WO2019245039A1 - 感染性免疫寛容を惹起するための組成物 - Google Patents
感染性免疫寛容を惹起するための組成物 Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Definitions
- the present disclosure relates to a novel technology relating to immune tolerance. More particularly, the present disclosure relates to pharmaceutical compositions comprising anergy T cells, production of the pharmaceutical compositions, and quality control of the pharmaceutical compositions.
- Liver transplantation has been widely used as a definitive treatment for patients with end-stage liver failure. Every year, there are more than 20,000 cases outside Japan and more than 500 cases in Japan.
- Transplantation is one of the main treatments of choice for organ failure such as end-stage renal, heart, liver, pancreas, etc., and despite significant progress in treating transplant rejection in recent years, there has been no immunosuppressive regimen Most transplants are eventually rejected.
- Current immunosuppressive regimens that rely on continuous drug therapies have been shown to provide organ transplant patients with infectious disease and cancer because the drug suppresses not only the directed response to transplant but also all immune responses. It is likely to cause an increase in sensitivity.
- JP-T-2002-504120 Japanese Patent Publication No. 2007-131598 Japanese Patent Publication No. 2016-520081
- the present inventors have conducted intensive studies and as a result, administered a cell preparation containing cells in which anergy was induced by an inhibitor such as an antibody that inhibits the interaction between CD80 / CD86 and CD28 to an organ transplant patient (recipient).
- an inhibitor such as an antibody that inhibits the interaction between CD80 / CD86 and CD28
- the present inventors have demonstrated that such a cell preparation can elicit immune tolerance against immune rejection caused by allergy, iPS cells or the like or cells, tissues or organs derived therefrom.
- a composition for inducing a permanent immune tolerance (infectious immune tolerance) to a donor in a subject comprising: Immune tolerance by mixing an inhibitor capable of inhibiting the interaction of CD28 with CD80 and / or CD86, cells from the subject, and antigens from the donor or inclusions of the antigens Induced cells
- a composition comprising: (2) The composition according to the above item, wherein the immune tolerance is induced in CD8-positive T cells in the subject.
- the inhibitory factor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars, and combinations thereof.
- composition according to any of the above items, wherein the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.
- the composition according to any one of the above items, wherein the variant of the antibody is an antigen-binding fragment.
- the composition according to any one of the above items, wherein the variant of the cell surface molecule is a fusion protein.
- the inhibitor is a group consisting of an anti-CD80 antibody, an anti-CD86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, and a CD28-Ig fusion protein.
- composition according to any of the preceding items wherein the composition is selected from: (8) The composition according to any one of the above items, wherein the CTLA4-Ig fusion protein is abatacept or veratacept.
- a method for preventing or treating a disease, disorder or condition in a subject comprising: 1) An inhibitor capable of inhibiting the interaction between CD80 and / or CD86 and CD28, cells derived from the subject, antigens derived from the subject, antigens not derived from the subject, or Administering to the subject a formulation containing cells from which the anergy has been induced by mixing with the ingredients.
- the preparation comprises CD4 positive anergy cells, CD8 positive anergy cells, or a combination thereof.
- the preparation contains CD8-positive anergy cells.
- the confirmation of the anergy state includes a step of confirming that the preparation containing the cells disappears.
- the disease, disorder or condition is transplant immune rejection, allergy, autoimmune disease, graft-versus-host disease, and immunity caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived therefrom.
- the method according to any of the preceding items selected from the group consisting of rejection reactions.
- the method according to any of the preceding items wherein the disease, disorder or condition includes allergy.
- the disease, disorder or condition includes an immune rejection reaction caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived therefrom.
- the inhibitory factor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars, and combinations thereof.
- the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.
- the variant of the antibody is an antigen-binding fragment.
- the variant of the cell surface molecule is a fusion protein.
- the inhibitor is a group consisting of an anti-CD80 antibody, an anti-CD86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, and a CD28-Ig fusion protein.
- composition for treating or preventing an allergy in a subject comprising: By mixing an inhibitor capable of inhibiting the interaction between CD80 and / or CD86 and CD28, cells from the subject, and an antigen causing the allergy or a content of the antigen Cells with induced tolerance
- a composition comprising: (23) The composition according to any one of the above items, wherein the antigen causing the allergy is selected from food, pollen, medicine, and metal.
- compositions for suppressing or preventing immune rejection caused by iPS cells or ES cells or cells, tissues or organs derived therefrom in a subject comprising: Mixing an inhibitor capable of inhibiting the interaction between CD80 and / or CD86 and CD28, cells derived from the subject, antigens derived from the iPS cells or ES cells, or contents of the antigens Cells to which tolerance has been induced
- a composition comprising: (25) Anergy-induced T cells that are not regulatory T cells.
- a method for producing a T cell, which is not a regulatory T cell and has been induced to be anergy to a subject A) A step of mixing an inhibitor capable of inhibiting the interaction between CD80 and / or CD86 and CD28, a cell derived from the subject, an antigen not derived from the subject, or a content of the antigen When, B) culturing the mixture to obtain T cells; C) stimulating the T cells with an antigen not derived from the subject or a content of the antigen, if necessary, to confirm that the T cells do not react;
- a method comprising: (27) The method according to any one of the above items, wherein the inhibitor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars, and combinations thereof.
- the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.
- the variant of the antibody is an antigen-binding fragment.
- the variant of the cell surface molecule is a fusion protein.
- (31) a group consisting of an anti-CD80 antibody, an anti-CD86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, and a CD28-Ig fusion protein
- (33) The T cell or the method according to any of the above items, wherein the T cell includes a CD4 positive cell and / or a CD8 positive cell.
- a composition for inducing a non-reactivity or a low response of CD8-positive T cells in a subject to a specific antigen (5) an inhibitor capable of inhibiting the interaction between CD80 and / or CD86 and CD28, cells derived from the subject, antigens derived from the subject or antigens not derived from the test sample or the antigens
- the medicament according to any of the above items, wherein the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.
- the medicament according to any one of the above items, wherein the variant of the antibody is an antigen-binding fragment.
- the medicament according to any of the above items, wherein the variant of the cell surface molecule is a fusion protein.
- the inhibitor is a group consisting of an anti-CD80 antibody, an anti-CD86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, and a CD28-Ig fusion protein.
- the medicament according to any of the above items selected from the group consisting of: (41) The medicament according to any one of the above items, wherein the CTLA4-Ig fusion protein is abatacept or veratacept. (42) The disease, disorder or condition is transplant immune rejection, allergy, autoimmune disease, graft-versus-host disease, and immunity caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived therefrom. The medicament according to any of the above items, selected from the group consisting of rejection reactions. (43) The medicament according to any of the above items, wherein the disease, disorder or condition includes allergy.
- A2 The method according to the above item, wherein the immune tolerance is induced in CD8-positive T cells in the subject.
- A3 The method according to any of the preceding items, wherein the inhibitor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars, and combinations thereof.
- A4 The method according to any of the preceding items, wherein the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.
- A5) The method according to any one of the above items, wherein the variant of the antibody is an antigen-binding fragment.
- A6 The method according to any one of the above items, wherein the variant of the cell surface molecule is a fusion protein.
- the inhibitor is a group consisting of an anti-CD80 antibody, an anti-CD86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, and a CD28-Ig fusion protein.
- a method for treating or preventing a subject's allergy comprising: an inhibitor capable of inhibiting an interaction between CD80 and / or CD86 and CD28; and a cell derived from the subject.
- the antigen causing the allergy is selected from food, pollen, medicine, and metal.
- a method for suppressing or preventing immune rejection caused by iPS cells or ES cells or cells, tissues or organs derived therefrom in a subject the method comprising CD80 and / or CD86 and CD28 Immunotolerance was induced by mixing an inhibitor capable of inhibiting the interaction with, a cell derived from the subject, an antigen derived from the iPS cell or ES cell or a content of the antigen Administering a cell to said subject in an effective amount.
- a method for treating or preventing a disease, disorder or condition caused by an antigen not derived from a subject comprising an inhibitor capable of inhibiting an interaction between CD80 and / or CD86 and CD28. And cells in which immunotolerance has been induced by mixing cells derived from the subject with an antigen derived from the subject or an antigen not derived from the test sample or a content of the antigen, to the subject in an effective amount.
- a method comprising the step of administering.
- A14 The method according to any one of the above items, wherein the inhibitor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars, and combinations thereof.
- the method according to any of the preceding items, wherein the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.
- the method according to any one of the above items, wherein the variant of the antibody is an antigen-binding fragment.
- the variant of the cell surface molecule is a fusion protein.
- the inhibitor is a group consisting of an anti-CD80 antibody, an anti-CD86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, and a CD28-Ig fusion protein.
- the disease, disorder or condition is transplant immune rejection, allergy, autoimmune disease, graft-versus-host disease, and immunity caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived therefrom.
- the method according to any of the preceding items selected from the group consisting of rejection reactions.
- A21 The method according to any of the preceding items, wherein the disease, disorder or condition includes allergy.
- the disease, disorder or condition includes an immune rejection reaction caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived therefrom.
- the interaction between CD80 and / or CD86 and CD28 can be inhibited for the manufacture of a medicament for raising permanent immune tolerance to a donor (infectious immune tolerance).
- B2 The use according to the preceding items, wherein the immune tolerance is induced in CD8-positive T cells in the subject.
- (B3) The use according to any of the preceding items, wherein the inhibitor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars, and combinations thereof.
- the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.
- the variant of the antibody is an antigen-binding fragment.
- B6 The use according to any of the preceding items, wherein the variant of the cell surface molecule is a fusion protein.
- the inhibitor comprises a group consisting of an anti-CD80 antibody, an anti-CD86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, and a CD28-Ig fusion protein.
- a CTLA4-Ig fusion protein Use according to any of the preceding items, selected from: (B8) The use according to any of the preceding items, wherein the CTLA4-Ig fusion protein is abatacept or veratacept.
- (B9) an inhibitor capable of inhibiting the interaction between CD80 and / or CD86 and CD28 for the manufacture of a medicament for preventing or treating a disease, disorder or condition in a subject;
- the use of the method wherein an anergy state of T cells of the body is confirmed, and if the anergy state can be confirmed, no further treatment is performed, and if the anergy state cannot be confirmed, a medicament containing the cells is administered again.
- the medicament comprises CD4-positive anergy cells, CD8-positive anergy cells, or a combination thereof.
- the medicament comprises CD8-positive anergy cells.
- the confirmation of the anergy state includes a step of confirming that the preparation containing the cells disappears.
- the disease, disorder or condition is transplant immune rejection, allergy, autoimmune disease, graft-versus-host disease, and immunity caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived therefrom.
- any of the preceding items selected from the group consisting of rejection reactions.
- B14 The use according to any of the preceding items, wherein said disease, disorder or condition includes allergy.
- B15 The use according to any of the preceding items, wherein the disease, disorder or condition includes immune rejection caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived therefrom.
- the inhibitor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars, and combinations thereof.
- the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.
- the inhibitor comprises a group consisting of an anti-CD80 antibody, an anti-CD86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, and a CD28-Ig fusion protein.
- CTLA4-Ig fusion protein is abatacept or veratacept.
- B22 An inhibitor capable of inhibiting the interaction between CD80 and / or CD86 and CD28, and a cell derived from the subject, for producing a medicament for treating or preventing allergy of the subject. Use of cells whose immune tolerance has been induced by mixing with an antigen causing the allergy or a substance containing the antigen.
- B23 The use according to any of the preceding items, wherein the antigen causing the allergy is selected from food, pollen, medicine, and metal.
- (B24) CD80 and / or CD86 and CD28 for producing a medicament for suppressing or preventing immune rejection caused by iPS cells or ES cells or cells, tissues or organs derived therefrom in a subject.
- Immunotolerance was induced by mixing an inhibitor capable of inhibiting the interaction with, a cell derived from the subject, an antigen derived from the iPS cell or ES cell or a content of the antigen Use of cells.
- the inhibitor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars, and combinations thereof.
- the inhibitor is a group consisting of an anti-CD80 antibody, an anti-CD86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, and a CD28-Ig fusion protein.
- CTLA4-Ig fusion protein is abatacept or veratacept.
- the disease, disorder or condition is transplant immune rejection, allergy, autoimmune disease, graft-versus-host disease, and immunity caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived therefrom.
- B33 The use according to any of the preceding items, wherein said disease, disorder or condition includes allergy.
- the disease, disorder or condition includes an immune rejection reaction caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived therefrom.
- C1 an inhibitor capable of inhibiting the interaction between CD80 and / or CD86 and CD28 to elicit permanent immune tolerance (infectious tolerance) to a donor in a subject; A cell in which immune tolerance has been induced by mixing a body-derived cell with an antigen derived from the donor or a substance containing the antigen.
- C2 The cells according to the above items, wherein the immune tolerance is induced in CD8-positive T cells in the subject.
- (C3) The cell according to any of the above items, wherein the inhibitory factor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars, and combinations thereof.
- the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.
- the variant of the antibody is an antigen-binding fragment.
- C6 The cell according to any one of the above items, wherein the variant of the cell surface molecule is a fusion protein.
- the inhibitor is a group consisting of an anti-CD80 antibody, an anti-CD86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, and a CD28-Ig fusion protein.
- (C9) an inhibitor capable of inhibiting the interaction between CD80 and / or CD86 and CD28 for preventing or treating a disease, disorder or condition in a subject, and a cell derived from the subject;
- C12 The cell according to any one of the above items, wherein the confirmation of the anergy state includes a step of confirming that the cell disappears.
- the disease, disorder or condition is transplant immune rejection, allergy, autoimmune disease, graft-versus-host disease, and immunity caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived therefrom.
- C14 The cell according to any one of the above items, wherein the disease, disorder or condition includes allergy.
- the inhibitory factor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars, and combinations thereof.
- the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.
- the variant of the antibody is an antigen-binding fragment.
- the inhibitory factor is a group consisting of an anti-CD80 antibody, an anti-CD86 antibody, a bispecific antibody to CD80 and CD86, an anti-CD28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, a CD28-Ig fusion protein
- the cell according to any one of the above items which is selected from the group consisting of: (C21) The cell according to any one of the above items, wherein the CTLA4-Ig fusion protein is abatacept or veratacept.
- C22 An inhibitor capable of inhibiting the interaction between CD80 and / or CD86 and CD28 for treating or preventing allergy in a subject, cells derived from the subject, and a cause of allergy. Use of a cell in which immunological tolerance has been induced by mixing an antigen or a substance containing the antigen.
- C23 The cell according to any one of the above items, wherein the antigen causing the allergy is selected from food, pollen, medicine, and metal.
- CD80 and / or CD86 Inhibiting the interaction of CD80 and / or CD86 with CD28 to suppress or prevent immune rejection caused by iPS cells or ES cells or cells, tissues or organs derived therefrom in a subject Use of a cell in which immunotolerance has been induced by mixing an inhibitor which can be administered, a cell derived from the subject, and an antigen derived from the iPS cell or ES cell or a content of the antigen.
- the inhibitory factor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars, and combinations thereof.
- the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.
- C29 The cell according to any one of the above items, wherein the variant of the cell surface molecule is a fusion protein.
- the inhibitor is a group consisting of an anti-CD80 antibody, an anti-CD86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, and a CD28-Ig fusion protein.
- the cell according to any one of the above items which is selected from the group consisting of: (C31) The cell according to any of the above items, wherein the CTLA4-Ig fusion protein is abatacept or veratacept. (C32) The disease, disorder or condition is transplant immune rejection, allergy, autoimmune disease, graft-versus-host disease, and immunity caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived therefrom. The cell according to any of the above items, selected from the group consisting of rejection reactions. (C33) The cell according to any one of the above items, wherein the disease, disorder or condition includes allergy.
