, ES cells, and embryonic germ stem cells (EG cells).
  • the pluripotent stem cells used in this disclosure are preferably iPS cells or ES cells, and more preferably iPS cells.
  • the pluripotent stem cells used in the present disclosure are mammalian pluripotent stem cells, and are preferably pluripotent stem cells derived from a mammalian individual of the same species as the individual in which induction of immune tolerance is desired.
  • the mammals in this disclosure include humans and non-human mammals, and examples of non-human mammals include rodents including mice, rats, hamsters, and guinea pigs, primates including monkeys and chimpanzees, livestock including pigs, cows, goats, horses, and sheep, and pets including dogs and cats.
  • rodents including mice, rats, hamsters, and guinea pigs, primates including monkeys and chimpanzees, livestock including pigs, cows, goats, horses, and sheep, and pets including dogs and cats.
  • a preferred mammal is a mouse or a human, and more preferably a human.
  • iPS cells can be generated by reprogramming methods known to those skilled in the art.
  • a reprogramming method is a method in which genes called reprogramming factors or proteins encoded by them, such as four types of genes Oct 3/4, Sox 2, Klf 4, and Myc (c-Myc, N-Myc, L-Myc), are introduced into somatic cells, such as fibroblasts or blood cell lines (e.g. peripheral blood-derived mononuclear cells), and then the cells are cultured under appropriate conditions to induce pluripotent cells.
  • somatic cells such as fibroblasts or blood cell lines (e.g. peripheral blood-derived mononuclear cells)
  • Other examples of reprogramming methods include a method in which the above genes are replaced with drugs such as low molecular weight compounds (e.g. Hou P. et al., 2013, Science, 9; 341(6146), 651-4) or a method using microRNA (Miyoshi N. et al., Cell Stem Cell, 2011,
  • ES cells can be produced, for example, by culturing the inner cell mass present in a pre-implantation blastocyst-stage embryo on feeder cells or in a medium containing LIF or FGF2.
  • Examples of human embryonic stem cells include those established from human embryos within 14 days of fertilization.
  • pluripotent stem cells provided by cell banks and commercially available pluripotent stem cells can also be used.
  • human induced pluripotent stem cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, and 1231A3 cells established at Kyoto University are available from Kyoto University and iPS Academia Japan, Inc.
  • Pluripotent stem cells can be produced, maintained and expanded in the presence of feeder cells.
  • Feeder cells are cells other than the pluripotent stem cells that are used when maintaining the undifferentiated state of pluripotent stem cells in culture, and examples of such cells include mouse fibroblasts (MEF), human fibroblasts, and SNL cells. Feeder cells are preferably used after treatment with a growth inhibitor such as mitomycin C or with gamma ray irradiation.
  • Pluripotent stem cells can also be generated, maintained, and expanded in the absence of feeder cells (feeder-free).
  • Feeder-free refers to conditions in which feeder cells are not added or are substantially free of feeder cells.
  • Media for feeder-free culture include, for example, Essential 8 medium, Essential 6 medium, TeSR medium, mTeSR medium, mTeSR-E8 medium, Stabilized Essential 8 medium, StemFit (registered trademark), etc.
  • the cells disclosed herein can be cultured using BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, ⁇ MEM medium, DMEM medium, F-12 medium, DMEM/F12 medium, IMDM/F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, or a mixture of these mediums, etc.
  • the pluripotent stem cells used in this disclosure maintain their undifferentiated state and have the ability to form teratomas.
  • the undifferentiated state of pluripotent stem cells can be confirmed by detecting the expression of undifferentiated markers.
  • undifferentiated markers include Oct3/4, Nanog, TRA-1-60, SSEA1, SSEA4, TRA-1-81, TRA-2-49, Rex-1, etc., and when the expression of one or more, preferably two or more, more preferably three or more of these markers is detected, the pluripotent stem cells can be determined to maintain their undifferentiated state.
  • marker expression can be confirmed by detecting or measuring the expression of a marker gene or marker protein.
  • Gene expression can be detected and measured by common methods such as hybridization, PCR, and RNA-Seq that utilize the base sequence information of the target gene.
  • Protein expression can be detected and measured by common methods such as ELISA, Western blotting, and flow cytometry that use a specific antibody against the target protein.
  • the undifferentiated state of pluripotent stem cells can also be confirmed by evaluating their ability to differentiate into the three germ layers (endoderm, mesoderm, and ectoderm). For example, if an embryoid body (EB) formed in vitro from a pluripotent stem cell contains endoderm, mesoderm, and ectoderm cells, it can be determined that the pluripotent stem cell has the ability to differentiate into the three germ layers and therefore maintains its undifferentiated state.
  • EB embryoid body
  • pluripotent stem cells are seeded in vitro into endoderm differentiation medium, mesoderm differentiation medium, and ectoderm differentiation medium, and differentiation into endoderm, mesoderm, and ectoderm cells is observed, it can be determined that the pluripotent stem cell has the ability to differentiate into the three germ layers and therefore maintains its undifferentiated state.
  • the presence of cells of each germ layer can be confirmed by observing cell morphology and detecting ectodermal markers (B-III tubulin, PAX6, MAP2, TUJ1, NESTIN, etc.), mesodermal markers (Brachyury, MSX1, ⁇ SMA, NCAM, etc.), and endodermal markers (SOX17, AFP, FOXA2, etc.).
  • ectodermal markers B-III tubulin, PAX6, MAP2, TUJ1, NESTIN, etc.
  • mesodermal markers Brainury, MSX1, ⁇ SMA, NCAM, etc.
  • endodermal markers SOX17, AFP, FOXA2, etc.
  • pluripotent stem cells can be confirmed by transplanting the pluripotent stem cells into immunodeficient animals such as NOD-Scid mice or Scid mice, or into animals that are MHC compatible with the pluripotent stem cells, and evaluating whether the tumor that is formed contains endoderm, mesoderm, and ectoderm cells.
  • Pluripotent stem cells can be determined to have teratoma-forming ability when they cause tumors containing cells of all three germ layers to form in the transplanted animal.
  • the immune tolerance induction composition and immune tolerance inducer may also contain a teratoma formed from pluripotent stem cells that maintain undifferentiated state and have teratoma-forming ability.
  • the contained teratoma may be in the form of a teratoma cell mass formed from pluripotent stem cells as is, or in the form of a part of a teratoma cell mass harvested from the teratoma cell mass, or in the form of tissue or cells separated from the teratoma cell mass.
  • the pluripotent stem cells express CD24, i.e., are CD24 positive.
  • the teratoma expresses CD24, i.e., are CD24 positive.