- (C34) The cell according to any of the above items, wherein the disease, disorder or condition includes an immune rejection reaction caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived therefrom.
- the present disclosure also provides: (D1) A composition for inducing a permanent immune tolerance (infectious immune tolerance) to a donor in a subject, wherein the composition inhibits an interaction between CD80 and / or CD86 and CD28. A cell from which immunotolerance has been induced by mixing an antibody capable of being administered, cells from the subject, and an antigen from the donor or a substance containing the antigen.
- (D2) The composition according to the above item, wherein the immune tolerance is induced in CD8-positive T cells in the subject.
- (D3) A method for preventing or treating a disease, disorder or condition in a subject, comprising: 1) an antibody capable of inhibiting the interaction between CD80 and / or CD86 and CD28; Administering to the subject a preparation containing cells from which anergy has been induced by mixing cells from the subject with an antigen derived from the subject or an antigen not derived from the subject or a substance containing the antigen; And 2) confirming the T cell anergy status of the subject, performing no further treatment if the anergy status can be confirmed, and re-administering a preparation containing the cells if the anergy status cannot be confirmed. ,Method.
- (D3A) a medicament for preventing or treating a disease, disorder or condition in a subject, the medicament comprising an antibody capable of inhibiting the interaction between CD80 and / or CD86 and CD28;
- the medicament is obtained by administering the medicament and then confirming the T cell anergy state of the subject; if the anergy state can be confirmed, perform no further treatment; if the anergy state cannot be confirmed, the medicament containing the cell Wherein the medicament is administered again.
- the disease, disorder or condition is transplant immune rejection, allergy, autoimmune disease, graft-versus-host disease, and immunity caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived therefrom.
- the method, medicament or composition according to any one of the preceding items selected from the group consisting of rejection reactions.
- a composition for treating or preventing allergy in a subject the composition comprising an antibody capable of inhibiting the interaction of CD80 and / or CD86 with CD28, and a composition derived from the subject.
- a composition comprising cells in which immunological tolerance has been induced by mixing the cells of the above with an antigen causing the allergy or a substance containing the antigen.
- D11 The composition according to any one of the above items, wherein the antigen causing the allergy is selected from food, pollen, medicine, and metal.
- D11A The composition according to items D10 or D11, further comprising the features described in any one or more of items D1 to D9.
- compositions for suppressing or preventing immune rejection caused by iPS cells or ES cells or cells, tissues or organs derived therefrom in a subject wherein the composition comprises CD80 and / or Immunotolerance is achieved by mixing an antibody capable of inhibiting the interaction between CD86 and CD28, cells from the subject, and antigens from the iPS cells or ES cells or the contents of the antigens.
- Induced cells A composition comprising: (D12A) The composition according to item D12, further comprising the features described in any one or more of items D1 to D11. (D13) Anergy-induced T cells that are not regulatory T cells.
- composition according to item D13 further comprising the features described in any one or more of items D1 to D11, D11A, D12 or D12A.
- D14 A method for producing a T cell that is not a regulatory T cell and has an anergy induced in a subject, wherein A) the interaction between CD80 and / or CD86 and CD28 can be inhibited. Mixing the antibody, cells from the subject, and an antigen not derived from the subject or a content of the antigen; B) culturing the mixture to obtain T cells; Stimulating the T cells with an antigen not derived from the subject or a content of the antigen, and confirming that the T cells do not respond.
- (D15) The T cell according to any one of the above items or the method according to any one of the above items, wherein the T cell includes a CD4 positive cell and / or a CD8 positive cell.
- (D15A) The method of any one of the preceding items, further comprising the features of any one or more of items D1-D11, D11A, D12, D12A or D13.
- (D16) A composition for inducing non-reactivity or low response of CD8-positive T cells in a subject to a specific antigen.
- (D16A) The composition of any one of the preceding items, further comprising the features of any one or more of items D1-D1, D11A, D12, D12A, D13, D14, D15 or D15A.
- the disease, disorder or condition is transplant immune rejection, allergy, autoimmune disease, graft-versus-host disease, and immunity caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived therefrom.
- the medicament according to any one of the above items, selected from the group consisting of rejection reactions.
- (D19) The medicament according to any one of the above items, wherein the disease, disorder or condition includes allergy.
- D20 The medicament according to any one of the above items, wherein the disease, disorder or condition includes an immune rejection reaction caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived therefrom.
- D20A Any one of the above items further including the features described in any one or more of items D1 to D11, D11A, D12, D12A, D13, D14, D15, D15A, D16, D16A, D17 to D19 The medicament according to item.
- compositions of the present disclosure can elicit permanent immune tolerance to a donor (infectious immune tolerance).
- the quality of the medicament can be controlled using CD8-positive cells as an index.
- the compositions of the present disclosure can treat allergies and suppress immune rejection of iPS cells or ES cells or cells, tissues or organs derived therefrom.
- FIG. 1 shows the scheme of the experiment performed in Example 1 (left panel, right panel lower) and the result (right panel upper).
- the results on the right panel show thymidine incorporation, from the left the proliferation response of responder cells stimulated by stimulator cells, and the second from the left the case where fresh responder cells and stimulator cells were placed in the upper row.
- the second row from the right shows the case where an anergy cell and a stimulator cell were placed in the upper row, and the right end shows the case where the anergy cell was placed in the same row as the stimulator / responder cell.
- FIG. 2 is an experimental result showing that immune tolerance continues even when cells derived from the cell preparation disappear from the recipient. In FIG. 2a, the experiment was performed by the method shown in the left panel.
- FIG. 2b shows the results of examining whether or not the transferred anergy cells were detected in peripheral blood (PBMC), lymph nodes (MLN), spleen (Splen), and transplanted heart (Transplant Heart).
- Tolerogenic BALB / c heart transplantation shows B6 mice transplanted with hearts of allo BALB / c mice transfected with anergy cells, and tolerant B6 heart transplantation transplanted hearts of syngeneic B6 mice with transfected anergy cells Shows B6 mice.
- the transferred anergy cells were not detected by fluorescence expression not only in the peripheral blood, lymph nodes, spleen, but also in the transplanted heart.
- Figure 3 shows that using a more sensitive detection technique that amplifies the GFP gene in the genomic gene by PCR, immunotolerance continues even if cells derived from the cell preparation disappear from the recipient. is there.
- FIG. 3a shows, from the left, molecular weight markers, 1: no template, 2: PC @ GFP mouse genomic DNA, 3: 1/100 diluted GFP mouse genomic DNA, 4: 1/1000 diluted GFP mouse genomic DNA, 5: 1 / 10,000 The diluted GFP mouse genomic DNA, 6: 1/1000000 diluted GFP mouse genomic DNA, and 7: wild type mouse genomic DNA are shown.
- Example 2 was performed using PCR conditions for the GFP gene in the genomic gene, which was detectable even at 0.1% (presence of 1/1000 diluted GFP mouse genomic DNA: lane 4). The results are shown in FIG. 3b.
- FIG. 3b shows PBMC and spleen from the upper left, and lymphocytes (MLN) and heart (Heart) from the lower left.
- Each lane is a molecular weight marker (M) from the left, 1: No NC template, 2: PC (positive control: GFP mouse lymphocyte genomic DNA) 3: 3 days after transplantation, 4: 1 week after transplantation, 5: 4 weeks after transplantation , 6: 7 weeks after transplantation, 7: 15 weeks after transplantation (tolerance).
- FIG. 4 is a diagram showing that immunosuppression by anergy cells is specific to an antigen.
- FIG. 4 shows the survival rate of hearts after transfer of B6 mouse-derived anergy cells stimulated with Balb / C mouse spleen cells in the presence of anti-CD80 / 86 antibody into B6 mice.
- FIG. 5 shows an experiment showing infectious immune tolerance performed in Example 4.
- FIG. 6 shows the results of infectious immune tolerance shown in FIG. The left panel shows the engraftment rate (tolerance) due to the transfer of CD4-positive T cells obtained from mice whose immune tolerance was induced into the transplanted heart by the transfer of anergy cells.
- the right panel shows the engraftment rate (tolerance) by transfer of CD8 positive T cells obtained from mice in which tolerance was induced into the transplanted heart by transfer of anergy cells.
- FIG. 7 shows a technique of sub-transfer of allo-specific inhibitory anergy cells in a human in vitro model performed in Example 5.
- FIG. 8 shows the evaluation of the immunosuppressive ability performed in Example 5.
- FIG. 8a shows a schematic diagram thereof.
- FIG. 8b shows CPM values of 3 H-thymidine incorporation.
- the ratio of the responder cells: anazy cells to the naive responder cells was 1: 1, 1: 0.5, 1: 0.25, 1: 0 under the stimulation by allo cells.
- .125 shows the CPM value when anergy cells were added.
- FIG. 8c shows the production of IL-2 (left) and IL-10 (right).
- CD4-positive naive cells allo cell stimulation From the left in each graph na ⁇ ve responder cells alone, CD4-positive naive cells allo cell stimulation, 1 st anergy cells added CD4-positive naive cells allo cells by stimulation upon, the 2 nd anergy cells during addition of CD4-positive naive cells allo cell stimulation, indicating a stimulation by 3 rd anergy CD4-positive naive cells allo cells upon cell addition.
- FIG. 8 shows the evaluation of the immunosuppressive ability performed in Example 5.
- FIG. 8a shows a schematic diagram thereof.
- FIG. 8b shows CPM values of 3 H-thymidine incorporation.
- the ratio of the responder cells: anazy cells to the naive responder cells was 1: 1, 1: 0.5, 1: 0.25, 1: 0 under the stimulation by allo cells.
- .125 shows the CPM value when anergy cells were added.
- FIG. 8c shows the production of IL-2 (left) and IL-10 (right).
- CD4-positive naive cells allo cell stimulation From the left in each graph na ⁇ ve responder cells alone, CD4-positive naive cells allo cell stimulation, 1 st anergy cells added CD4-positive naive cells allo cells by stimulation upon, the 2 nd anergy cells during addition of CD4-positive naive cells allo cell stimulation, indicating a stimulation by 3 rd anergy CD4-positive naive cells allo cells upon cell addition.
- FIG. 8 shows the evaluation of the immunosuppressive ability performed in Example 5.
- FIG. 8a shows a schematic diagram thereof.
- FIG. 8b shows CPM values of 3 H-thymidine incorporation.
- the ratio of the responder cells: anazy cells to the naive responder cells was 1: 1, 1: 0.5, 1: 0.25, 1: 0 under the stimulation by allo cells.
- .125 shows the CPM value when anergy cells were added.
- FIG. 8c shows the production of IL-2 (left) and IL-10 (right).
- CD4-positive naive cells allo cell stimulation From the left in each graph na ⁇ ve responder cells alone, CD4-positive naive cells allo cell stimulation, 1 st anergy cells added CD4-positive naive cells allo cells by stimulation upon, the 2 nd anergy cells during addition of CD4-positive naive cells allo cell stimulation, indicating a stimulation by 3 rd anergy CD4-positive naive cells allo cells upon cell addition.
- FIG. 9 shows the results confirming the reactivity of CD8 positive cells and the necessity of CD4 positive T cells. From the left, regulatory T cells (also referred to herein as regT cells) by anti-CD4 antibody in early (3-24 days), middle (25-42 days) and late (80-100 days), respectively.
- FIG. 4 shows the survival rate of heart after transplantation (the rate at which no cardiac rejection occurred) when CD4-positive cells containing the same were removed.
- FIG. 10 shows the results showing the response of CD8-positive T cells to the donor.
- FIG. 10a shows an experimental procedure showing the response of CD8 positive T cells to the donor.
- FIG. 10b shows that CSFE-labeled cells were irradiated with BALB / c-derived irradiated spleen cells at 2 ⁇ 10 4 in a 12-well plate (Corning, Cat. No. 3513) or a 24-well plate (corning Cat. No. 3526).
- FIG. 10c shows a comparison of the CD8 MLR proliferation ratio compared to anti-CD3 CD28 antibody stimulation.
- the left group shows no stimulation with anti-CD3 / CD28 antibody
- the middle group shows the results of stimulation with irradiated autologous cells
- the right group shows the results of stimulation with irradiated donor cells.
- CD8 of naive B6 cells from the left was used. The reaction of a positive T cell, a CD8 positive T cell derived from a mouse whose transplanted heart was rejected, and a CD8 positive T cell of an immunotolerant mouse are shown.
- FIG. 10 shows the results showing the response of CD8-positive T cells to the donor.
- FIG. 10a shows an experimental procedure showing the response of CD8 positive T cells to the donor.
- FIG. 10b shows that CSFE-labeled cells were irradiated with BALB / c-derived irradiated spleen cells at 2 ⁇ 10 4 in a 12-well plate (Corning, Cat. No. 3513) or a 24-well plate (corning Cat. No. 3526).
- This is a result obtained by mixing and culturing at a mixing ratio of 1: 1 at 6 cells / mL, and examining the expression of CSFE of CD8-positive T cells on day 5 of culture by a flow cytometer (BD verse). From the left, donor cells, tolerized mice, and naive mice are shown. The upper part shows the result by dot plot, and the lower part shows the result by histogram.
- FIG. 10c shows a comparison of the CD8 MLR proliferation ratio compared to anti-CD3 CD28 antibody stimulation.
- the left group shows no stimulation with anti-CD3 / CD28 antibody
- the middle group shows the results of stimulation with irradiated autologous cells
- the right group shows the results of stimulation with irradiated donor cells.
- CD8 of naive B6 cells from the left was used. The reaction of a positive T cell, a CD8 positive T cell derived from a mouse whose transplanted heart was rejected, and a CD8 positive T cell of an immunotolerant mouse are shown.
- FIG. 10 shows the results showing the response of CD8-positive T cells to the donor.
- FIG. 10a shows an experimental procedure showing the response of CD8 positive T cells to the donor.
- FIG. 10b shows that CSFE-labeled cells were irradiated with BALB / c-derived irradiated spleen cells at 2 ⁇ 10 4 in a 12-well plate (Corning, Cat. No. 3513) or a 24-well plate (corning Cat. No. 3526).
- This is a result obtained by mixing and culturing at a mixing ratio of 1: 1 at 6 cells / mL, and examining the expression of CSFE of CD8-positive T cells on day 5 of culture by a flow cytometer (BD verse). From the left, donor cells, tolerized mice, and naive mice are shown. The upper part shows the result by dot plot, and the lower part shows the result by histogram.
- FIG. 10b shows that CSFE-labeled cells were irradiated with BALB / c-derived irradiated spleen cells at 2 ⁇ 10 4 in a 12-well plate (Corning, Cat. No. 3513) or
- FIG. 10c shows a comparison of the CD8 MLR proliferation ratio compared to anti-CD3 CD28 antibody stimulation.
- the left group shows no stimulation with anti-CD3 / CD28 antibody
- the middle group shows the results of stimulation with irradiated autologous cells
- the right group shows the results of stimulation with irradiated donor cells.
- CD8 of naive B6 cells from the left was used. The reaction of a positive T cell, a CD8 positive T cell derived from a mouse whose transplanted heart was rejected, and a CD8 positive T cell of an immunotolerant mouse are shown.
- FIG. 11 shows the procedure of immune tolerance in an allergic pneumonia (asthma) model shown in Example 8.
- FIG. 1 shows the procedure of immune tolerance in an allergic pneumonia (asthma) model shown in Example 8.
- FIG. 12 shows the results of experiments on immune tolerance in the allergic pneumonia (asthma) model shown in FIG.
- FIG. 12a shows the number of leukocytes contained in bronchial exudate (BAL)
- FIG. 12b shows the number of eosinophils contained in BAL.
- FIG. 12c shows the amount of IL-4 contained in the BAL.
- mice receiving only phosphate buffered saline (PBS control), mice stimulated with ovalbumin (OVA challenge), and mice receiving ovalbumin stimulation and anergy cells of the present disclosure (cell therapy) are shown.
- FIG. 13 shows the procedure for inducing immune tolerance in a food allergy model, as described in Example 9.
- FIG. 13 shows the procedure for inducing immune tolerance in a food allergy model, as described in Example 9.
- FIG. 14 shows the results of immunofluorescent staining of FoxP3 / CD4 of a typical intestinal tract as a result of inducing tolerance in the food allergy model shown in FIG.
- the left panel (2 rows) shows the results of two mice (11, 12) that induced food allergy (food allergy mice).
- the middle panel (2 rows) shows the mice that induced food allergy and transferred anergy cells.
- the right end shows the results of two normal mice (untreated normal mice) (31, 32). Food-allergic mice and mice transfected with anergy-transfected food allergic cells showed three independent tissue-stained images (three vertically).
- FIG. 15 shows the results of induction of immune tolerance in the food allergy model shown in FIG. FIG.
- FIG. 15a shows typical intestinal HE staining of each mouse (mouse 11 and 12 (food allergic mouse), 21 and 22 (anagy cell-transfected food allergic mouse), 31 and 32 (untreated normal mouse) in FIG. 14). The photograph of is shown.
- FIG. 15b shows the average number of regulatory T cells (CD4 + FOXP3 +) per 1 mm 2 obtained from the analysis result of the image shown in FIG. 14, and from the left of the graph, food allergy mice (allergy) and anergy cell transfer Food allergy mice (transferred anergy cells) and untreated normal mice (untreated normal).
- a graph showing the average number of eosinophils per 1 mm 2 obtained from the analysis result of the image shown in FIG. 15a is FIG. 15c, and as in FIG.
- FIG. 15 shows the results of induction of immune tolerance in the food allergy model shown in FIG.
- FIG. 15a shows typical intestinal HE staining of each mouse (mouse 11 and 12 (food allergic mouse), 21 and 22 (anagy cell-transfected food allergic mouse), 31 and 32 (untreated normal mouse) in FIG. 14). The photograph of is shown.
- FIG. 15b shows the average number of regulatory T cells (CD4 + FOXP3 +) per 1 mm 2 obtained from the analysis result of the image shown in FIG. 14, and from the left of the graph, food allergy mice (allergy) and anergy cell transfer Food allergy mice (transferred anergy cells) and untreated normal mice (untreated normal).
- FIG. 15c A graph showing the average number of eosinophils per 1 mm 2 obtained from the analysis result of the image shown in FIG. 15a is FIG. 15c, and as in FIG. 15b, allergy, anergy cell transfer, and untreated normal Is shown.
- FIG. 15 shows the results of induction of immune tolerance in the food allergy model shown in FIG.
- FIG. 15a shows typical intestinal HE staining of each mouse (mouse 11 and 12 (food allergic mouse), 21 and 22 (anagy cell-transfected food allergic mouse), 31 and 32 (untreated normal mouse) in FIG. 14). The photograph of is shown.
- FIG. 15b shows the average number of regulatory T cells (CD4 + FOXP3 +) per 1 mm 2 obtained from the analysis result of the image shown in FIG.
- FIG. 15c shows the results of inducing tolerance in iPS cells.
- the graph shows the increase in lymphocytes as a CPM value of 3 H-thymidine uptake.
- tolerance refers to a state in which a specific immune response to a specific antigen is not exhibited or a specific immune response is suppressed.
- Immune tolerance refers to whether immune cells (particularly T cells) do not show a specific immune response to a specific antigen, or whether the specific immune response is suppressed and whether humans show a specific immune response to a specific antigen. And / or one in which the specific immune response is suppressed. Attention has been paid to the fact that the induction of immune tolerance enables treatment for immune rejection and treatment for allergy.
- the term “anagy” means a state in which no co-stimulation is input when an antigen is presented from an antigen-presenting cell, so that the cell cannot respond even if stimulated under the next co-stimulation condition. I do.
- anagive cells refers to cells that have become tolerant (immune unresponsive)
- anagive T cells are T cells that have developed tolerance (immune unresponsive).
- T cells that are not activated and that are unresponsive when exposed to the same antigen again.
- PBMC (or T cell) induced immune tolerance” and “anagically PBMC (or T cell)” have the same meaning. It can be confirmed by confirming CD44 positivity, but is not limited to this.
- subject refers to domestic animals (eg, cows, sheep, cats, dogs, horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents. Includes teeth (eg, mice and rats). In certain embodiments, the subject is a human.
- permanent tolerance refers to a specific antigen against an antigen by an inhibitor such as an antibody capable of inhibiting the interaction of CD80 and / or CD86 with CD28.
- an inhibitor such as an antibody capable of inhibiting the interaction of CD80 and / or CD86 with CD28.
- the cells from which the anergy was induced disappeared from the subject for at least a certain period of time (for example, several months, one year, or three years). Years or more), the state of tolerance to a specific antigen is maintained.
- drug As used herein, “drug”, “agent” or “factor” (each of which corresponds to “agent” in English) is used interchangeably in a broad sense, and refers to any substance that can achieve the intended purpose. It may also be a substance or other element (eg, energy such as light, radioactivity, heat, electricity, etc.).
- Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, other small organic compounds, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (eg, small molecule ligands, etc.) ) And the like, but are not limited to these complex molecules.
- proteins proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids,
- the term “inhibitor” refers to any type of small molecule, protein, nucleic acid, lipid, sugar, etc., that can inhibit a predetermined action (eg, interaction, signal transmission, etc.). While not wishing to be bound by any particular theory, the present disclosure discloses that by blocking the interaction of CD28 with CD80 and / or CD86 on the cell surface with CD28 and inhibiting the CD28 costimulatory signal, T cells can be anogenously synthesized. To induce.
- the inhibitor used to block the interaction of CD28 with CD80 and / or CD86 is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars, and combinations thereof. .
- the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.
- the variant of the antibody is an antigen-binding fragment.
- the variant of the cell surface molecule is a fusion protein.
- the inhibitor comprises an anti-CD80 antibody, an anti-CD86 antibody, a bispecific antibody to CD80 and CD86, an anti-CD28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, a CD28-Ig fusion protein. Selected from the group
- the CTLA4-Ig fusion protein is abatacept or veratacept. It is also envisioned that factors that indirectly inhibit the above-mentioned interaction (eg, factors that inhibit upstream or downstream of signal transduction) may be used in combination.
- antibody in a broad sense refers to a molecule or a population thereof capable of specifically binding to a specific epitope on an antigen.
- the “antibody” in the broad sense herein may be a full-length antibody (ie, an antibody having an Fc portion) or an antibody lacking the Fc portion.
- the antibody lacking the Fc portion only needs to be able to bind to the target antigen.
- Examples of such an antibody include Fab antibody, F (ab ') 2 antibody, Fab' antibody, Fv antibody, scFv antibody and the like. But not limited thereto.
- the antibody may be any type of antibody, ie, an immunoglobulin known in the art.
- the antibody is of the isotype IgA, IgD, IgE, IgG, or IgM class.
- the antibodies described herein comprise one or more alpha, delta, epsilon, gamma, and / or mu heavy chains.
- the antibodies described herein comprise one or more kappa or light chains.
- the antibody is an IgG antibody and is one of four human subclasses: IgG1, IgG2, IgG3, and IgG4.
- Antibodies contemplated for use in the present disclosure include camelid-derived antibodies (eg, VHH antibodies), shark-derived antibodies (eg, single-chain antibodies), peptibodies, peptibodies, nanobodies, and monobodies.
- camelid-derived antibodies eg, VHH antibodies
- shark-derived antibodies eg, single-chain antibodies
- peptibodies peptibodies
- nanobodies and monobodies.
- One domain antibody One domain antibody
- minibody, minibody, multispecific antibody eg, bispecific antibody, diabody, diabody, triabody, tetrabody, tandem di-scFV, tandem tri-scFv
- multispecific antibody eg, bispecific antibody, diabody, diabody, triabody, tetrabody, tandem di-scFV, tandem tri-scFv
- Antibodies include modified antibodies or unmodified antibodies.
- the modified antibody may be a combination of an antibody and various molecules such as polyethylene glycol.
- the modified antibody can be obtained by chemically modifying the antibody using a known technique. See also Biochemistry (2016) 88: 3 380-385 for artificially created antibodies and various methods of modifying / altering antibodies.
- the term "antibody” refers to an immunoglobulin or a population thereof capable of specifically binding to a specific epitope on an antigen, and a variant thereof is referred to as a "antibody variant".
- the “antibody” in the narrow sense herein may be a full-length antibody (ie, an antibody having an Fc portion), and the “variant of the antibody” herein is a variant lacking the Fc portion of the antibody. possible. Therefore, in the present specification, an antibody in a narrow sense can also be referred to as a full-length antibody, and a variant of the antibody can also be referred to as a full-length antibody variant.
- the variant lacking the Fc portion only needs to be able to bind to the target antigen.
- variants of antibodies include modified antibodies or unmodified antibodies.
- the modified antibody may be a combination of an antibody and various molecules such as polyethylene glycol.
- the modified antibody can be obtained by chemically modifying the antibody using a known technique.
- a “polyclonal antibody” is used in mammals (eg, rats, mice, rabbits, cows, monkeys, etc.), birds, etc., for example, to induce the production of polyclonal antibodies specific for an antigen. It can be produced by administering an immunogen containing the antigen of interest. Administration of the immunogen may involve infusion of one or more immunizing agents and, if desired, an adjuvant. Adjuvants are sometimes used to increase the immune response and may include Freund's adjuvant (complete or incomplete), mineral gels (such as aluminum hydroxide), or surfactants (such as lysolecithin) and the like. . Immunization protocols are known in the art and may be performed by any method that elicits an immune response, depending on the host organism selected (Protein Experiment Handbook, Yodosha (2003): 86-91). .).
- “monoclonal antibodies” are those in which the individual antibodies that comprise the population correspond to substantially the same epitope, except for those antibodies that have mutations that can occur in small amounts in nature. Including the case of a specific antibody. Alternatively, the individual antibodies that make up the population may be substantially identical, except for those that have mutations that can occur in small amounts naturally. Monoclonal antibodies are highly specific, different from ordinary polyclonal antibodies, which typically include different antibodies corresponding to different epitopes and / or typically include different antibodies corresponding to the same epitope. different. In addition to their specificity, monoclonal antibodies are useful in that they can be synthesized from hybridoma cultures that are not contaminated with other immunoglobulins.
- a monoclonal antibody may indicate a characteristic of being obtained from a substantially homogeneous population of antibodies, but does not imply that the antibodies must be produced in any particular manner.
- a monoclonal antibody may be prepared by a method similar to the hybridoma method described in "Kohler G, ⁇ Milstein ⁇ C., Nature. ⁇ 1975 ⁇ August 7; 256 (5517): 495-497.”
- monoclonal antibodies may be made by methods similar to recombinant methods as described in US Pat. No. 4,816,567.
- the monoclonal antibody is "Clackson et al., Nature.
- a “chimeric antibody” is one in which the variable region of an antibody between heterologous organisms and the constant region of the antibody are linked, and can be constructed by genetic recombination technology.
- the mouse-human chimeric antibody can be prepared, for example, by the method described in "Roguska et al., Proc Natl Acad Sci U U S A 1994 Feb 1; 91 (3): 969-973.”
- Basic methods for producing mouse-human chimeric antibodies include, for example, encoding the mouse leader and variable region sequences present in the cloned cDNA, encoding the human antibody constant regions already present in mammalian cell expression vectors. Ligated to the sequence.
- the mouse leader sequence and variable region sequence present in the cloned cDNA may be ligated to a sequence encoding a human antibody constant region, and then ligated to a mammalian cell expression vector.
- Fragments of the human antibody constant region can be those of the H chain constant region of any human antibody and the L chain constant region of a human antibody, such as C ⁇ 1, C ⁇ 2, C ⁇ 3 or C ⁇ 4 for human H chains.
- C ⁇ or C ⁇ can be mentioned, respectively.
- a “humanized antibody” has, for example, one or more CDRs from a non-human species, and a framework region (FR) from a human immunoglobulin, and a constant region from a human immunoglobulin. And an antibody that binds to the desired antigen.
- Humanization of antibodies can be performed using various techniques known in the art (Almagro et al., Front Biosci. 2008 Jan Jan 1; 13: 1619-1633.). For example, CDR grafting (Ozaki et al., Blood.
- amino acid residues in the human FR region may be replaced with corresponding residues from a CDR donor antibody.
- This FR substitution can be performed by methods known in the art (Riechmann et al., Nature. 1988 Mar 24; 332 (6162): 323-327.).
- FR residues important for antigen binding may be identified by modeling the interaction between CDRs and FR residues.
- abnormal FR residues at specific positions may be identified by sequence comparison.
- human antibody refers to, for example, the variable region and constant region of the heavy chain and the light chain variable region and the constant region constituting the antibody are derived from a gene encoding human immunoglobulin.
- the main production methods include a transgenic mouse method for producing a human antibody, a phage display method, and the like.
- a transgenic mouse method for producing a human antibody when a functional human Ig gene is introduced into a mouse in which endogenous Ig has been knocked out, a human antibody having various antigen-binding abilities is produced instead of a mouse antibody. If the mouse is further immunized, a human monoclonal antibody can be obtained by the conventional hybridoma method.
- phage display a foreign gene is typically fused to the N-terminal end of a coat protein (g3p, g10p, etc.) of filamentous phage such as M13 or T7, which is one of Escherichia coli viruses, so as not to lose phage infectivity
- a coat protein g3p, g10p, etc.
- filamentous phage such as M13 or T7, which is one of Escherichia coli viruses
- the term “cells derived from a subject” refers to cells obtained from a subject to which the composition of the present disclosure is administered or cells derived from cells obtained from the subject.
- subject-derived antigen refers to an antigen produced by a subject that produces an immune response, for example, a subject that produces an autoimmune disease in a subject having an autoimmune disease.
- Antigen As used herein, “antigen not derived from a subject” refers to a foreign antigen that can generate an immune response.
- “antigen-containing material” which is not derived from a subject refers to any substance or a collection of substances containing an antigen that is not derived from a subject. Cells, cell populations, tissues, etc., which express antigens that do not.
- transplant immune rejection refers to a condition in which a subject's immune system attacks a transplanted organ, tissue or cell in a subject who has received a transplant of the organ, tissue or cell and damages the organ, tissue or cell. Or to destroy.
- allergy refers to a state in which an immune response excessively occurs to an antigen not derived from a subject.
- An antigen that is not derived from a subject that causes an allergy is also called an allergen, and includes, for example, mite antigen, egg white antigen, milk antigen, wheat antigen, peanut antigen, soybean antigen, buckwheat antigen, sesame antigen, rice antigen, crustacean antigen, kiwi Antigen, apple antigen, banana antigen, peach antigen, tomato antigen, tuna antigen, salmon antigen, mackerel antigen, beef antigen, chicken antigen, pork antigen, cat skin antigen, insect antigen, pollen antigen, dog skin antigen, fungal antigen , Bacterial antigens, latex, haptens, metals and the like, but are not limited thereto.
- autoimmune disease refers to any disease in which the immune system produces an undesirable immune response against its own cells, tissues or organs.