  • the immune tolerance induction composition and immune tolerance inducer may contain a CD24 positive pluripotent stem cell population, and may also contain a CD24 positive teratoma cell population.
  • the CD24 positive cell population contains CD24 positive cells at 70% or more, preferably 80% or more, and more preferably 90% or more of the entire cell population. The proportion of CD24 positive cells in the entire cell population can be determined by analyzing the cell population by flow cytometry.
  • CD24 also known as heat-stable antigen (HSA) or nectadrin
  • HSA heat-stable antigen
  • nectadrin a cell adhesion protein that is mainly expressed in immune cells, epithelial cells, nerve cells and muscle cells.
  • NCBI National Center for Biotechnology Information
  • NP_001278666.1, NP_001278667.1, NP_001346013.1 and NP_037362.1 The amino acid sequences of the human CD24 isoform a preproprotein have been registered with the National Center for Biotechnology Information (NCBI) as NP_001278666.1, NP_001278667.1, NP_001346013.1 and NP_037362.1
  • mRNA sequences have been registered as NM_001291737.1, NM_001291738.1, NM_001359084.1 and NM_013230.3.
  • the amino acid sequence of human CD24 isoform b has been registered in NCBI as NP_001278668.1, and the mRNA nucleotide sequence as NM_001291739.1.
  • Mouse CD24 (called Cd24a) has been registered in NCBI as NP_033976.1 and the mRNA nucleotide sequence as NM_009846.2 (both searched on April 26, 2023).
  • the teratoma has the ability to produce erythropoietin.
  • the erythropoietin production ability of a teratoma can be confirmed by detecting or measuring the expression of the erythropoietin gene or protein in the teratoma.
  • the expression level of the erythropoietin gene or protein in the teratoma is greater than the expression level in negative control cells, tissues, or organs known not to produce erythropoietin, or is comparable to the expression level in positive control cells, tissues, or organs known to produce erythropoietin, such as the kidneys and ovaries
  • the teratoma can be determined to have the ability to produce erythropoietin.
  • Erythropoietin is a glycoprotein hormone that regulates red blood cell production and hemoglobin synthesis. It is known to induce immune tolerance to allogeneic donor-derived transplants (Purroy C. et al., 2017, J. Am. Soc. Nephrol, 28(8):2377-2392.).
  • the amino acid sequence of the human erythropoietin precursor has been registered in NCBI as NP_000790.2, and the nucleotide sequence of the mRNA has been registered as NM_000799.4.
  • amino acid sequence of the precursor of mouse erythropoietin has been registered in NCBI as NP_001299804.1 and NP_031968.1, and the mRNA nucleotide sequence has been registered as NM_001312875.1 and NM_007942.2 (both searched on April 27, 2023).
  • the teratoma has the ability to accumulate regulatory T cells (a type of CD4-positive helper T cell, a cell population with immunosuppressive ability) around the teratoma or to infiltrate into the teratoma (also referred to as the "ability to accumulate or infiltrate regulatory T cells").
  • regulatory T cells a type of CD4-positive helper T cell, a cell population with immunosuppressive ability
  • This ability can be confirmed by administering pluripotent stem cells to an MHC-compatible mammalian individual to form a teratoma, or by transplanting a teratoma into an MHC-compatible mammalian individual to allow it to take root, and then detecting regulatory T cells around or inside the teratoma.
  • Known means for detecting regulatory T cells include, for example, immunohistological staining or flow cytometry analysis using an antibody that binds to a known cell marker of regulatory T cells (e.g., CD25, etc.), and gene expression analysis of known cell markers of regulatory T cells by RNA-seq, etc.
  • Immunological tolerance induction compositions and immune tolerance inducers containing pluripotent stem cells can induce immune tolerance with or without teratoma formation in mammalian individuals that have MHC compatibility with the contained pluripotent stem cells. Furthermore, immune tolerance induction compositions and immune tolerance inducers containing teratomas can induce immune tolerance in mammalian individuals that have MHC compatibility with the contained teratomas.
  • Immune tolerance refers to a state in which the immune system is unresponsive to antigens that may cause an immune response.
  • immune tolerance There are two types of immune tolerance: self-tolerance, which is immune unresponsiveness to self-antigens, and acquired tolerance (also called induced immune tolerance), which is immune unresponsiveness to non-self antigens.
  • Acquired tolerance is known to be induced by administering non-self antigens under certain conditions, and is thought to involve the suppression of immune responses to non-self antigens by regulatory T cells.
  • autoimmune diseases and allergic diseases are thought to be caused by the breakdown of immune tolerance to self and non-self, respectively.
  • induction of immune tolerance refers to inducing a state of immune unresponsiveness to a specific antigen, and includes, for example, delaying the onset or progression of an immune response, weakening the strength of an immune response, etc.
  • immune tolerance may be directed to either a self antigen or a non-self antigen.
  • the immune tolerance induction composition and immune tolerance inducer of the present disclosure are used in mammalian individuals in whom induction of immune tolerance is desired.
  • the immune tolerance induction composition and immune tolerance inducer can be used, for example, in mammalian individuals who will be recipients of cell, tissue, or organ transplants to induce immune tolerance to donor-derived grafts that are non-self antigens, and can also be used, for example, in mammalian individuals with autoimmune diseases to control, and preferably suppress, immune responses to self antigens.
  • the mammalian individuals in which the immune tolerance induction composition and immune tolerance inducer are used have MHC compatibility with the pluripotent stem cells contained in the immune tolerance induction composition and immune tolerance inducer, or with the pluripotent stem cells from which the contained teratomas are derived.
  • MHC compatible means that the donor mammalian individual and the donor-derived graft do not have different alleles at some or all of the major MHC loci from the recipient mammalian individual.
  • the pluripotent stem cells do not have different alleles at some or all of the major MHC loci from the mammalian individual (recipient) in which the immune tolerance induction composition and immune tolerance inducer are used, the mammalian individual is MHC compatible with the pluripotent stem cells.
  • the mammalian individual when both MHC haplotypes of the pluripotent stem cells are included in the MHC haplotype of the mammalian individual (recipient), i.e., when the pluripotent stem cells are MHC homozygous and the recipient has the same MHC haplotype as the pluripotent stem cells, or when the MHC duplotype of the pluripotent stem cells is identical to the MHC duplotype of the recipient, the mammalian individual is MHC compatible with the pluripotent stem cells.
  • MHC incompatible means that the donor mammalian individual has different alleles at all major MHC loci than the recipient mammalian individual.
  • MHC compatibility can be routinely confirmed by histocompatibility tests such as HLA typing and lymphocyte crossmatch, which check the compatibility between donor and recipient.
  • Major MHC loci in mice include H-2K, H-2D, H-2L, IA and IE, and may further include any one, preferably all, of Qa-2 and Qa-1. Note that mouse MHC haplotypes are designated according to the specific set of alleles at the major loci.
  • MHC haplotype b corresponds to a haplotype having the allele set H- 2Kb , H- 2Db , H- 2Lnull , IAb , and IEnull
  • MHC haplotype d corresponds to a haplotype having the allele set H- 2Kd , H- 2Dd , H- 2Ld , IAd , and IEd
  • MHC haplotype k also called H-2k
  • H-2k corresponds to a haplotype having the allele set H- 2Kk , H- 2Dk , H- 2Lnull , IAk , and IEk .
  • the major MHC loci in humans include HLA-A, HLA-B, and HLA-DR, and may further include any one, and preferably all, of HLA-C, HLA-DQ, and HLA-DP.
  • MHC compatibility in humans would be a case where a graft with homozygous genotypes at the three loci of HLA-A, HLA-B, and HLA-DR in human HLA types is transplanted to a recipient with a combination of at least one allele at the corresponding locus that matches the allele at that locus, but this is not limited to three loci.
  • a specific example would be an HLA half-matched transplant, where cells contained in peripheral blood or umbilical cord blood of the donor with homozygous HLA-A, HLA-B, and HLA-DR of *24:02-*52:01-*15:02, *33:03-*44:03-*13:02, *24:02-*07:02-*01:01, or *24:02-*54:01-*04:05 are transplanted.
  • HLA-A, HLA-B, HLA-DR of *24:02-*52:01-*15:02, *33:03-*44:03-*13:02, *24:02-*07:02-*01:01, or *24:02-*54:01-*04:05.
  • HLA-A, HLA-B, and HLA-DR *24:02-*52:01-*15:02, *33:03-*44:03-*13:02, *24:02-*07:02-*01:01, or *24:02-*54:01-*04:05 are known to be frequent HLAs in Japan (see WO2015/125941 and the iPS cell stock cells (HLA homozygous donor) Kyoto University iPS Cell Research Foundation (https://www.cira-foundation.or.jp/j/research-institution/ips-stock-project/homozygous.html)).
  • the pluripotent stem cells may be prepared using cells taken from the transplant donor, or may be prepared using cells taken from an individual of the same species as the recipient and transplant donor but different from either. In the latter case, it is preferable that the pluripotent stem cells have an MHC haplotype that completely matches that of the transplant donor, or an MHC duplotype that completely matches that of the transplant donor.
  • the immune tolerance induction composition is not limited as long as it contains pluripotent stem cells or teratomas and a liquid in which they can survive, and can be in the form of a cell suspension, for example.
  • the liquid in which pluripotent stem cells and teratomas can survive may be any liquid commonly used in cell preparations, such as water, saline, phosphate-buffered saline, cell culture medium, etc.
  • the immune tolerance induction composition may contain cells other than pluripotent stem cells and teratomas, and may also contain pharma- ceutical acceptable ingredients such as adjuvants, buffers, antioxidants, preservatives, excipients, carriers, and other active pharmaceutical ingredients. Pharmaceutically acceptable ingredients are well known to those skilled in the art, and those skilled in the art can select and use them as appropriate within the scope of their ordinary practice.
  • the immune tolerance induction composition and immune tolerance inducer can be administered to a mammalian individual in which induction of immune tolerance is desired in an amount effective to induce immune tolerance in the individual, and can be administered one or more times so as to maintain the immune tolerance state for the period for which immune tolerance is desired.
  • the amount effective to induce immune tolerance means an amount effective to form or engraft a teratoma.
  • the maintenance of the immune tolerance state can be evaluated using as an indicator whether the formed teratoma is maintained.
  • the immune tolerance induction composition and immune tolerance inducer can be administered, for example, by subcutaneous or intradermal inoculation.
  • the immune tolerance induction composition and immune tolerance inducer are administered to a recipient in order to induce immune tolerance to a donor-derived graft in the recipient in allogeneic transplantation.
  • the allogeneic transplant is preferably MHC compatible.
  • the donor of the allogeneic transplant does not have alleles different from the recipient at all major MHC loci (HLA loci).
  • the immune tolerance induction composition and immune tolerance inducer can induce immune tolerance in a recipient even in a transplant that has MHC compatibility but a large degree of mismatch in minor histocompatibility antigens, so matching of minor histocompatibility antigens is not required in allogeneic transplantation.
  • the transplant may be any of cells (e.g., but not limited to, retinal pigment epithelium, dopamine neural progenitor cells, glial cells, etc.), tissues (e.g., but not limited to, skin, bone, pancreatic islets, heart valves, blood vessels, cornea, retina, etc.), and organs (e.g., but not limited to, heart, kidney, liver, etc.).
  • cells e.g., but not limited to, retinal pigment epithelium, dopamine neural progenitor cells, glial cells, etc.
  • tissues e.g., but not limited to, skin, bone, pancreatic islets, heart valves, blood vessels, cornea, retina, etc.
  • organs e.g., but not limited to, heart, kidney, liver, etc.
  • the immune tolerance induction composition and immune tolerance inducer are administered to a patient with an autoimmune disease to induce immune tolerance to a self-antigen.
  • the immune tolerance induction composition and immune tolerance inducer can be a therapeutic agent for an autoimmune disease.
  • treatment includes all medical interventions for delaying or halting the progression of an autoimmune disease, regressing or eliminating lesions, and alleviating or curing symptoms.
  • autoimmune diseases include lymphoproliferative disorders, systemic lupus erythematosus, Sjögren's syndrome, rheumatoid arthritis, polymyositis, dermatomyositis, scleroderma, hyperthyroidism, hypothyroidism, autoimmune adrenal insufficiency, true red cell anemia, multiple sclerosis, autoimmune hepatitis, and type 1 diabetes.
  • the immune tolerance induction composition and immune tolerance inducer can be used in combination with treatments to suppress the immune system, examples of which include administration of immunosuppressants, radiation exposure, and T cell depletion treatments.
  • immunosuppressants include cyclosporine, tacrolimus, mycophenolate mofetil, steroids (e.g., methylprednisolone), and T cell activation inhibitors.