- Autoimmune diseases include, for example, rheumatoid arthritis, multiple sclerosis, type 1 diabetes, inflammatory bowel disease (eg, Crohn's disease or ulcerative colitis), systemic lupus erythematosus, psoriasis, scleroderma, autoimmune thyroid Diseases, alopecia areata, Graves' disease, Guillain-Barre syndrome, celiac disease, Sjogren's syndrome, rheumatic fever, gastritis, autoimmune atrophic gastritis, autoimmune hepatitis, pancreatitis, ovitis, orchitis, uveitis, Lens-induced uveitis, myasthenia gravis, primary myxedema, pernicious anemia, autoimmune hemolytic anemia, Add
- graft-versus-host disease refers to a situation where a transplanted organ, tissue or cell attacks, damages or destroys a transplanted subject's cell, tissue or organ by an immune response.
- immune rejection caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived therefrom refers to an antigen of iPS cells or ES cells, or iPS cells or ES cells.
- composition for eliciting infectious immune tolerance The present inventors have developed a technique for inducing immune tolerance by administering to an organ transplant patient (recipient) a cell preparation containing cells in which anergy has been induced by an inhibitory factor that inhibits the interaction between CD80 / CD86 and CD28. Unexpectedly found that immunotolerance continued even after cells derived from the cell preparation disappeared (no longer detected) from the recipient (infectious immune tolerance).
- the present disclosure provides for permanent immunological tolerance (infectiousness) in a subject against a donor (antigen from the donor or inclusion of an antigen from the donor, eg, a cell, tissue or organ of the donor).
- a composition for inducing an immune tolerance comprising: an inhibitor capable of inhibiting the interaction of CD28 with CD80 and / or CD86; CD28; cells from the subject;
- a composition comprising cells in which immune tolerance (anaginess) has been induced by mixing an antigen derived from a donor or a substance containing the antigen.
- the contents of the antigen can be cells and can be irradiated to prevent proliferation and activation of the cells.
- composition of the present disclosure can permanently suppress or prevent transplant immune rejection without continuously administering cell preparations, once immunological tolerance is induced and transplant immune rejection is suppressed.
- the transplant immune rejection is kidney, liver, heart, skin, lung, pancreas, esophagus, stomach, small intestine, large intestine, nerve, blood, blood cells including immune system cells, bone, cartilage, blood vessels.
- the cornea, the eyeball or the bone marrow is transplanted.
- the tolerance is generated in CD4-positive T cells and / or CD8-positive T cells, and preferably, the tolerance is raised in at least CD8-positive T cells.
- the compositions of the present disclosure can include CD4 positive anergy T cells and / or CD8 positive anergy T cells.
- the compositions of the present disclosure may further include regulatory T cells, for example, FOXP3-positive CD4-positive CD25-positive T cells.
- the cells in which this tolerance has been induced can be induced by an inhibitor such as an antibody capable of inhibiting the interaction between CD80 and / or CD86 and CD28.
- an inhibitor such as an antibody capable of inhibiting the interaction between CD80 and / or CD86 and CD28.
- Such inhibitors are selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars, and combinations thereof.
- the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.
- the variant of the antibody is an antigen-binding fragment.
- the variant of the cell surface molecule is a fusion protein.
- the inhibitor comprises an anti-CD80 antibody, an anti-CD86 antibody, a bispecific antibody to CD80 and CD86, an anti-CD28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, a CD28-Ig fusion protein. Selected from the group
- the CTLA4-Ig fusion protein is abatacept or veratacept.
- CD80 and / or CD86 are expressed by antigen presenting cells and CD28 is expressed by T cells.
- the inhibitor capable of inhibiting the interaction of CD28 with CD80 and / or CD86 can be an anti-CD80 and / or anti-CD86 antibody or a CTLA4-Ig fusion protein.
- Inhibitors envisioned for use in the present disclosure include CTLA-4 ⁇ Ig fusion proteins, as described above.
- the CTLA-4 ⁇ Ig fusion protein competes with CD28, a costimulatory receptor on T cells, for binding to CD80 / CD86 on antigen presenting cells, and thus functions to inhibit T cell activation. I do.
- Abatacept Orencia®
- Veratacept or Maxy-4 is assumed as the CTLA-4 ⁇ Ig fusion protein.
- Veratacept contains two amino acid substitutions (L104E and A29Y) that significantly increase the binding avidity for CD80 and CD86 (Davies @ JK et al., Cell @ Transplant. (2012); 21 (9): 2047-61; Adams @ AB et al. J.
- CD28-Ig fusion proteins are also examples of inhibitors that are expected to have the same effects as CTLA4-Ig fusion proteins.
- the inhibitors of the present disclosure can also be used in the form of nucleic acids. As an example, it is conceivable that a nucleic acid encoding a CTLA4-Ig fusion protein is introduced into a cell via an adenovirus vector or the like and expressed. See, for example, Jin @ YZ et al., Transplant @ Proc. (2003); 35 (8): 3156-9.
- the disclosure is a method of preventing or treating a disease, disorder or condition in a subject, the method comprising: (1) inhibiting the interaction between CD80 and / or CD86 and CD28.
- Re-administering a formulation comprising cells comprising cells.
- the present disclosure also provides a medicament for preventing or treating a disease, disorder or condition in a subject, wherein the medicament inhibits an interaction between CD80 and / or CD86 and CD28.
- a mixture of an inhibitor such as an antibody, a cell derived from the subject, and an antigen derived from the subject or an antigen not derived from the subject, or a cell in which anergy is induced by mixing the content of the antigen ( For example, T cells), wherein the medicament confirms the anergy status of the subject's T cells, does not take further treatment if the anergy status is ascertainable, and removes the cells if the anergy status is not ascertainable. It is characterized in that the preparation containing the composition is administered again.
- the formulation or medicament comprises CD4-positive anergy cells, CD8-positive anergy cells, or a combination thereof. In certain embodiments, the formulation or medicament comprises CD8 positive anergy cells.
- Confirmation of the anergy state can be performed by confirming that the preparation containing cells from which the anergy was induced disappears. Confirmation of the disappearance of the preparation containing cells from which anergy has been induced is typically confirmed by obtaining peripheral blood mononuclear cells (PBMC) from a subject (eg, a recipient), collecting DNA, and performing a method such as PCR. Analyzing the expressed MHC and detecting the presence or absence of MHC expressed in cells not derived from the subject (eg, cells derived from the donor) and not expressed in the subject (eg, cells derived from the recipient), etc. Can be.
- PBMC peripheral blood mononuclear cells
- PBMC peripheral blood mononuclear cells
- the disease, disorder or condition is allergy, an autoimmune disease, graft-versus-host disease, and immune rejection caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived therefrom. Selected from the group consisting of:
- the anergy cells are the inhibitory factor, recipient-derived cells (PBMC or spleen cells), donor-derived antigens or donor-derived antigens.
- PBMC recipient-derived cells
- donor-derived antigens donor-derived antigens.
- the anergy cell is obtained by mixing the inhibitory factor, a cell (PBMC or spleen cell) derived from the subject, and an antigen not derived from the subject causing the allergy. By doing so.
- the anergy cells may contain the inhibitory factor, a cell (PBMC or spleen cell) derived from the subject, and a subject derived from the subject causing the autoimmune disease.
- PBMC a cell derived from the subject
- a subject derived from the subject causing the autoimmune disease By mixing with an antigen.
- the anergy cell comprises the above-mentioned inhibitory factor, PBMC or spleen cells of the donor providing the graft, and antigen or the antigen derived from the recipient.
- PBMC inhibitory factor
- spleen cells of the donor providing the graft
- antigen or the antigen derived from the recipient By mixing with the content of The contents of the antigen from the recipient can be PBMCs, spleen cells or cells around or derived from the site where the organ is transplanted.
- an anergy cell is tested with the above-mentioned inhibitory factor, It can be induced by mixing cells derived from the body (PBMC or spleen cells) with cells used for transplantation differentiated from iPS cells or ES cells.
- the present disclosure provides a method for treating or preventing an allergy and / or an autoimmune disease using the medicament of the present disclosure, and a medicament, a composition, or a cell mixture used therein.
- allergy and autoimmune diseases macrophages obtained from the peripheral blood of a patient are differentiated into dendritic cells with high antigen presenting ability (macrophage-derived dendritic cells) by a conventional method, These cells are presented with an antigen causing an overreaction in an allergic or autoimmune disease, and a group of T cells obtained from the same patient's peripheral blood is combined with an anti-CD80 antibody and / or an anti-CD86 antibody or a CTLA4-Ig fusion protein.
- anergy cells specific to the antigen causing the allergy or autoimmune disease co-culture for 1-2 weeks in the presence of the inhibitor to obtain anergy cells specific to the antigen causing the allergy or autoimmune disease.
- the administration of these anergy cells to a patient induces immune tolerance specific to an antigen causing allergy and autoimmune disease, and is used for prevention and treatment of allergy and autoimmune disease.
- the number of administrations may be plural.
- the present disclosure provides a method for treating or preventing graft-versus-host disease using the medicament of the present disclosure, and a medicament, a composition, or a cell mixture used therein.
- graft-versus-host disease in contrast to treatment for transplant immune rejection, cells that can cause graft-versus-host disease, such as PBMCs or T cells of the donor providing the graft, are exposed to radiation ( ⁇ -rays).
- PBMCs or T cells of the donor providing the graft are exposed to radiation ( ⁇ -rays).
- Host-specific anergy cells co-cultured with the irradiated host-derived PBMC or other cells for 1-2 weeks in the presence of an inhibitor such as an anti-CD80 antibody and / or an anti-CD86 antibody or a CTLA4-Ig fusion protein Get.
- the response of the graft to the host caused by the graft-versus-host disease is suppressed (inducing immune tolerance), and the graft-versus-host disease is prevented and treated.
- the number of administrations may be multiple.
- the present disclosure relates to transplantation of iPS cells or ES cells and cells, tissues or organs derived from those cells in the prevention or treatment with iPS cells or ES cells using the medicament of the present disclosure.
- the present invention provides a method for treating or preventing immune rejection or other side effects caused by the above, and a medicament, a composition, or a cell mixture used for the method.
- immunorejection reactions caused by transplantation of iPS cells or ES cells and cells, tissues or organs derived from those cells are typically exemplified as the treatment target.
- irradiation is applied to cells used for transplantation or dendritic cells differentiated from iPS cells or ES cells.
- the cells are then co-cultured with the PBMC or T cell population of the patient receiving the transplant for 1-2 weeks in the presence of an inhibitor such as an anti-CD80 antibody and / or an anti-CD86 antibody or a CTLA4-Ig fusion protein, and the iPS cells or An anergy cell specific to a cell differentiated from an ES cell is obtained.
- the anergy cells By administering the anergy cells to a host, specific immune tolerance is induced in transplanted cells, tissues, and organs derived from iPS cells or ES cells, and rejection to them is prevented and treated. Depending on whether the treatment is prophylactic or therapeutic, the tissue to be transplanted, its size, and the condition of the symptoms, the number of administrations may be several.
- the present disclosure provides a method for treating or preventing an allergy using the medicament of the present disclosure, and a medicament, a composition, and a cell mixture used for the same.
- the present inventors have demonstrated that a preparation containing cells in which anergy is induced by an inhibitor such as an antibody that inhibits the interaction between CD80 / CD86 and CD28 can induce immune tolerance to allergy.
- the disclosure is a composition for treating or preventing an allergy in a subject, wherein the composition inhibits the interaction of CD28 with CD80 and / or CD86.
- a composition comprising cells that have been induced immune tolerance by mixing an inhibitor such as an antibody, and cells derived from the subject with an antigen that causes allergy or a substance containing the antigen. I will provide a.
- the immune tolerance to an allergy can be permanent tolerance (infectious tolerance).
- Antigens that cause allergies include food, pollen, drugs, and metals, and more specifically, mite antigen, egg white antigen, milk antigen, wheat antigen, peanut antigen, soybean antigen, buckwheat antigen, sesame antigen Antigen, rice antigen, crustacean antigen, kiwi antigen, apple antigen, banana antigen, peach antigen, tomato antigen, tuna antigen, salmon antigen, mackerel antigen, beef antigen, chicken antigen, pork antigen, cat dander antigen, insect antigen, Examples include, but are not limited to, pollen antigens, canine dander antigens, fungal antigens, bacterial antigens, latex, haptens, metals and the like.
- the present disclosure provides a method for suppressing or preventing immune rejection caused by iPS cells or the like, and provides a medicament, a composition, or a cell mixture for suppressing or preventing the same.
- the present inventors have proposed that a preparation containing cells in which anergy is induced by an inhibitor such as an antibody that inhibits the interaction between CD80 / CD86 and CD28 is produced by iPS cells or the like or cells, tissues or organs derived therefrom. It has been demonstrated that immune tolerance to immune rejection can be induced.
- the present disclosure is a composition for suppressing or preventing immune rejection caused by iPS cells or ES cells or cells, tissues or organs derived therefrom in a subject.
- the composition comprises an inhibitor such as an antibody capable of inhibiting the interaction of CD80 and / or CD86 with CD28, cells from the subject, antigens from the iPS cells or ES cells or Provided is a composition comprising cells whose immune tolerance has been induced by mixing with a content of the antigen.
- the immune tolerance to immune rejection caused by iPS cells or the like or cells, tissues or organs derived therefrom can be permanent immune tolerance (infectious immune tolerance).
- Cells, tissues or organs derived from (differentiated from) iPS cells or ES cells include, for example, nerve cells or tissues, corneal cells or tissues, cardiomyocytes or tissues, liver or tissues, chondrocytes or tissues, skin cells or Tissue, kidney or tissue, and the like.
- cells, tissues or organs derived from (differentiated from) iPS cells or ES cells include nerve cells or tissues, cardiomyocytes or tissues, chondrocytes or tissues and skin cells or tissues.
- T cell that is not a regulatory T cell but has an activity of inducing anergy and method for producing the same The present inventors have demonstrated that late in transplantation (eg, 80 days after transplantation), CD8-positive cells lose their reactivity and removal of CD4-positive T cells induces immune tolerance. .
- a T cell that is not a regulatory T cell and has an activity of inducing anergy there is provided a T cell that is not a regulatory T cell and has an activity of inducing anergy.
- the present disclosure is a method of producing a T cell that is not a regulatory T cell and has an activity of inducing anergy in a subject, the method comprising: (A) combining CD80 and / or CD86 with CD28.
- verifying that the T cells do not respond may include verifying that they do not proliferate in response to the stimulus.
- the T cells include CD4 positive cells and / or CD8 positive cells. In certain embodiments, said T cells comprise CD8 positive cells.
- the present disclosure provides a composition for inducing a non-responsive or low-responsive CD8-positive T cell in a subject to a specific antigen.
- the present disclosure provides an inhibitor, such as an antibody, that can inhibit the interaction of CD28 with CD80 and / or CD86, cells from a subject, antigens from the subject, or antigens from the subject.
- an inhibitor such as an antibody
- a medicament for treating or preventing a disease, disorder or condition caused by an antigen not derived from the subject, including cells in which immunotolerance is induced by mixing an antigen not derived from the sample or a substance containing the antigen. provide.
- the disease, disorder or condition treated by the medicaments of the present disclosure is transplant immune rejection, allergy, autoimmune disease, graft versus host disease, and iPS cells or ES cells or derived therefrom. It is selected from the group consisting of immune rejection reactions caused by transplantation of cells, tissues or organs.
- anergy T cells is performed after organ transplantation (eg, liver transplantation) from a donor is performed on the recipient.
- organ transplantation eg, liver transplantation
- donor-derived mononuclear cells which are the source of autologous anergy T cells, are also contained in a state without virus removal. , Liver) is transplanted into the recipient. Therefore, donor-derived mononuclear cells used as a material for autologous anergy T cells are not considered to be biologically-derived materials.
- cell preparations can be made as follows. The various numerical values and the like illustrated below are representative examples, and those skilled in the art can appropriately modify and manufacture cell preparations. 1) About 19 days before the administration, apheresis is performed on the donor at a medical institution, the donor apheresis product is irradiated with 30 Gy of radiation, and the cell proliferation ability is lost.
- the donor mononuclear cells are separated and collected by a density gradient centrifugation method in a cell culture processing facility, frozen in two parts, and stored at -80 ⁇ 10 ° C. 3) About 14 days before administration, apheresis is performed on the recipient at a medical institution, and the recipient apheresis product is sent to a cell culture processing facility that performs cell processing.
- the recipient mononuclear cells were separated by density gradient centrifugation, collected, and thawed the donor mononuclear cells and the anti-CD80 antibody and anti-CD86 antibody or CTLA4-Ig fusion. Co-culture with inhibitors such as proteins. 5) Change the medium about 7 days before administration. Intermediate products cultured for 7 days are collected and co-cultured with thawed donor monocytes and inhibitors such as anti-CD80 and anti-CD86 antibodies or CTLA4-Ig fusion proteins. 6) On the day of administration, the cell processed product is collected by density gradient centrifugation, washed, and filled with a physiological saline solution. 7) Dispatch to a medical institution and administer to the recipient at the medical institution.
- in-process control test In one embodiment, an in-process control test described in Table 2 can be performed in the manufacturing process. Various numerical values and the like and procedures illustrated below are typical examples, and those skilled in the art can appropriately perform the in-process control test.
- Standard Test, Characteristic Analysis Test In one embodiment, the standard test described in Table 3 can be performed using the final product.
- the procedure illustrated in Table 3 is a representative example, and a person skilled in the art can perform a standard test and a characteristic analysis test with appropriate modifications.
- the standard exemplified in the eleventh embodiment can be cited. If the result is not clear at the time of administration of the inducible suppressive T cell, the shipment of the trial product can be determined by referring to the result of the in-process control test.
- the characteristic analysis test described in Table 3 can be performed using the cells in the manufacturing process and the final product.
- the above-mentioned clinical trial product standard test and characteristic analysis test are as described in the present specification, and the clinical trial product standard test includes appearance, cell number, viable cell rate, cell surface markers (CD3, CD4, CD8, CD25, CD44). , CD45RA / CD45RO), manufacturing process-related impurities (donor-derived cells, medium components, anti-CD80 antibody, anti-CD86 antibody, cell cryoprotectant solution components, specific gravity separation solution components), virus negative test, sterility test, mycoplasma negative test, and It may include endotoxin.
- the efficacy test may include a cytokine production or tritium uptake test by a mixed lymphocyte test (MLR) using cultured cells.
- MLR mixed lymphocyte test
- the CD3-positive cell ratio can be typically set to 50% or more in the table as a reference value, or, for example, 30% or more, 35% or more, 40% or more , 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, or a numerical value between these values (can be set in 1%, 0.5%, etc.) Good.
- the ratio of CD8-positive CD44-positive cells in CD3-positive cells can be typically set to 10% or more in the table as a reference value, or, for example, 1% or more, 2% or more, 3% or more, 4% or more 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, etc. .
- the ratio of CD4 + CD44 + cells in the CD3 + cells may not be set, or may be, for example, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more.
- the ratio of CD8-positive CD45RA-negative cells in CD3-positive cells may not be set, or may be, for example, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more. , 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more % Or more may be set as the reference value.
- the ratio of CD8-positive CD45RA-negative CD45RO-positive cells in CD3-positive cells may not be set, or may be, for example, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more. % Or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, or the like may be set as the reference value.
- the ratio of CD4-positive CD45RA-negative CD45RO-positive cells in CD3-positive cells may not be set, or may be, for example, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more. % Or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, or the like may be set as the reference value.
- the ratio of CD4 + CD25 cells in CD3 + cells can be typically 5% or more in the table as a reference value, or, for example, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more.
- the number of cells may be typically 1 ⁇ 10 9 or more, or alternatively, for example, 1 ⁇ 10 8 or more, 5 ⁇ 10 8 or more, 1 ⁇ 10 9 or more, 2 ⁇ 10 9 The number may be more than 3 ⁇ 10 9 or more.
- the viable cell rate can be typically taken as 70% or more, or alternatively, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more. % Or more, or 90% or more can be adopted as a reference.
- composition of the final product is composed of, for example, the components shown in the following table. Those skilled in the art can also change these compositions as appropriate to configure products such as regenerative medicine.
- Method of administering products such as regenerative medicine
- the product is administered once 14 days after organ transplantation.
- a person skilled in the art can specifically implement the method of administration, the period of administration, and the like, as necessary, while appropriately taking into account the technical matters described in the present specification.
- the prior confirmation can be performed as follows.
- the various numerical values, reagents, procedures, and the like illustrated below are representative examples, and those skilled in the art can make appropriate changes to perform confirmation beforehand.
- the separation of donor lymphocytes can be manufactured as follows.
- the various values, reagents, procedures, and the like illustrated below are representative examples, and those skilled in the art can appropriately separate donor lymphocytes.
- centrifuge accelerator slow and brake slow -Discard the supernatant and transfer the cell suspension containing the lymphocyte layer to another centrifuge tube (for example, two 50 mL centrifuge tubes).
- another centrifuge tube for example, two 50 mL centrifuge tubes.
- physiological saline to the centrifuge tube containing the cell suspension (for example, an appropriate amount until the total volume becomes 50 mL), and aspirate and discharge with a syringe (for example, a 50 mL syringe with an 18G injection needle) or a pipette. Repeat and mix well.
- an ALyS505N-0 culture solution (plasma institute (CSTI) 1020P10) containing plasma previously collected from the donor to the cell pellet (for example, an appropriate amount until the total volume becomes 31 mL), Repeat the suction and discharge with a pipette to mix well.
- a syringe for example, a 1 mL syringe equipped with an 18G injection needle
- a pipette withdraw an appropriate amount (for example, 0.3 mL), and confirm the number of cells and the number of viable cells.
- cryopreservation of donor lymphocytes can be performed as follows.
- the various numerical values, reagents, procedures, and the like illustrated below are representative examples, and those skilled in the art can appropriately modify the cryopreservation of donor lymphocytes.
- Frozen bag eg, Frozen Bag F-050 25mL Freezing Bag Nipro 89-101
- a syringe e.g., a 30 mL syringe with an 18G injection needle
- ACD solution (2 mL per 15 mL of cell suspension, Terumo TP-A05ACD, for example) Add to the frozen bag containing the cell suspension, and chill for about 10 minutes with a cooling agent cooled at 4 ° C. 7.
- CP-1 Keloto Pharmaceutical Co., Ltd. 551-27202-4 cell cryoprotection solution CP-1) cooled at 4 ° C. using a syringe (eg, a 20 mL syringe fitted with an 18G injection needle); 5 mL) to the freezing bag over a period of about one and a half minutes. At this time, the frozen bag is slowly stirred. Using a syringe, remove all air from the cryobag and its port. Seal the freezing bag using a tube sealer, first cool at 4 ° C for about 5-10 minutes, then store in a -80 ° C freezer.
- thawing of donor lymphocytes can be performed as follows.
- the various values, reagents, procedures, and the like exemplified below are representative examples, and those skilled in the art can appropriately change the thawing of the donor lymphocytes.
- the subsequent operation is preferably performed under aseptic conditions.
- a syringe eg, a 50 mL syringe with an 18G syringe needle
- withdraw the cell suspension from the thawed frozen bag and transfer to a centrifuge tube eg, 12.5 mL for each of two 50 mL centrifuge tubes.
- a centrifuge tube eg, 12.5 mL for each of two 50 mL centrifuge tubes.
- a 5% albumin solution 123146364 Nippon Pharmaceutical Co., Ltd., blood donation 5% intravenous 12.5 g / 250 mL
- centrifuge at 600 G for 10 minutes at 22 ° C (for example, preferably, set the accelerator of the centrifuge to fast and the brake to slow).
- centrifuge at 600 G for 10 minutes at 22 ° C for example, preferably, set the accelerator of the centrifuge to fast and the brake to slow.
- ALyS505N culture solution eg, 10 mL for a 50 mL centrifuge tube).
- -An anti-human CD80 antibody for example, m2D10.4; Cat. No. 16-0809-85
- a culture bag for example, Nipro 87598 Nipromedium AlyS505NB10 containing an ALyS505N-0 culture solution or a solution equivalent thereto.
- EBioscience an anti-human CD86 antibody (eg, IT2.2; Cat. No. 16-0869-85, eBioscience), respectively, at a final concentration of, for example, 10 ⁇ g / mL (or a CTLA4-Ig fusion protein (eg, Veratacept).
- Is added to the culture bag by injecting the above cell suspension with a syringe for example, a 20 mL syringe with an 18G injection needle.
- a syringe for example, a 20 mL syringe with an 18G injection needle.
- the total volume in the culture bag is about 840 mL.
- the separation of patient lymphocytes can be performed as follows.
- the various numerical values, reagents, procedures, and the like illustrated below are representative examples, and those skilled in the art can appropriately modify patient lymphocytes.
- the plasma collected from the patient is heated in a thermostat, for example, at 56 ° C. for 30 minutes to be inactivated. If not used immediately, store frozen.
- Peripheral blood collected from a patient is placed in a centrifuge tube containing an appropriate amount of a suitable medium, for example, Ficoll-Paque (for example, 20 mL), and centrifuged at, for example, 860 G for 20 minutes at 22 ° C.
- a suitable medium for example, Ficoll-Paque (for example, 20 mL)
- centrifuge for example, preferably, Set the accelerator of the centrifuge to slow and the brake to slow.
- -Add physiological saline to the centrifuge tube containing the cell suspension for example, an appropriate amount until the total volume becomes 50 mL
- repeat the suction and discharge with a pipette for example, centrifuge at 500 G for 10 minutes at 22 ° C (the accelerator of the centrifuge may be set to fast and the brake may be set to fast).
- -Discard the supernatant add physiological saline again (for example, an appropriate amount until the total volume becomes 50 mL), and mix the cell pellet well by repeatedly aspirating and discharging with a pipette. For example, centrifuge at 500 G for 5 minutes at 22 ° C (the accelerator of the centrifuge may be set to fast and the brake may be set to fast).
- -Discard the supernatant and suspend the cell pellet by adding, for example, an ALyS505N-0 culture solution (for example, 10 mL) to prepare a cell suspension (for example, adding the ALyS505N-0 culture solution until the total volume becomes 20 mL).
- the cell suspension is withdrawn, and the number of cells, the number of living cells, and the expression of the surface antigen are confirmed.
- a culture bag containing inhibitory factors such as donor cells and antibodies in the ALyS505N-0 culture solution prepared in "3. Thawing donor lymphocytes".
- the above-mentioned patient-derived cell suspension is injected into the culture bag with a syringe (for example, a 20 mL syringe fitted with an 18G injection needle) and added thereto, and the culture bag is sealed using a tube sealer.
- the total volume in the culture bag is about 1000 mL.
- Culture in a 37 ° C. incubator for example, for one week.
- the medium exchange can be performed as follows.
- the various values, reagents, procedures, and the like illustrated below are representative examples, and those skilled in the art can appropriately change the medium to replace the medium.
- centrifuge tubes for example, four 225 mL centrifuge tubes.
- centrifuge at 600 G for 10 minutes at 22 ° C for example, preferably, the accelerator of the centrifuge may be set to fast and the brake may be set to fast).
- the cell pellet • Discard the supernatant gently, and suspend the cell pellet by adding, for example, an ALyS505N-0 culture solution to prepare a cell suspension (for example, add the ALyS505N-0 culture solution to a total volume of 20 mL).
- an ALyS505N-0 culture solution for example, add the ALyS505N-0 culture solution to a total volume of 20 mL.
- the cell suspension is injected into a culture bag containing the ALyS505N-0 culture solution with a syringe (eg, a 20 mL syringe fitted with an 18G injection needle) and added.
- a syringe eg, a 20 mL syringe fitted with an 18G injection needle
- the dilution of the anti-human CD80 antibody (for example, 2D10.4) and the dilution of the anti-human CD86 antibody (for example, IT2.2) are respectively adjusted to, for example, a final concentration of 10 ⁇ g / mL (or a CTLA4-Ig fusion protein (AZA4-Ig fusion protein (AZA4-Ig fusion protein).
- An inhibitor such as, for example, veratacept
- a syringe eg, a 20 mL syringe with an 18G syringe needle.
- the thawing and restimulating antigen of donor lymphocytes-start of secondary culture can be performed as follows.
- the various numerical values, reagents, procedures and the like exemplified below are representative examples, and those skilled in the art can appropriately change the thawing and restimulation of antigen from the donor lymphocyte to the start of secondary culture.
- the subsequent operation is preferably performed under aseptic conditions.
- a syringe eg, a 50 mL syringe with an 18G syringe needle
- withdraw the donor cell suspension from the thawed frozen bag and transfer to a centrifuge tube (eg, two 50 mL centrifuge tubes).
- a centrifuge tube eg, two 50 mL centrifuge tubes.
- albumin solution for example, about 50 mL in total for two 50 mL centrifuge tubes
- it is left still for about 5 minutes. For example, centrifuge at 600 G for 10 minutes at 22 ° C. (set the accelerator of the centrifuge to fast and set the brake to slow).
- saline solution containing albumin for washing for example, prepared from 25 mL of a 5% albumin solution and 19 mL of a saline solution
- a saline solution containing albumin for washing for example, prepared from 25 mL of a 5% albumin solution and 19 mL of a saline solution
- a syringe (for example, 10 mL) of thawed inactivated plasma from a patient is placed in a culture bag containing an inhibitor such as an antibody or a patient cell in the ALyS505N culture solution prepared in “3. Thawing donor lymphocytes”.
- a 20 mL syringe with an 18G injection needle is injected and added, and the above cell suspension is further injected into the culture bag with a syringe (for example, a 20 mL syringe with an 18G injection needle).
- the total volume in the culture bag is about 1000 mL.
- testing during subculture can be performed as follows.
- the various numerical values, reagents, procedures, and the like exemplified below are representative examples, and those skilled in the art can appropriately perform the tests during the secondary culture.
- a small amount of the culture solution is withdrawn from the culture bag on the third day (10 days in total culture) from the start of the secondary culture, and inspected for mycoplasma contamination.
- Collection and filling of cultured lymphocytes can be performed as follows.
- the various values, reagents, procedures, and the like exemplified below are representative examples, and those skilled in the art can appropriately collect and fill the cultured lymphocytes.
- Secondary Packaging can be performed as follows. Various numerical values, reagents, procedures, and the like illustrated below are representative examples, and those skilled in the art can perform secondary packaging with appropriate modifications. ⁇ Typically, enter the subject ID, serial number, and expiration date on the label based on appropriate standards (typically, NUHCPC-M-12-ATREG), print the label, and affix the label to the container. • Issue “Dosage / Dose / Efficacy / Efficacy and Precautions for Use or Handling” based on appropriate criteria (typically, NUHCPC-PMF-ATREG14). ⁇ The test sample and “usage / dose / effect / effect and precautions for use or handling” are stored in a plastic bag with a zipper. ⁇ Store in a transfer container and store in the monitoring unit until shipment.