  • T cell activation inhibitors include drugs that inhibit the binding between the costimulatory molecule CD28 on T cells and CD80/CD86 on antigen-presenting cells, such as CTLA-4Ig (abatacept), a fusion protein of the extracellular portion of CTLA-4 and the Fc portion of IgG; drugs that inhibit the binding between the costimulatory molecule CD40L8 on T cells and CD40 on antigen-presenting cells, such as anti-CD40L (CD154) antibodies; and drugs that inhibit the binding between the IL-2 receptor ⁇ chain (CD25) on T cells and IL-2, such as anti-CD25 monoclonal antibodies (basiliximab).
  • T cell activation inhibitors are also called co-stimulatory blockades (CBs).
  • CBs co-stimulatory blockades
  • the radiation irradiation may be any type of irradiation that is normally performed as one of the pre-treatments given to the recipient prior to transplantation therapy, with total body irradiation of the recipient or irradiation of the thymus being preferred.
  • the amount of irradiation can be adjusted as appropriate, for example, to a single dose of 1 Gy or more for total body irradiation in humans, and 3 Gy or more for irradiation of the thymus, with the aim of not reaching a lethal dose.
  • the T cell removal procedure may be any procedure that is normally performed as one of the pretreatments administered to the recipient when performing transplantation therapy, and may include administration of an anti-T cell antibody or a steroid.
  • anti-T cell antibodies include anti-CD4 antibody, anti-CD8 antibody, anti-CD3 antibody, anti-TCR antibody, and anti-thymocyte globulin (ATG), and it is preferable to administer one or more of these in combination to the recipient.
  • the anti-T cell antibody is a combination of anti-CD4 antibody and anti-CD8 antibody, which are preferably administered to the recipient intravenously or intraperitoneally simultaneously or consecutively.
  • the amount administered may be an amount sufficient to remove T cells in the recipient's body.
  • the recipient is preferably subjected to radiation or T cell depletion treatment, and more preferably to both radiation and T cell depletion treatment, before administration of the immune tolerance induction composition or immune tolerance inducer.
  • immune tolerance induction composition or immune tolerance inducer By combining the immune tolerance induction composition or immune tolerance inducer with these immune system suppression treatments, immune tolerance to the donor-derived graft can be efficiently induced in the recipient, thereby improving the survival rate of the donor-derived graft in the recipient. It also becomes possible to avoid administration of immunosuppressants to the recipient or to reduce the dosage.
  • Radiation and T cell ablation treatment can be performed once or multiple times just before transplantation if it is desired to induce immune tolerance before transplantation, or at the desired timing to induce tolerance if it is desired to induce immune tolerance after transplantation, i.e., to induce delayed tolerance induction.
  • the present disclosure provides a method for inducing immune tolerance in a mammalian individual, comprising administering an effective amount of pluripotent stem cells capable of forming teratomas, or teratomas derived from the pluripotent stem cells, to a mammalian individual in which immune tolerance is desired.
  • the present disclosure also provides a method for improving the survival rate of a donor-derived graft in a recipient, comprising administering (e.g., subcutaneously or intradermally) to the recipient an effective amount of pluripotent stem cells having teratoma-forming ability or a teratoma derived from the pluripotent stem cells, and transplanting a donor-derived graft into the recipient.
  • the pluripotent stem cells are preferably pluripotent stem cells derived from a donor or from a mammalian individual of the same biological species as the recipient and the donor but different from both, and it is also preferable that the recipient has MHC compatibility with the donor.
  • the administration of the pluripotent stem cells or teratoma and the transplantation of the donor-derived graft may be performed simultaneously or with a time interval.
  • the administration of the pluripotent stem cells or teratoma and the transplantation of the donor-derived graft may be performed with an interval of less than one day, for example, less than 12 hours, less than 6 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, less than 10 minutes, or less than 5 minutes.
  • the donor-derived graft can be transplanted at an interval of more than one day, for example, 3 days or more, 5 days or more, 7 days or more, 10 days or more, 20 days or more, 30 days or more, or 40 days or more.
  • the present disclosure further provides a method for treating an autoimmune disease, comprising administering an effective amount of pluripotent stem cells capable of forming teratomas, or teratomas derived from the pluripotent stem cells, to a mammalian individual having the autoimmune disease.
  • the present disclosure provides a medical transplant kit that includes an effective amount of pluripotent stem cells capable of forming teratomas, or teratomas derived from the pluripotent stem cells, and a donor-derived transplant.
  • a medical transplant kit that includes an effective amount of pluripotent stem cells capable of forming teratomas, or teratomas derived from the pluripotent stem cells, and a donor-derived transplant.
  • Such a kit can be suitably used in the above-mentioned method for improving the survival rate of donor-derived transplants.
  • the present disclosure provides the use of pluripotent stem cells capable of forming teratsomas, or teratomas derived from the pluripotent stem cells, for the manufacture of a composition for inducing immune tolerance, particularly a pharmaceutical composition for inducing immune tolerance.
  • the present disclosure provides the use of pluripotent stem cells capable of forming teratsomas, or teratomas derived from the pluripotent stem cells, for inducing immune tolerance.
  • the present disclosure provides the use of pluripotent stem cells capable of forming teratsomas, or teratomas derived from the pluripotent stem cells, for transplantation medicine, particularly for improving the survival rate of donor-derived transplants in recipients in transplantation medicine.
  • the present disclosure provides the use of pluripotent stem cells capable of forming teratsomas, or teratomas derived from the pluripotent stem cells, for the treatment or prevention of autoimmune diseases or allergic diseases.
  • mice C57BL/6J (B6, MHC haplotype H-2b/b), CBA/N (MHC haplotype H-2k/k), 129X1/SvJ Jms Slc (129, MHC haplotype H-2b/b), C3H/He (C3H, MHC haplotype H-2k/k), and BALB/c (MHC haplotype H-2d/d) were purchased from Japan SLC Co., Ltd.
  • C3129F1 (MHC haplotype H-2k/b) mice were obtained by mating C3H females with 129 males.
  • H-2b/b indicates homozygous for mouse MHC H2 haplotype b
  • H-2d/d indicates homozygous for H2 haplotype d
  • H-2k/k indicates homozygous for H2 haplotype k
  • H-2k/b and H-2b/k indicate heterozygous for H2 haplotypes k and b.
  • iPS cells Fibroblasts were isolated from the ear skin tissue of adult B6, CBA/N, and 129 mice in a standard manner, and four factors, Oct4, Sox2, Klf4, and c-Myc, were introduced into the fibroblasts using a retroviral vector to establish iPS cells derived from B6 mice, CBA/N mice, and 129 mice (Takahashi et al., Cell, 2006, Vol. 126, pp. 663-676. DOI 10.1016/j.cell.2006.07.024).
  • Mouse iPS cells were maintained on laminin-coated culture dishes using advanced-DMEM/F-12 (1:1 mix, Thermo, SIGMA) supplemented with 0.5x NeuroBrew-21 (Miltenyi), 0.5x N2 supplement (Wako), 10 U/ml penicillin, 100 ⁇ g/ml streptomycin, 0.1 mM 2-mercaptoethanol (Nacalai), 0.03% L-glutamine (Gibco), 3 ⁇ M CHIR99021 (Adooq), 1 ⁇ M PD0325901 (Tocris) and recombinant human leukemia inhibitory factor (in house) (hereafter referred to as maintenance medium).
  • maintenance medium recombinant human leukemia inhibitory factor
  • iPS cells were passaged as follows. The culture supernatant was removed from the culture fluid cultured in maintenance medium, and the remaining culture fluid was washed with PBS, after which 1000 ⁇ l of trypsin (0.5 g/l trypsin/0.53 mmol/l EDTA solution, Nacalai Tesque) was added and the cells were allowed to stand in an incubator. After 5 minutes, 5 ml of maintenance medium was added to neutralize the trypsin. The cells were sprayed with medium to produce a single-cell suspension, which was then collected in a tube through a 40 ⁇ m filter and centrifuged at 1500 rpm at 4°C for 5 minutes.
  • trypsin 0.5 g/l trypsin/0.53 mmol/l EDTA solution, Nacalai Tesque
  • the supernatant was removed and the cells were resuspended in maintenance medium.
  • the cell concentration of the suspension was measured, and the required number of cells were reseeded onto a laminin-coated dish and cultured again using maintenance medium.
  • ES cells Adult CBA/N mice were mated with the same strain, and blastocysts were obtained from the uterus 3.5 days after mating. Only the inner cell mass-like cell mass was scraped from the hatched blastocysts and seeded on a laminin-coated dish for culture. SSEA1-positive cells, a pluripotent stem cell marker, were isolated and their teratoma formation ability was confirmed by a teratoma formation assay in nude mice (Marta Vila-Cejudo et al., J. Vis. Exp., 2017, 20;(126):56171. DOI: doi:10.3791/56171). ES cells were passaged in the same manner as iPS cells. The same medium (maintenance medium) was also used.
  • pluripotent stem cells iPS cells, ES cells
  • SSEA1 the undifferentiated marker
  • the luciferase gene was then introduced into the iPS cells using a lentiviral vector (pHIV-Luc-ZsGreen (Addgene)), to generate luciferase-expressing CBA/N mouse-derived iPS cells (Luc-CBA-iPS).
  • the day on which the grafted skin regressed to less than 30% of the size (length of the major axis) of the grafted skin on the day of grafting was defined as the rejection date, and the period from the grafting date to the rejection date was defined as the survival period of the grafted skin.
  • the survival rate of the grafts was calculated by the formula: (total number of grafted skin - number of rejected skin) / total number of grafted skin x 100.
  • CBA/N mice H-2k/k
  • B6C3F1 mice H-2b/k
  • BALB/c mice H-2d/d
  • transplantation of Luc-CBA-iPS into CBA/N mice and B6C3F1 mice is MHC compatible
  • transplantation of Luc-CBA-iPS into BALB/c is MHC incompatible.
  • Figure 1 shows the time course of luciferin luminescence from the site of Luc-CBA-iPS inoculation in the recipient mice. Strong luminescence was detected in the syngenic CBA/N mice 20 days after inoculation, while among the allogeneic recipients, luminescence was detected in the MHC half-matched B6C3F1 mice 30 days after inoculation, although weaker than in the CBA/N mice, and only slight luminescence was detected in the MHC-mismatched BALB/c mice 30 days after inoculation.
  • CBA/N-iPS C57BL/6 mouse-derived iPS cells
  • C57BL/6-iPS C57BL/6 mouse-derived iPS cells
  • 129X1 mouse-derived iPS 129X1-iPS
  • CBA-ES CBA/N mouse-derived ES cells
  • CBA/N mouse-derived iPS cells (CBA-iPS) were subcutaneously inoculated into B6C3F1 mice, and tumors formed at the inoculation site were harvested more than 60 days after inoculation. Formalin-fixed paraffin-embedded sections of the tumors were prepared and stained with hematoxylin and eosin for pathological analysis. Tri-germline structures (ectodermal keratin pearls, mesodermal striated muscle, and endodermal ciliated epithelium) were observed inside the tumors ( Figure 2), confirming that the tumors were teratomas.
  • teratomas were confirmed to have formed at the inoculation site 10 days after iPS cell inoculation, both visually and tactilely (by touching with the hands).
  • the recipient B6C3F1 mice (H-2b/k) are equivalent to allogeneic recipients that are half-MHC matched and minor-mismatched to the transplanted CBA-iPS (H-2k/k). Therefore, transplantation of CBA-iPS into B6C3F1 mice is MHC compatible.
  • the recipient B6C3F1 mouse corresponds to a syngenic recipient for the B6C3F1 skin fragment (H-2b/k), an MHC half-matched and minor-mismatched allogeneic recipient for the CBA/N skin fragment (H-2k/k), and an MHC-mismatched and minor-mismatched allogeneic recipient for the BALB/c skin fragment (H-2d/d).
  • the survival period of the grafted skin pieces is shown in Figure 3.
  • the B6C3F1 skin pieces survived completely in both the CBA-iPS transplantation group and the untreated control group (administered physiological saline).
  • the CBA/N skin pieces were completely rejected about 40 days after skin grafting in the untreated control group, but the skin pieces survived completely in the CBA-iPS transplantation group.
  • the BALB/c skin pieces were completely rejected within 20 days after skin grafting in both the CBA-iPS transplantation group and the untreated control group.
  • teratomas were confirmed to have formed at the inoculation site 10 days after inoculation, both visually and tactilely (by touching with the hands).
  • the recipient B6C3F1 mice (H-2b/k) are equivalent to allogeneic recipients that are half-MHC matched and minor-mismatched to the transplanted CBA-ES (H-2k/k). Therefore, transplantation of CBA-ES into B6C3F1 mice is MHC compatible.
  • the recipient B6C3F1 mouse corresponds to a syngenic recipient for the B6C3F1 skin fragment (H-2b/k), to an MHC half-matched and minor-mismatched allogeneic recipient for the CBA/N skin fragment (H-2k/k) and 129X1 skin fragment (H-2b/b), and to an MHC-mismatched and minor-mismatched allogeneic recipient for the BALB/c skin fragment (H-2d/d).