- Example 1 Immune tolerance induction experiment
- Lymphocyte separation Media Cat. No. C-44010
- Ficoll-Paque PREMIUM GE Healthcare # 17-5442-02
- Lymphocyte separation using a Lymphocyte separation produced by Promocell
- PBMC Mononuclear cells
- a culture solution containing the irradiation stimulator PBMC and the anti-CD80 antibody / anti-CD86 antibody was added under the same conditions as at the start of the culture. Two days later (day 7), the cells were collected and the culture was washed out by centrifugation to obtain anergy cells.
- 3 H-thymidine (10 ⁇ L) was added, and on the fifth day of the culture (16 to 20 hours after the addition of 3 H-thymidine), the cultured cells were collected by Cell Harvester (Molecular Devices) and scintillated. 3 H-thymidine incorporation was measured by a counter.
- Example 2 Mouse experiment showing the effect of adoptive transfer of ex vivo-induced anergy cells in mouse heart transplantation and that immune tolerance is maintained even after cells disappear
- a mouse model was used to demonstrate that immune tolerance continues even when cells derived from the cell preparation disappear from the recipient. The procedure is described below.
- C57BL6 genetically altered to express GFP (hereinafter B6) removing the spleen from mouse-derived and BALB / c mice, after obtaining spleen cells were hemolyzed erythrocytes (lymphocytes), 4 ⁇ 10 6 Cells / ml in RPMI1640 medium (Sigma; R8758-500MK) containing 10% inactivated fetal calf serum (FCS) (SIGMA # 172012-500ML Lot 11D257 or biosera # FB-1380 / 500 Lot.015BS482) It was adjusted.
- B6 C57BL6 genetically altered to express GFP
- BALB / c spleen cells serving as stimulators were irradiated with 30 Gy of radiation ( ⁇ -ray), mixed with B6 spleen cells at a ratio of 1: 1 and hamster anti-mouse CD80 antibody (16-10A1) manufactured by eBioscience (Cat. No. .16-0801-82) and a rat anti-mouse CD86 antibody (GL1) (Cat. No. 14-0862-82) were added to a final concentration of 10 ⁇ g / mL, and the mixture was added to a 12-well plate (Corning, # 3513). ) (1-2.5 ml), 6-well plate (Corning, Cat. No.
- the cells were cultured in a Petri dish (Corning, Cat. No. 430167) (10 to 15 ml) at 37 ° C. in a 5% CO 2 incubator for 14 days. On day 7 after the start of the culture, the culture solution was removed by centrifugation, and a culture solution containing BALB / c-irradiated spleen cells and an anti-CD80 antibody / anti-CD86 antibody was newly added under the same conditions as at the start of the culture. After 14 days, the cells were collected to obtain anergy cells.
- the GFP-expressing B6 mouse-derived anergy cells were transferred to a wild-type B6 mouse transplanted with a BALB / c mouse heart 3 days after irradiation with 2 Gy on 6 ⁇ 10 6 , 4 ⁇ 10 6 or 2 ⁇ 10 6 cells. Each was administered via the tail vein and cardiac rejection was observed. Recipient mice transfected with 6 ⁇ 10 6 anergy cells more than 3 months after transplantation (attained immune tolerance) were sacrificed, and the spleen, lymph node, and transplanted together with peripheral blood mononuclear cells (PBMC) Lymphocytes were obtained from the heart and the presence of the transferred anergy cells was examined by fluorescence of GFP using a flow cytometer.
- PBMC peripheral blood mononuclear cells
- the same analysis was performed using a B6 recipient mouse in which the heart of a syngeneic B6 mouse was transplanted and 6 ⁇ 10 6 anergy cells were transferred.
- 3 days, 7 days, 28 days, 50 days, and 100 days after BALB / c mouse heart transplantation the heart transplant recipient mice into which 6 ⁇ 10 6 anergy cells had been transferred were sacrificed, and peripheral blood mononuclear cells were transplanted. Lymphocytes were obtained from the extracted spleen, lymph nodes, and transplanted heart along with the spheres (PBMCs), and genomic genes were extracted from these.Then, the GFP gene was amplified by PCR. Checked for existence.
- the transfer of the anergy cells improved the survival rate of the transplanted heart in a cell number-dependent manner, and the transfer of 6 ⁇ 10 6 anergy cells transplanted all mice for more than 100 days.
- An engraftment of the transplanted heart was observed, that is, induction of immune tolerance to the transplanted heart was observed.
- the transferred anergy cells were not detected by fluorescence expression not only in peripheral blood, lymph nodes and spleen, but also in the transplanted heart.
- FIG. 2a the transfer of the anergy cells improved the survival rate of the transplanted heart in a cell number-dependent manner, and the transfer of 6 ⁇ 10 6 anergy cells transplanted all mice for more than 100 days.
- An engraftment of the transplanted heart was observed, that is, induction of immune tolerance to the transplanted heart was observed.
- the transferred anergy cells were not detected by fluorescence expression not only in peripheral blood, lymph nodes and s
- Example 3 Immunosuppression by anergy cells is specific for antigen
- anergy cells have specificity and do not suppress rejection of an antigen derived from the third party.
- Example 4 Experiment showing infectious immune tolerance
- Anergy cells were obtained in the same manner as in Example 2, and the experiment shown in FIG. 5 was performed. Briefly, spleen cells obtained from B6 mice genetically engineered to express the fluorescent dye GFP in all cells were stimulated with irradiated spleen cells from BALB / c mice in the presence of anti-CD80 / 86 antibody and anergy was performed. Cells were obtained. Four days after the irradiation of 2 Gy, 5 ⁇ 10 6 of the anergy cells were administered to a wild-type B6 mouse transplanted with the heart of a BALB / c mouse via the tail vein.
- FIG. 6 shows the results.
- Example 5 Passage transfer of allo-specific inhibitory anergy cells in a human in vitro model
- human-derived anergy cells were obtained (1-4). Briefly, mononuclear cells were obtained from human peripheral blood of four volunteers (two as stimulators and two as responders) using Lymphocyte separation Media (Cat. No. C-44010) manufactured by Promo cell. (PBMC) was separated and adjusted to 4 ⁇ 10 6 cells / mL with Bioly's ALyS505N-0 medium supplemented with 2% human AB type serum (pool). The stimulator PBMC was irradiated with 30 Gy of radiation and mixed 1: 1 with the responder PBMC. A final concentration of 10 ⁇ g each of mouse anti-human CD80 antibody (Cat. No.
- a culture solution containing the irradiation stimulator PBMC and the anti-CD80 antibody / anti-CD86 antibody was added under the same conditions as at the start of the culture. Two days later (day 7), the cells were collected and the culture was washed out by centrifugation to obtain anergy cells (1st), which were labeled with a fluorescent dye CFSE.
- the irradiated same stimulator PBMC and responder are mixed.
- Cells were added to a 1: 1 mixed culture (day 7).
- the irradiation stimulator PBMC was supplemented again.
- JSAN the only responder cells derived from the CFSE negative to this day 7 of culture (day14) was recovered by sorting use the (Bay Bioscience) (2 nd anergy cells).
- the 1st anergy cells and the 2nd anergy cells were collected on the last day of each induction, diluted so that the ratio with the responder PBMC became 1: 1 to 1: 0.125, and the same stimulator was newly collected with the responder PBMC. and PBMC irradiated the origin of radiation (gamma rays) 30 Gy to about 1: 96-well plates (Corning Inc., Cat.No.3799) containing 1 in cell number mixed culture system in (each 2 ⁇ 10 5 / 200 ⁇ L / Well, 4 wells per well) and cultured at 37 ° C. in a 5% CO 2 incubator.
- 3 H-thymidine (10 ⁇ L) was added, and on the fifth day of the culture (16 to 20 hours after the addition of 3 H-thymidine), the cultured cells were collected by Cell Harvester (Molecular Devices) and scintillated. 3 H-thymidine incorporation was measured by a counter.
- FIG. 8b shows CPM values of 3 H-thymidine incorporation, indicating that the 1 st anergy cells and the 2 nd anergy cells exhibit immunosuppressive ability.
- 1 st anergy cells, 2nd anergy cells, in 3 rd anergy cells both suppressed the production of cytokines IL-2 to induce cell proliferation from responder CD4-positive cells, while the typical suppressing an immune reaction It indicates that induces such production of cytokines IL-10, 1 st anergy cells, 2nd anergy cells, both 3 rd anergy cells, was shown to have an immunosuppressive action. From the above results, the immunosuppressive ability of anergy cells is passed on to naive cells whose response to the same antigen stimulation has been suppressed in the presence of the anergy cells. Indicates that it will be inherited.
- Example 6 Confirmation of reactivity of CD8 positive cells and necessity of CD4 positive T cells
- Example 6 Confirmation of reactivity of CD8 positive cells and necessity of CD4 positive T cells
- spleen cells obtained from wild-type B6 mice were stimulated with BALB / c cells in the presence of an anti-CD80 / 86 antibody to obtain anergy cells.
- Three days after irradiation with 2 Gy, 5 ⁇ 10 6 of the anergy cells were administered to a wild-type B6 mouse transplanted with the heart of a BALB / c mouse via the tail vein.
- An anti-mouse CD4 antibody (GK1.5) generated in the laboratory to remove CD4-positive T cells, including regT cells, from 3 to 24 days, 25 to 42 days, and 80 to 100 days after heart transplantation was intraperitoneally administered 200 ⁇ g every three days, and rejection of the transplanted heart was observed.
- FIG. 9 shows the results.
- CD4 positive cells including regT cells were removed at a later stage (80 to 100 days)
- rejection of the transplanted heart was not observed in all mice. That is, up to about 42 days later, CD4-positive T cells including regT cells are required for immunological tolerance, and depletion thereof induces rejection of transplanted hearts by CD8-positive T cells.
- the necessity of CD4 + T cells including regT cells was eliminated, suggesting that the reactivity of CD8 + cells was lost. In other words, this shows that the recipients who have received the cell therapy are ultimately (after 80 days) induced immune tolerance without the presence of immunosuppressive cells.
- Example 7 Response of CD8-positive T cells to donor
- the response of CD8-positive T cells to the donor was confirmed.
- spleen cells obtained from wild-type B6 mice were stimulated with BALB / c cells in the presence of an anti-CD80 / 86 antibody to obtain anergy cells.
- Three days after irradiation with 2 Gy, 5 ⁇ 10 6 of the anergy cells were administered to a wild-type B6 mouse transplanted with the heart of a BALB / c mouse via the tail vein.
- Spleen cells were obtained from immune-tolerant mice and naive mice over 100 days after transplanted hearts survived without rejection, and labeled with CSFE using the CFSE cell proliferation kit (Invitrogen C34554) ( Figure 10a).
- the CSFE-labeled cells were irradiated with BALB / c-derived irradiated spleen cells and 2 ⁇ 10 6 cells / mL in a 12-well plate (Corning, Cat. No. 3513) or a 24-well plate (Corning Cat. No. 3526).
- the cells were mixed at 1: 1 and cultured, and the expression of CSFE of the CD8-positive T cells on day 5 of the culture was examined using a flow cytometer (BD verse) (FIG. 10b).
- mice were reacted with PE fluorescence-labeled anti-mouse CD8 antibody (53-6.7; eBioscience, # 12-0081-85), and then reacted with anti-PE magnetic beads (Miltenyi Biotech # 1300-). 10-639), CD8-positive cells were separated by auto-MACS (Miltenyi Biotech).
- the cells and irradiated spleen cells derived from B6 and BALB / c mice were mixed at 1 ⁇ 10 6 cells / mL, respectively, on a 96-well plate (Corning, Cat. No. 3799) at 1: 1 and cultured.
- the culture volume was 200 ⁇ L
- 3 H-thymidine (10 ⁇ L) was added on the fourth day of the culture, and on the fifth day of the culture (16 to 20 hours after the addition of 3 H-thymidine), Cell Harvester (Molecular) was added. Devices), and the amount of 3 H-thymidine incorporation was measured with a scintillation counter.
- naive CD8-positive T cells divide (proliferate) upon stimulation of donor cells, resulting in a decrease in the CSFE expression level in many cells (right), but not proliferating with naive cells alone. Therefore, there is no cell where the fluorescence level of CSFE becomes low (left).
- CD8-positive T cells of mice in which tolerance has been induced hardly divide (proliferate) even when stimulated with donor cells, so that few cells have reduced CSFE expression (middle). That is, CD8-positive cells naturally respond to donor cells by themselves, but CD8-positive cells from mice to which tolerance has been induced have lost their reactivity alone. That is, CD8-positive T cells themselves are induced intolerant (unreactive) independent of the presence of suppressor cells.
- CD8-positive T cells of mice to which tolerance was induced had a proliferative response to donor stimulation compared to stimulation with anti-CD3 / anti-CD28 antibodies, and CD8-positive T cells of naive mice and transplanted cells.
- the rejection of the affected heart was lower than that of CD8-positive T cells in mice. This indicates that the number of cells that respond to and proliferate in the donor antigen-specific manner in all CD8-positive T cells is reduced. Indicates that clone anergy or clone elimination may be induced.
- the anergy cells of the present disclosure exert immunosuppressive ability by contacting naive cells of a recipient. 2.
- the transferred suppressive anergy T cells become undetectable. 3.
- the transferred suppressive anergy cells induce new immunosuppressive cells in the recipient organism. 4.
- the reactivity of the donor antigen-specific CD8-positive clone is significantly reduced. This indicates that clonal anergy or clonal elimination may have been induced.
- the transferred donor-specific anergy cells are rapidly eliminated from the recipient body, but continue to induce new donor-specific immunosuppressive cells in the recipient organism after the disappearance, and ultimately reject transplantation. It was suggested that donor-specific CD8-positive T cells themselves, which are thought to play a central role in, lose their reactivity.
- the management of treatment using the present disclosure indicates that the transferred inhibitory anergy T cells become undetectable, and / or that cells having a new immunosuppressive ability in the recipient organism. It is understood that it is useful to detect that is induced. Furthermore, it is understood that detection of a donor antigen-specific CD8-positive clone can be used to manage whether treatment is successful.
- Example 8 Immune tolerance in an allergic pneumonia (asthma) model
- OVA-specific anergy cells 100 ⁇ g of OVA (Sigma-Aldrich # O1641 or MBL Life Science TS-5001-P etc.) and 250 ⁇ g of each anti-mouse CD80 / 86 antibody were simultaneously intraperitoneally administered, and BVA mice sensitized to OVA were sacrificed 5 days later. Then, the spleen was removed and spleen cells were obtained by hemolysis.
- the spleen cells were adjusted to 4 ⁇ 10 6 cells / mL in RPMI1640 medium containing 10% FCS, and the OVA was adjusted to 100 ⁇ g / mL and the CD80 / 86 antibody was adjusted to a final concentration of 10 ⁇ g / mL, respectively.
- This spleen cell suspension was cultured in a 5% CO 2 incubator at 37 ° C. for 7 days to obtain OVA-specific anergy cells.
- a model in which PBS was administered intranasally was used.
- the induced anergy cells were administered 1 ⁇ 10 7 via the tail vein on day 14, and bronchial lavage fluid (BAL: Bronchoalveolar Lavage) was collected by tracheal lavage using 1 ml of saline and leached therefrom on day 24.
- BAL Bronchoalveolar Lavage
- the leukocyte count (CD45-positive cells) and eosinophil count (calculated as the ratio of CD45-positive CD11b-positive SiglecF-positive cells to CD45-positive cells) were measured.
- IL-4 in BAL was measured by ELISA (eBioscience # 88-704-88).
- FIG. 12 shows the results.
- FIG. 12a shows the number of leukocytes contained in bronchial exudate (BAL)
- FIG. 12b shows the number of eosinophils contained in BAL.
- the transfer of anergy cells reduces the number of leukocytes and eosinophils that exude.