  • Figure 4 shows the survival period of grafted skin in recipient mice subcutaneously inoculated with CBA-ES. B6C3F1 and CBA/N skin fragments survived completely, whereas 129X1 and BALB/c skin fragments were completely rejected within 30 days after skin grafting. These results confirmed that donor-specific immune tolerance was induced in MHC-half-matched minor-mismatched allogeneic recipients by transplantation of donor-derived ES cells.
  • CBA/N-iPS, C57BL/6-iPS, and 129X1-iPS were subcutaneously inoculated into C3129F1 and B6C3F1 mice, respectively, and cell engraftment and tumor formation were evaluated.
  • Table 1 teratoma formation at the inoculation site 10 days after iPS cell inoculation and extended survival of grafted skin were observed, confirming the induction of donor-specific immune tolerance in MHC half-matched minor mismatched allogeneic recipients.
  • the survival period of the grafted skin pieces is shown in Figures 5 and 6.
  • a CBA/N skin piece was grafted on the same day as CBA-iPS inoculation, it only survived for about the same period as the untreated control ( Figures 5A and B).
  • the survival period increased the longer the period between CBA-iPS inoculation and skin grafting ( Figure 5C, Figures 6A-C).
  • Example 6 Effect of teratoma resection on immune tolerance induction
  • CBA-iPS cells were subcutaneously inoculated into B6C3F1 mice.
  • teratomas were confirmed to have formed at the inoculation site 10 days after iPS cell inoculation, both visually and tactilely.
  • the survival period of the grafted skin is shown in Figure 7.
  • Figure 7A When the teratoma was excised 20 days before CBA/N skin grafting, the inoculated CBA-iPS did not extend the survival period ( Figure 7A).
  • Figure 7B When the teratoma was excised 20 days after CBA/N skin grafting, the inoculated CBA-iPS extended the survival period ( Figure 7B), but the extension was shorter than when the teratoma was not excised ( Figures 3 and 6C).
  • Luc-CBA-iPS cells were inoculated subcutaneously into B6C3F1 mice, and 200 days after inoculation, the formed teratomas, spleens, teratoma-draining lymph nodes (dLNs), and teratoma-distal lymph nodes (non-dLNs) were harvested and the presence of Luc-CBA-iPS-derived cells was analyzed by in vivo imaging.
  • dLNs teratoma-draining lymph nodes
  • non-dLNs teratoma-distal lymph nodes
  • Example 8 Cell surface antigen analysis of iPS cells and teratomas
  • CBA-iPS cells were inoculated subcutaneously into B6C3F1 mice, and 40 days after inoculation, the formed teratomas were harvested.
  • Single-cell suspensions prepared from teratomas using Horizon TM Dri Tumor and Tissue Dissociation Reagent (BD Biosciences) and single-cell suspensions of CBA-iPS cells were subjected to flow cytometry.
  • Flow cytometry was performed using a BD FACSCelesta (Becton Dickinson) and Flowjo software (Tree Star).
  • Live and dead cells were distinguished by forward scatter, side scatter, and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) incorporation.
  • DAPI 4',6-diamidino-2-phenylindole dihydrochloride
  • the fluorescent dye-conjugated monoclonal antibodies used are shown in Table 2. Isotype control antibodies or Fluorescence minus one (FMO) were used as controls.
  • the rate of CD24 positive cells in the CD45 negative live cell population was calculated for single cell suspensions prepared from teratomas and single cell suspensions of CBA-iPS.
  • the results of flow cytometry analysis are shown in Figure 9. CD24 expression was confirmed in both CBA-iPS and teratoma cells formed from CBA-iPS.
  • CD24 CRISPR Plasmids mouse which contained guide RNA to knock out the mouse Cd24a gene, was introduced into CBA-iPS cells according to the manufacturer's protocol to generate CBA-iPS cells in which the Cd24a gene was knocked out (CD24 ko CBA-iPS cells).
  • the survival period of the grafted skin is shown in Figure 10.
  • the CBA/N skin fragments survived with a 75% probability when CBA-iPS cells were subcutaneously inoculated, whereas the survival rate was only 50% when CD24 ko CBA-iPS cells were subcutaneously inoculated.
  • the teratomas had disappeared at the time of skin fragment rejection. This result suggested that CD24 contributes to the induction of immune tolerance by iPS cell transplantation.
  • Example 10 Analysis of erythropoietin (EPO) expression in iPS cells and teratomas
  • CBA-iPS cells were subcutaneously inoculated into B6C3F1 mice, and teratomas formed 20 days after inoculation were harvested. Teratomas, ovaries, and kidneys were harvested 40 days after inoculation. The ovaries and kidneys are known to produce erythropoietin, and were used as positive controls in this example.
  • the amount of erythropoietin gene expression is shown in Figure 11, and the immunostaining image is shown in Figure 12. Teratomas were confirmed to produce erythropoietin, just like the ovaries and kidneys.
  • Example 11 Effect of erythropoietin on immune tolerance induction
  • B6C3F1 mice were intraperitoneally administered 5000 U/kg body weight erythropoietin (Espoo Injection 750, Kyowa Kirin) on the day before (Day -1), the day after (Day 0), and the day after skin transplantation (Day 1), and then intraperitoneally administered 1000 U/kg body weight erythropoietin three times a week. Mice that received the same volume of saline intraperitoneally instead of erythropoietin served as controls.
  • the survival period of the grafted skin is shown in Figure 13.
  • Administration of erythropoietin extended the survival period of the CBA/N skin graft. This result suggests that erythropoietin contributes to immune control in MHC-half-matched minor-mismatched allogeneic recipients.
  • Example 12 Induction of immune tolerance by iPS cell transplantation (autoimmune disease)]
  • autoimmune disease autoimmune disease
  • C3H/lpr mice are known to spontaneously develop lymphadenopathy and produce autoantibodies such as anti-dsDNA antibodies.
  • mice subcutaneously inoculated with iPS cells teratomas were confirmed to have formed at the inoculation site 10 days after iPS cell inoculation, both visually and tactilely (by touching with the hand).
  • teratomas were confirmed to have formed at the inoculation site 10 days after iPS cell inoculation, both visually and tactilely (by touching with the hand).
  • blood was collected and anti-dsDNA IgG in the serum was measured using a LevisR anti-dsDNA-mouse ELISA kit (Fujifilm Wako Shibayagi Co., Ltd.).
  • the draining lymph nodes of the teratomas were harvested and weighed.
  • the C3H/lpr mouse corresponds to an allogeneic recipient that is MHC-matched but minor-mismatched to the transplanted CBA-iPS (H-2k/k). Therefore, transplantation of CBA-iPS into C3H/lpr mice is MHC compatible.
  • Anti-dsDNA IgG and lymph node weight are shown in Figure 14.
  • data from male C3H mice of the same age are also shown in Figure 14.
  • Both anti-dsDNA IgG and lymph node weight were high in untreated controls, but were reduced in C3H/lpr mice inoculated with CBA-iPS.
  • These results confirmed that immune tolerance can be induced even in autoimmune diseases by transplantation of iPS cells.
  • no weight loss was observed in C3H/lpr mice inoculated with CBA-iPS, and no abnormalities were observed in their appearance or behavioral health.
  • Treg Regulatory T cells infiltrating into teratomas were classified into three clusters, Treg-1, Treg-2, and Treg-3, based on gene expression information (Fig. 15A).
  • Marker genes for Treg-1 are trp53inp1, Lrig1, Zfp260, Itgae, and Myo3b
  • marker genes for Treg-2 are Slamf8, Ifi205, Mgl2, Cd209a, and Htr7
  • marker genes for Treg-3 are Pbk, Pclaf, Cenph, Neil3, and Kif14 (Fig. 15B).
  • Marker genes for Treg-1, Lrig1 and Itgae are known as marker genes for Treg with high immunosuppressive ability.
  • Treg-1 also strongly expressed known immunosuppressive factors Il10, Il2ra, Clta4, Entpd1, and Nt5e (Fig. 15C).
  • Example 14 Effect of regulatory T cells on immune tolerance induction
  • CBA-iPS cells were subcutaneously inoculated into B6C3F1 mice, and anti-CD25 antibody (clone:PC61) or isotype control Control IgG was intraperitoneally administered at 30 ⁇ g/mouse once every 3 days for 60 days from the day of inoculation.
  • CD25 antibody was used to remove Tregs.
  • Ten days after iPS cell inoculation teratomas were confirmed to have formed at the inoculation site in all mice by both visual and tactile inspection. Forty days after iPS cell inoculation, skin fragments derived from CBA/N mice were grafted.
  • the survival period of the grafted skin is shown in Figure 16.
  • the survival rate of CBA/N skin grafts was significantly reduced by administration of anti-CD25 antibody compared to control IgG, suggesting the contribution of Tregs to the induction of immune tolerance accompanied by teratoma formation.
  • Example 15 Induction of immune tolerance at an early stage after iPS cell transplantation (skin transplantation)] The same experiment as the same day transplantation experiment of iPS cells and skin fragments performed in Example 5 was repeated three times. Specifically, CBA-iPS were subcutaneously inoculated into B6C3F1 mice, and skin fragments derived from CBA/N mice were transplanted within 4 hours after inoculation. In all mice subcutaneously inoculated with iPS cells, it was confirmed by visual and tactile inspection that teratomas had been formed at the inoculation site 10 days after iPS cell inoculation.
  • the survival period of the grafted skin fragments is shown in Figure 17.
  • the test conducted in Example 5 is shown as Trial 1, and the additional tests conducted in this Example are shown as Trials 2 to 4.
  • Trials 2 to 4 showed long-term survival of the skin fragments even when iPS cells and skin fragments were transplanted on the same day.
  • immune tolerance induction compositions and immune tolerance inducers containing pluripotent stem cells can induce immune tolerance without forming teratomas by transplanting pluripotent stem cells into a mammalian individual that has MHC compatibility with the pluripotent stem cells contained therein. This is presumably because immune tolerance can be acquired in a mammalian individual that has MHC compatibility with either (1) the transplanted pluripotent stem cells themselves, (2) any cells that have progressed in differentiation from the transplanted pluripotent stem cells into non-teratomas, or (3) cells that are in the process of differentiating from the transplanted pluripotent stem cells into teratomas.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Microbiology (AREA)
  • Public Health (AREA)
  • Reproductive Health (AREA)
  • Biochemistry (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Gynecology & Obstetrics (AREA)
  • Transplantation (AREA)
  • Virology (AREA)
  • Epidemiology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une composition destinée à induire une tolérance immunitaire, la composition contenant : des cellules souches pluripotentes ayant la capacité de former des cellules de tératome ; ou des cellules de tératome issues des cellules souches pluripotentes. La présente invention concerne également une méthode pour induire une tolérance immunitaire chez un individu mammifère, cette méthode consistant à administrer une quantité efficace de cellules souches pluripotentes ou de cellules tératome à un individu mammifère pour lequel une tolérance immunitaire est souhaitée. L'induction d'une tolérance immunitaire chez un individu mammifère au moyen de la composition induisant une tolérance immunitaire et d'un inducteur de tolérance immunitaire permet de traiter des maladies auto-immunes et de réaliser une allotransplantation dans des conditions où une quantité réduite d'immunosuppresseur est utilisée par rapport à la quantité utilisée traditionnellement, ou dans des conditions où aucun immunosuppresseur n'est utilisé.