- IL-4 which is involved in the allergic reaction contained in BAL, is also reduced. This indicates that in an OVA-induced allergic pneumonia (asthma) model, the transfer of anergy cells induced by co-culture of the anti-CD80 / 86 antibody and OVA induces immune tolerance and attenuates the allergic reaction.
- Example 9 Induction of immune tolerance in a food allergy model
- alum adjuvant Thermo Fisher # 77161
- CD4 positive Foxp3 positive cell density CD4 positive Foxp3 positive cell density, fluorescence microscope Axioplan 2 imaging (Zeiss), after acquiring a fluorescent immunostaining image with CCD camera AxioCam HRc (Zeiss), using the image analysis software KS400 (Zeiss), the area of the measurement target area The number of positive cells existing in the same region as in the measurement was counted, and the number of positive cells / mm 2 was calculated.
- Eosinophil density can be measured using an optical microscope Axioskop 2 plus (Zeiss), using a CCD camera AxioCam MRc (Zeiss) to obtain HE (hematoxylin eosin) stained images, and then using the image analysis software KS400 (Zeiss) to measure the eosinophil density. Eosin strongly positive eosinophils present in the same area as the area measurement were counted and the number of positive cells / mm 2 was calculated (result). The results are shown in FIGS. FIG. 14 shows a typical result of immunostaining of FoxP3 / CD4.
- FIG. 15a shows one photograph of typical HE staining of each mouse. Digitizing analysis results of the image shown in FIG. 14 is a Figure 15b that the graph the number of regT cells per 1 mm 2, and quantify the analysis results of the image shown in FIG. 15a, 1 mm 2 per FIG. 15c is a graph showing the number of eosinophils.
- RegAs shown in FIG. 15b the infiltration of regT cells was increased in the anergy cell-administered group as compared to the allergic group, and was present in the intestinal tract wall more than in normal mice. Consistent with this, as shown in FIG. 15c, the number of eosinophils, which is considered to be high during allergy, has been reduced to the same level as in normal mice by the transfer of anergy cells. From the above results, it was demonstrated that even in food allergy, immune tolerance was induced by the transfer of anergy cells, resulting in reduction of eosinophil infiltration and induction of regT infiltration, and alleviation of symptoms.
- Example 10 Induction of tolerance in iPS cells
- an experiment was performed to demonstrate whether serious immune rejection that occurred in a transplantation experiment using iPS cells and cells / tissues differentiated therefrom can be cured using the anergy cells of the present disclosure. Immune tolerance was induced as follows. This will be described below.
- iPS cells were differentiated into neurons.
- the nerve cells were irradiated with 30 Gy of radiation ( ⁇ -ray) and used as stimulator cells.
- Mononuclear cells (PBMC) obtained from human peripheral blood of volunteers having different MHC were used as the responder cells, and 2% human AB was used so that the stimulator cells and the responder cells each became 2 ⁇ 10 6 cells / mL.
- PBMC Mononuclear cells
- a 96-well plate (Corning, Cat. No. 3799) containing nerve cells irradiated with 30 Gy of radiation ( ⁇ -ray) derived from the same iPS cell and newly collected PBMC of the same volunteer at a cell number of about 1: 1. ) (4 ⁇ 4 ⁇ 2 ⁇ 10 5 cells / 200 ⁇ L / well) and cultured at 37 ° C. in a 5% CO 2 incubator.
- neurons differentiated from iPS cells stimulate PBMC of MHC-mismatched volunteers to induce proliferation.
- Anergy cells induced by co-culture of anti-CD80 / 86 antibody and neurons differentiated from iPS cells suppress the proliferative response of lymphocytes in response to neurons differentiated from iPS cells in vitro. It has been shown. This demonstrates that the present disclosure can attenuate immune rejection that occurs in transplantation using iPS cell-derived cells and tissues.
- Example 11 Induction of immune tolerance using various inhibitors
- This example shows that various inhibitors can be used to induce immune tolerance.
- Recipient PBMCs and donor PBMCs are either freshly isolated from human peripheral blood, or those that have been cryopreserved at ⁇ 80 ° C. and rapidly thawed, and these cells are used together with autologous plasma or autologous plasma. 4 ⁇ 10 6 cells / mL in RPMI 1640 medium (Sigma; R8758-500MK) containing 10% inactivated fetal calf serum (FCS) (SIGMA # 172012-500ML Lot 11D257 or biosera # FB-1380 / 500 Lot.015BS482).
- FCS inactivated fetal calf serum
- the donor PBMC is irradiated with 20 Gy radiation.
- the recipient PBMC and the donor PBMC are mixed 1: 1 and the mixture is mixed with an inhibitor (eg, for anti-CD80 / anti-CD86 antibodies each at a final concentration of 10 ⁇ g / ml, for veratacept (or abatacept), (Final concentration 10 ⁇ g / ml to 40 ⁇ g / ml) is added.
- the culture was performed at 37 ° C. in a 6 cm Petri dish (Greiner CELLSTAR (registered trademark) dish, Cat. No. 628160) (culture volume: 3 to 6 mL) or a 10 cm petri dish (Corning, Cat. No. 430167) (culture volume: 10 to 15 mL). Perform in a 5% CO 2 incubator for 7 days (the cell density at the start of culture is 4 ⁇ 10 6 cells / mL).
- the cultured recipient PBMC On day 7 from the start of the culture, the cultured recipient PBMC is collected by centrifugation, and adjusted to 4 ⁇ 10 6 cells / mL in the above medium.
- a freshly prepared irradiated donor PBMC was added at a cell ratio of 2: 1. ml-10 ⁇ g / ml, for veratacept (or abatacept) a final concentration of 10 ⁇ g / ml-40 ⁇ g / ml) is also added. Culture is performed for 7 days under the same conditions as described above (cell density: 4 ⁇ 10 6 cells / mL).
- the induced cells were collected by centrifugation on the 14th day from the start of the culture, and a lymphocyte mixing test was performed basically according to the method already described in the literature (3).
- the cell suspension is co-cultured at 37 ° C., 5% CO 2 incubator.
- 3H-thymidine (10 ⁇ l) was added.
- 3H-thymidine in the culture solution was removed. By measuring the amount of uptake, the ability to suppress the immune response can be confirmed.
- Example 12 Quality control of cell preparation
- Example 12 Quality control of cell preparation
- CD3 FITC fluorescently labeled anti-human CD3 antibody (UCHT1; eBioscience # 11-0038-42) or Pacific Blue fluorescently labeled anti-human CD3 antibody (UCHT1; Invitrogen # CD0328)
- CD4 PE fluorescently labeled anti-human CD4 antibody (RPA-T4; eBioscience # 25-0049-42)
- CD8 APC fluorescently labeled anti-human CD8 antibody (RPA-T8; eBioscience # 17-0088-42)
- CD25 PerCP fluorescently labeled anti-human CD25 antibody (MEM-181; eBioscience # A15802)
- CD44 PE-Cy7 fluorescently labeled anti-human CD44 antibody (IM7; eBioscience # 25-0441-82)
- CD45 Brilliant Violet fluorescently labeled anti-human CD45 antibody
- the ratio of CD45 + cells in living cells the ratio of CD8 + CD44 + cells in all living CD3 + cells, the ratio of CD4 + CD44 + cells, the ratio of CD8 + CD45RA negative cells, the ratio of CD8 +
- the ratio of CD45RA-negative CD45RO-positive cells, the ratio of CD4-positive CD45RA-negative CD45RO-positive cells, and the ratio of CD4-positive CD25-positive cells are determined.
- CD45-positive cells that meet quality standards should be CD45-positive in 95% or more of live cells and do not contain significant amounts of impurities such as red blood cells and platelets. Furthermore, in the CD3-positive cell population, 5% or more of CD8-positive CD44-positive, 5% or more of CD4-positive CD44-positive, 5% or more of CD8-positive CD45RA-negative, and 5% or more of CD8-positive CD45RA negative CD45RO positive, 5% or more are CD4 positive CD45RA negative CD45RO positive, and 5% or more are CD4 positive CD25 positive cells.
- Sterility test method The anergy cell suspension is gently centrifuged, and the supernatant is subjected to a sterility test.
- the direct method which is one of the typical sterility tests in the Japanese Pharmacopoeia
- the supernatant is inoculated into soybean casein digest medium or liquid thioglycolic acid medium and incubated at 30-35 ° C or 20-25 ° C, respectively. Incubate for at least one day. The culture is then observed several times during the culture period.
- the membrane filter method the supernatant was diluted with a sterile diluent (eg, 1 g / L meat or casein peptone solution (pH 7.1 ⁇ 0.2)).
- the membrane filter is placed in each of the two types of media described above, and cultured for 14 days or more. For products that meet the quality specifications, there is no visible microbial growth in the medium during and on the last day of the culture.
- Endotoxin test method The anergy cell suspension is appropriately diluted with physiological saline to adjust the pH to 6.0 to 8.0. Then, it is mixed with the lysate reagent, and the color formation due to the gel formation of the lysate reagent (gel method) or the turbidity change in the gelation process of the lysate reagent as the index (turbidimetry) or hydrolysis of the synthetic substrate As an index (colorimetric method), the endotoxin concentration in the sample is quantitatively determined. If necessary, conduct a preliminary test to confirm the labeling sensitivity of the lysate reagent. For products that meet the quality specifications, the endotoxin concentration must be less than 0.25 EU / mL.
- Mycoplasma negative test The culture solution or the suspension of anergy cells is gently centrifuged, and the supernatant is subjected to a mycoplasma negative test.
- a sample is inoculated on an agar plate medium and placed in a nitrogen gas containing 5 to 10% carbon dioxide under an appropriate humidity. Incubate at ⁇ 37 ° C for 14 days or more, or inoculate the sample into a container containing liquid medium, incubate at 35-37 ° C, observe the color change of the liquid medium, or for a certain period from the start of culture.
- a DNA staining method using an indicator cell typically, a Vero cell as an indicator cell and a designated mycoplasma strain are used.
- indicator cells are inoculated in a culture dish or the like in which a cover glass is submerged, and grown at 35 to 38 ° C. in air containing 5% carbon dioxide gas for one day.
- a sample (culture solution or supernatant) is added, and culturing is continued for 3 to 6 days under the same conditions.
- DNA fluorescence staining is performed with a staining agent such as bisbenzimide, and the specimen is inspected with a fluorescence microscope (400 to 600 times or higher magnification), and a negative (non-inoculated) control and a mycoplasma positive control are performed.
- a staining agent such as bisbenzimide
- the number of anergy cells suspended in physiological saline is measured under a microscope using a hemocytometer or by an automatic cell counter.
- the number of anergy cells suitable for administration that meets the quality standard is 1 ⁇ 10 8 to 30 ⁇ 10 8 (for example, in 100 mL of physiological saline). Should.
- -Viable cell ratio Anergy cells suspended in physiological saline are mixed with 0.3-0.5% trypan blue staining solution (for example, catalog # 35525-02, Nacalai Tesque), and a hemocytometer is used. Viable cells are counted under a microscope or by an automatic cell counter. For products that meet quality specifications, 70% or more of the cells should be viable.
- the present disclosure provides a pharmaceutical composition comprising cells in which specific tolerance to a particular antigen has been induced. Also, as infectious immune tolerance has been confirmed, the present disclosure may be used in fields requiring therapeutic management. Techniques that can be used in industries (pharmaceuticals) related to pharmaceuticals and the like based on such techniques are provided.