PCT/JP2024/016734 2023-04-30 2024-04-30 Composition destinée à induire une tolérance immunitaire WO2024228384A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2023-075353 2023-04-30
JP2023075353 2023-04-30

Publications (1)

Publication Number Publication Date
WO2024228384A1 true WO2024228384A1 (fr) 2024-11-07

Family

ID=93332967

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2024/016734 WO2024228384A1 (fr) 2023-04-30 2024-04-30 Composition destinée à induire une tolérance immunitaire

Country Status (1)

Country Link
WO (1) WO2024228384A1 (fr)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013507974A (ja) * 2009-10-29 2013-03-07 マックマスター ユニバーシティー 線維芽細胞からの誘導多能性幹細胞および前駆細胞の作製法
JP2013532989A (ja) * 2010-07-27 2013-08-22 ウニヴェルシタ・デッリ・ストゥーディ・ディ・ペルージャ 羊水由来多能性幹細胞の単離及び自己免疫疾患の処置又は予防におけるその使用
JP2015502747A (ja) * 2011-12-01 2015-01-29 ザ ニューヨーク ステム セル ファウンデーション 人工多能性幹細胞または分化細胞を作製するための自動化されたシステム
US20150038371A1 (en) * 2009-09-01 2015-02-05 Mcmaster University Transformed human pluripotent stem cells and associated methods
JP2016504324A (ja) * 2012-12-14 2016-02-12 ラトガース，ザ ステート ユニバーシティ オブ ニュー ジャージー 幹細胞の免疫制御作用を調節する方法
JP2016187354A (ja) * 2001-12-07 2016-11-04 アステリアス バイオセラピューティクス インコーポレイテッド ヒト胚性幹細胞に由来する造血細胞
KR102280509B1 (ko) * 2020-09-04 2021-07-22 건국대학교 글로컬산학협력단 전분화능 줄기세포 기반 자가면역성 및 염증성 피부질환 예방 또는 치료용 조성물 및 이의 제조방법
WO2023176986A1 (fr) * 2022-03-18 2023-09-21 国立大学法人北海道大学 Composition d'induction de l'immunotolérance et son procédé de production

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016187354A (ja) * 2001-12-07 2016-11-04 アステリアス バイオセラピューティクス インコーポレイテッド ヒト胚性幹細胞に由来する造血細胞
US20150038371A1 (en) * 2009-09-01 2015-02-05 Mcmaster University Transformed human pluripotent stem cells and associated methods
JP2013507974A (ja) * 2009-10-29 2013-03-07 マックマスター ユニバーシティー 線維芽細胞からの誘導多能性幹細胞および前駆細胞の作製法
JP2013532989A (ja) * 2010-07-27 2013-08-22 ウニヴェルシタ・デッリ・ストゥーディ・ディ・ペルージャ 羊水由来多能性幹細胞の単離及び自己免疫疾患の処置又は予防におけるその使用
JP2015502747A (ja) * 2011-12-01 2015-01-29 ザ ニューヨーク ステム セル ファウンデーション 人工多能性幹細胞または分化細胞を作製するための自動化されたシステム
JP2016504324A (ja) * 2012-12-14 2016-02-12 ラトガース，ザ ステート ユニバーシティ オブ ニュー ジャージー 幹細胞の免疫制御作用を調節する方法
KR102280509B1 (ko) * 2020-09-04 2021-07-22 건국대학교 글로컬산학협력단 전분화능 줄기세포 기반 자가면역성 및 염증성 피부질환 예방 또는 치료용 조성물 및 이의 제조방법
WO2023176986A1 (fr) * 2022-03-18 2023-09-21 国立大学法人北海道大学 Composition d'induction de l'immunotolérance et son procédé de production

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
KAMATANI, TOMOKI ET AL. : "S7-2 Elucidating the Mechanisms of Transplantation Tolerance Induced by iPS Cells", ORGAN BIOLOGY, NIHON ZOKI HOZON SEIBUTSU IGAKKAI, TOKYO, JP, vol. 30, no. 3, 1 October 2023 (2023-10-01), JP , pages 81, XP009559300, ISSN: 1340-5152 *
KIMURA, REIKO ET AL. : "O2-2 Do Erythroblasts Contribute to Pluripotent Stem Cell-Based Transplantation Tolerance Induction?", ORGAN BIOLOGY, NIHON ZOKI HOZON SEIBUTSU IGAKKAI, TOKYO, JP, vol. 30, no. 3, 1 October 2023 (2023-10-01), JP , pages 106, XP009559301, ISSN: 1340-5152 *
LO TZU-CHI, CHENG JING-YAN, LEE CHIEN-WEI, HUNG JUNG-TUNG, LIN CHUN-CHENG, YEH HSIAO-FONG, YANG BEI-CHIA, HUANG YENLIN, WU HSIEN-M: "Priming of macrophage by glycosphingolipids from extracellular vesicles facilitates immune tolerance for embryo-maternal crosstalk", DEVELOPMENTAL CELL, CELL PRESS, US, vol. 58, no. 22, 1 November 2023 (2023-11-01), US , pages 2447 - 2459.e5, XP093230688, ISSN: 1534-5807, DOI: 10.1016/j.devcel.2023.09.012 *
OTSUKA RYO, WADA HARUKA, BAGHDADI MUHAMMAD, TSUJI HYUMA, SEINO KEN-ICHIRO: "Immune tolerance induction through using pluripotent stem cells", JAPANESE JOURNAL OF TRANSPLANTATION., vol. 52, no. 6, 1 January 2017 (2017-01-01), pages 489 - 494, XP093230690 *
ROUX CLÉMENCE, SAVIANE GAËLLE, PINI JONATHAN, BELAÏD NOURHÈNE, DHIB GIHEN, VOHA CHRISTINE, IBÁÑEZ LIDIA, BOUTIN ANTOINE, MAZURE NA: "Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo", FRONTIERS IN IMMUNOLOGY, FRONTIERS MEDIA, LAUSANNE, CH, vol. 8, Lausanne, CH , XP093230687, ISSN: 1664-3224, DOI: 10.3389/fimmu.2017.01991 *

Similar Documents

Publication Publication Date Title
JP6502544B2 (ja) Ｈｌａ ｇ改変された細胞および方法
US10933124B2 (en) Methods of transplantation and disease treatment
JP2022526218A (ja) 汎用ドナー幹細胞及び関連する方法
JP6920363B2 (ja) 多機能未成熟歯髄幹細胞および療法適用
Zhao et al. Heterelogous expression of mutated HLA-G decreases immunogenicity of human embryonic stem cells and their epidermal derivatives
KR20180066262A (ko) 만능 세포의 유전자 조작
US20150139994A1 (en) Compositions and methods for preventing allogeneic immune rejection
JP2023507118A (ja) 治療のための操作された細胞
US10751368B2 (en) Methods of transplantation and disease treatment
US20220112280A1 (en) Transplant tolerance induction with carbodiimide treated tolerizing vaccine
WO2010069008A9 (fr) Cellule à compétence germinale dérivée de tissu adulte
Gröschel et al. Efficient killing of murine pluripotent stem cells by natural killer (NK) cells requires activation by cytokines and partly depends on the activating NK receptor NKG2D
WO2023176986A1 (fr) Composition d'induction de l'immunotolérance et son procédé de production
WO2024228384A1 (fr) Composition destinée à induire une tolérance immunitaire
EP3556848A1 (fr) Agent d'élimination de cellules souches indifférenciées, et procédé d'élimination de cellules souches indifférenciées
US20230374448A1 (en) Hypoimmunogenic Cells and Methods and Compositions for Their Production
Drukker Immunological considerations for cell therapy using human embryonic stem cell derivatives
EP3893898B1 (fr) Procédé de traitement de maladies auto-immunes et inflammatoires en utilisant des cellules b
ALSHARIF Thymic recovery following chemotherapy damage–the impact of sex hormones and ageing
Pizzato Metabolic Control and Immune Barriers of Hematopoietic Stem Cells
CN119452076A (zh) 免疫细胞的产生
CN119487199A (zh) 用于增强免疫耐受性的组合物和方法
Thompson et al. Research Article F4/80 Host Macrophages Are a Barrier to Murine Embryonic Stem Cell-Derived Hematopoietic Progenitor Engraftment In Vivo

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24800115

Country of ref document: EP

Kind code of ref document: A1