Abstract
Description
(1)被験体において、ドナーに対する恒久的な免疫寛容(感染性免疫寛容)を惹起するための組成物であって、該組成物は、
CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、該ドナーに由来する抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞
を含む、組成物。
(2)前記免疫寛容は、前記被験体におけるCD8陽性T細胞において免疫寛容が惹起されている、上記項目に記載の組成物。
(3)前記阻害因子が、低分子、タンパク質、核酸、脂質、糖、およびこれらの組み合わせからなる群から選択される、上記項目のいずれかに記載の組成物。
(4)前記タンパク質が、抗体もしくはその改変体、または細胞表面分子もしくはその改変体である、上記項目のいずれかに記載の組成物。
(5)前記抗体の改変体が、抗原結合断片である、上記項目のいずれかに記載の組成物。
(6)前記細胞表面分子の改変体が、融合タンパク質である、上記項目のいずれかに記載の組成物。
(7)前記阻害因子は、抗CD80抗体、抗CD86抗体、CD80およびCD86に対する二重特異性抗体、抗CD28抗体もしくはこれらの抗原結合断片、CTLA4-Ig融合タンパク質、CD28-Ig融合タンパク質からなる群より選択される、上記項目のいずれかに記載の組成物。
(8)前記CTLA4-Ig融合タンパク質は、アバタセプトまたはベラタセプトである、上記項目のいずれかに記載の組成物。
(9)被験体において、疾患、障害または状態を予防または治療する方法であって、該方法は、
1) CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、該被験体由来の抗原あるいは該被験体に由来しない抗原または該抗原の含有物とを混合することによってアナジーが誘導された細胞を含む製剤を該被験体に投与する工程と、
2) 該被験体のT細胞のアナジー状態を確認し、アナジー状態が確認できる場合はさらなる処置をせず、アナジー状態が確認できない場合、該細胞を含む製剤を再度投与する工程と
を含む、方法。
(10)前記製剤は、CD4陽性アナジー細胞、CD8陽性アナジー細胞、またはそれらの組合せを含む、上記項目のいずれかに記載の方法。
(11)前記製剤は、CD8陽性アナジー細胞を含む、上記項目のいずれかに記載の方法。
(12)前記アナジー状態の確認は、前記細胞を含む製剤が消失することを確認する工程を含む、上記項目のいずれかに記載の方法。
(13)前記疾患、障害または状態は、移植免疫拒絶反応、アレルギー、自己免疫疾患、移植片対宿主病、およびiPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応からなる群より選択される、上記項目のいずれかに記載の方法。
(14)前記疾患、障害または状態はアレルギーを含む、上記項目のいずれかに記載の方法。
(15)前記疾患、障害または状態は、iPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応を含む、上記項目のいずれかに記載の方法。
(16)前記阻害因子が、低分子、タンパク質、核酸、脂質、糖、およびこれらの組み合わせからなる群から選択される、上記項目のいずれかに記載の方法。
(17)前記タンパク質が、抗体もしくはその改変体、または細胞表面分子もしくはその改変体である、上記項目のいずれかに記載の方法。
(18)前記抗体の改変体が、抗原結合断片である、上記項目のいずれかに記載の方法。
(19)前記細胞表面分子の改変体が、融合タンパク質である、上記項目のいずれかに記載の方法。
(20)前記阻害因子は、抗CD80抗体、抗CD86抗体、CD80およびCD86に対する二重特異性抗体、抗CD28抗体もしくはこれらの抗原結合断片、CTLA4-Ig融合タンパク質、CD28-Ig融合タンパク質からなる群より選択される、上記項目のいずれかに記載の方法。
(21)前記CTLA4-Ig融合タンパク質は、アバタセプトまたはベラタセプトである、上記項目のいずれかに記載の方法。
(22)被験体のアレルギーを治療または予防するための組成物であって、該組成物は、
CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、アレルギーの原因となっている抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞
を含む、組成物。
(23)前記アレルギーの原因となっている抗原は、食品、花粉、薬品、および金属から選択される、上記項目のいずれかに記載の組成物。
(24)被験体において、iPS細胞またはES細胞またはそれらに由来する細胞、組織もしくは臓器によって生じる免疫拒絶反応を抑制するまたは予防するための組成物であって、該組成物は、
CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、該iPS細胞またはES細胞に由来する抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞
を含む、組成物。
(25)制御性T細胞ではない、アナジーが誘導されたT細胞。
(26)制御性T細胞ではない、被験体に対してアナジーが誘導されたT細胞を製造する方法であって、
A)CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、該被験体に由来しない抗原または該抗原の含有物とを混合する工程と、
B)該混合物を培養してT細胞を得る工程と、
C)必要に応じて、該被験体に由来しない抗原または該抗原の含有物で該T細胞を刺激し、該T細胞が反応しないことを確認する工程と
を包含する、方法。
(27)前記阻害因子が、低分子、タンパク質、核酸、脂質、糖、およびこれらの組み合わせからなる群から選択される、上記項目のいずれかに記載の方法。
(28)前記タンパク質が、抗体もしくはその改変体、または細胞表面分子もしくはその改変体である、上記項目のいずれかに記載の方法。
(29)前記抗体の改変体が、抗原結合断片である、上記項目のいずれかに記載の方法。
(30)前記細胞表面分子の改変体が、融合タンパク質である、上記項目のいずれかに記載の方法。
(31)前記阻害因子は、抗CD80抗体、抗CD86抗体、CD80およびCD86に対する二重特異性抗体、抗CD28抗体もしくはこれらの抗原結合断片、CTLA4-Ig融合タンパク質、CD28-Ig融合タンパク質からなる群より選択される、上記項目のいずれかに記載の方法。
(32)前記CTLA4-Ig融合タンパク質は、アバタセプトまたはベラタセプトである、上記項目のいずれかに記載の方法。
(33)前記T細胞は、CD4陽性細胞および/またはCD8陽性細胞を含む、上記項目のいずれかに記載のT細胞または方法。
(34)特定の抗原に対する、被験体内のCD8陽性T細胞の無反応または低反応を誘導するための組成物。
(35)CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、被験体由来の細胞と、該被験体由来の抗原あるいは該験検体に由来しない抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞を含む、該被験体に由来しない抗原によって生じる疾患、障害または状態を処置または予防するための医薬。
(36)前記阻害因子が、低分子、タンパク質、核酸、脂質、糖、およびこれらの組み合わせからなる群から選択される、上記項目のいずれかに記載の医薬。
(37)前記タンパク質が、抗体もしくはその改変体、または細胞表面分子もしくはその改変体である、上記項目のいずれかに記載の医薬。
(38)前記抗体の改変体が、抗原結合断片である、上記項目のいずれかに記載の医薬。
(39)前記細胞表面分子の改変体が、融合タンパク質である、上記項目のいずれかに記載の医薬。
(40)前記阻害因子は、抗CD80抗体、抗CD86抗体、CD80およびCD86に対する二重特異性抗体、抗CD28抗体もしくはこれらの抗原結合断片、CTLA4-Ig融合タンパク質、CD28-Ig融合タンパク質からなる群より選択される、上記項目のいずれかに記載の医薬。
(41)前記CTLA4-Ig融合タンパク質は、アバタセプトまたはベラタセプトである、上記項目のいずれかに記載の医薬。
(42)前記疾患、障害または状態は、移植免疫拒絶反応、アレルギー、自己免疫疾患、移植片対宿主病、およびiPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応からなる群より選択される、上記項目のいずれかに記載の医薬。
(43)前記疾患、障害または状態はアレルギーを含む、上記項目のいずれかに記載の医薬。
(44)前記疾患、障害または状態は、iPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応を含む、上記項目のいずれかに記載の医薬。
(A1)被験体において、ドナーに対する恒久的な免疫寛容(感染性免疫寛容)を惹起する方法であって、該方法は、CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、該ドナーに由来する抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞を有効量で該被験体に投与する工程を含む、方法。
(A2)前記免疫寛容は、前記被験体におけるCD8陽性T細胞において免疫寛容が惹起されている、上記項目に記載の方法。
(A3)前記阻害因子が、低分子、タンパク質、核酸、脂質、糖、およびこれらの組み合わせからなる群から選択される、上記項目のいずれかに記載の方法。
(A4)前記タンパク質が、抗体もしくはその改変体、または細胞表面分子もしくはその改変体である、上記項目のいずれかに記載の方法。
(A5)前記抗体の改変体が、抗原結合断片である、上記項目のいずれかに記載の方法。
(A6)前記細胞表面分子の改変体が、融合タンパク質である、上記項目のいずれかに記載の方法。
(A7)前記阻害因子は、抗CD80抗体、抗CD86抗体、CD80およびCD86に対する二重特異性抗体、抗CD28抗体もしくはこれらの抗原結合断片、CTLA4-Ig融合タンパク質、CD28-Ig融合タンパク質からなる群より選択される、上記項目のいずれかに記載の方法。
(A8)前記CTLA4-Ig融合タンパク質は、アバタセプトまたはベラタセプトである、上記項目のいずれかに記載の方法。
(A9)被験体のアレルギーを治療または予防する方法であって、該方法は、CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、アレルギーの原因となっている抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞を有効量で該被験体に投与する工程を含む、方法。
(A10)前記アレルギーの原因となっている抗原は、食品、花粉、薬品、および金属から選択される、上記項目のいずれかに記載の方法。
(A11)被験体において、iPS細胞またはES細胞またはそれらに由来する細胞、組織もしくは臓器によって生じる免疫拒絶反応を抑制するまたは予防する方法であって、該方法は、CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、該iPS細胞またはES細胞に由来する抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞を有効量で該被験体に投与する工程を含む、方法。
(A12)特定の抗原に対する、被験体内のCD8陽性T細胞の無反応または低反応を誘導する方法。
(A13)被験体に由来しない抗原によって生じる疾患、障害または状態を処置または予防する方法であって、該方法は、CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、被験体由来の細胞と、該被験体由来の抗原あるいは該験検体に由来しない抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞を有効量で該被験体に投与する工程を含む、方法。
(A14)前記阻害因子が、低分子、タンパク質、核酸、脂質、糖、およびこれらの組み合わせからなる群から選択される、上記項目のいずれかに記載の方法。
(A15)前記タンパク質が、抗体もしくはその改変体、または細胞表面分子もしくはその改変体である、上記項目のいずれかに記載の方法。
(A16)前記抗体の改変体が、抗原結合断片である、上記項目のいずれかに記載の方法。
(A17)前記細胞表面分子の改変体が、融合タンパク質である、上記項目のいずれかに記載の方法。
(A18)前記阻害因子は、抗CD80抗体、抗CD86抗体、CD80およびCD86に対する二重特異性抗体、抗CD28抗体もしくはこれらの抗原結合断片、CTLA4-Ig融合タンパク質、CD28-Ig融合タンパク質からなる群より選択される、上記項目のいずれかに記載の方法。
(A19)前記CTLA4-Ig融合タンパク質は、アバタセプトまたはベラタセプトである、上記項目のいずれかに記載の方法。
(A20)前記疾患、障害または状態は、移植免疫拒絶反応、アレルギー、自己免疫疾患、移植片対宿主病、およびiPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応からなる群より選択される、上記項目のいずれかに記載の方法。
(A21)前記疾患、障害または状態はアレルギーを含む、上記項目のいずれかに記載の方法。
(A22)前記疾患、障害または状態は、iPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応を含む、上記項目のいずれかに記載の方法。
(B1)被験体において、ドナーに対する恒久的な免疫寛容(感染性免疫寛容)を惹起するための医薬を製造するための、CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、該ドナーに由来する抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞の使用。
(B2)前記免疫寛容は、前記被験体におけるCD8陽性T細胞において免疫寛容が惹起されている、上記項目に記載の使用。
(B3)前記阻害因子が、低分子、タンパク質、核酸、脂質、糖、およびこれらの組み合わせからなる群から選択される、上記項目のいずれかに記載の使用。
(B4)前記タンパク質が、抗体もしくはその改変体、または細胞表面分子もしくはその改変体である、上記項目のいずれかに記載の使用。
(B5)前記抗体の改変体が、抗原結合断片である、上記項目のいずれかに記載の使用。
(B6)前記細胞表面分子の改変体が、融合タンパク質である、上記項目のいずれかに記載の使用。
(B7)前記阻害因子は、抗CD80抗体、抗CD86抗体、CD80およびCD86に対する二重特異性抗体、抗CD28抗体もしくはこれらの抗原結合断片、CTLA4-Ig融合タンパク質、CD28-Ig融合タンパク質からなる群より選択される、上記項目のいずれかに記載の使用。
(B8)前記CTLA4-Ig融合タンパク質は、アバタセプトまたはベラタセプトである、上記項目のいずれかに記載の使用。
(B9)被験体において、疾患、障害または状態を予防または治療するための医薬を製造するための、CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、該被験体由来の抗原あるいは該被験体に由来しない抗原または該抗原の含有物とを混合することによってアナジーが誘導された細胞の使用であって、該細胞は、該被験体のT細胞のアナジー状態を確認し、アナジー状態が確認できる場合はさらなる処置をせず、アナジー状態が確認できない場合、該細胞を含む医薬を再度投与されることを特徴とする、使用。
(B10)前記医薬は、CD4陽性アナジー細胞、CD8陽性アナジー細胞、またはそれらの組合せを含む、上記項目のいずれかに記載の使用。
(B11)前記医薬は、CD8陽性アナジー細胞を含む、上記項目のいずれかに記載の使用。
(B12)前記アナジー状態の確認は、前記細胞を含む製剤が消失することを確認する工程を含む、上記項目のいずれかに記載の使用。
(B13)前記疾患、障害または状態は、移植免疫拒絶反応、アレルギー、自己免疫疾患、移植片対宿主病、およびiPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応からなる群より選択される、上記項目のいずれかに記載の使用。
(B14)前記疾患、障害または状態はアレルギーを含む、上記項目のいずれかに記載の使用。
(B15)前記疾患、障害または状態は、iPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応を含む、上記項目のいずれかに記載の使用。
(B16)前記阻害因子が、低分子、タンパク質、核酸、脂質、糖、およびこれらの組み合わせからなる群から選択される、上記項目のいずれかに記載の使用。
(B17)前記タンパク質が、抗体もしくはその改変体、または細胞表面分子もしくはその改変体である、上記項目のいずれかに記載の使用。
(B18)前記抗体の改変体が、抗原結合断片である、上記項目のいずれかに記載の使用。
(B19)前記細胞表面分子の改変体が、融合タンパク質である、上記項目のいずれかに記載の使用。
(B20)前記阻害因子は、抗CD80抗体、抗CD86抗体、CD80およびCD86に対する二重特異性抗体、抗CD28抗体もしくはこれらの抗原結合断片、CTLA4-Ig融合タンパク質、CD28-Ig融合タンパク質からなる群より選択される、上記項目のいずれかに記載の使用。
(B21)前記CTLA4-Ig融合タンパク質は、アバタセプトまたはベラタセプトである、上記項目のいずれかに記載の使用。
(B22)被験体のアレルギーを治療または予防するための医薬を製造するための、CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、アレルギーの原因となっている抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞の使用。
(B23)前記アレルギーの原因となっている抗原は、食品、花粉、薬品、および金属から選択される、上記項目のいずれかに記載の使用。
(B24)被験体において、iPS細胞またはES細胞またはそれらに由来する細胞、組織もしくは臓器によって生じる免疫拒絶反応を抑制するまたは予防するための医薬を製造するための、CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、該iPS細胞またはES細胞に由来する抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞の使用。
(B25)被験体に由来しない抗原によって生じる疾患、障害または状態を処置または予防するための医薬の製造のための、CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、被験体由来の細胞と、該被験体由来の抗原あるいは該験検体に由来しない抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞の使用。
(B26)前記阻害因子が、低分子、タンパク質、核酸、脂質、糖、およびこれらの組み合わせからなる群から選択される、上記項目のいずれかに記載の使用。
(B27)前記タンパク質が、抗体もしくはその改変体、または細胞表面分子もしくはその改変体である、上記項目のいずれかに記載の使用。
(B28)前記抗体の改変体が、抗原結合断片である、上記項目のいずれかに記載の医薬。
(B29)前記細胞表面分子の改変体が、融合タンパク質である、上記項目のいずれかに記載の使用。
(B30)前記阻害因子は、抗CD80抗体、抗CD86抗体、CD80およびCD86に対する二重特異性抗体、抗CD28抗体もしくはこれらの抗原結合断片、CTLA4-Ig融合タンパク質、CD28-Ig融合タンパク質からなる群より選択される、上記項目のいずれかに記載の使用。
(B31)前記CTLA4-Ig融合タンパク質は、アバタセプトまたはベラタセプトである、上記項目のいずれかに記載の使用。
(B32)前記疾患、障害または状態は、移植免疫拒絶反応、アレルギー、自己免疫疾患、移植片対宿主病、およびiPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応からなる群より選択される、上記項目のいずれかに記載の使用。
(B33)前記疾患、障害または状態はアレルギーを含む、上記項目のいずれかに記載の使用。
(B34)前記疾患、障害または状態は、iPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応を含む、上記項目のいずれかに記載の使用。
(C1)被験体において、ドナーに対する恒久的な免疫寛容(感染性免疫寛容)を惹起するための、CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、該ドナーに由来する抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞。
(C2)前記免疫寛容は、前記被験体におけるCD8陽性T細胞において免疫寛容が惹起されている、上記項目に記載の細胞。
(C3)前記阻害因子が、低分子、タンパク質、核酸、脂質、糖、およびこれらの組み合わせからなる群から選択される、上記項目のいずれかに記載の細胞。
(C4)前記タンパク質が、抗体もしくはその改変体、または細胞表面分子もしくはその改変体である、上記項目のいずれかに記載の細胞。
(C5)前記抗体の改変体が、抗原結合断片である、上記項目のいずれかに記載の細胞。
(C6)前記細胞表面分子の改変体が、融合タンパク質である、上記項目のいずれかに記載の細胞。
(C7)前記阻害因子は、抗CD80抗体、抗CD86抗体、CD80およびCD86に対する二重特異性抗体、抗CD28抗体もしくはこれらの抗原結合断片、CTLA4-Ig融合タンパク質、CD28-Ig融合タンパク質からなる群より選択される、上記項目のいずれかに記載の細胞。
(C8)前記CTLA4-Ig融合タンパク質は、アバタセプトまたはベラタセプトである、上記項目のいずれかに記載の細胞。
(C9)被験体において、疾患、障害または状態を予防または治療するための、CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、該被験体由来の抗原あるいは該被験体に由来しない抗原または該抗原の含有物とを混合することによってアナジーが誘導された細胞であって、該被験体のT細胞のアナジー状態を確認し、アナジー状態が確認できる場合はさらなる処置をせず、アナジー状態が確認できない場合、該細胞が再度投与されることを特徴とする、細胞。
(C10)前記細胞は、CD4陽性アナジー細胞、CD8陽性アナジー細胞、またはそれらの組合せを含む細胞混合物である、上記項目のいずれかに記載の細胞。
(C11)前記細胞は、CD8陽性アナジー細胞を含む、上記項目のいずれかに記載の細胞。
(C12)前記アナジー状態の確認は、前記細胞が消失することを確認する工程を含む、上記項目のいずれかに記載の細胞。
(C13)前記疾患、障害または状態は、移植免疫拒絶反応、アレルギー、自己免疫疾患、移植片対宿主病、およびiPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応からなる群より選択される、上記項目のいずれかに記載の細胞。
(C14)前記疾患、障害または状態はアレルギーを含む、上記項目のいずれかに記載の細胞。
(C15)前記疾患、障害または状態は、iPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応を含む、上記項目のいずれかに記載の細胞。
(C16)前記阻害因子が、低分子、タンパク質、核酸、脂質、糖、およびこれらの組み合わせからなる群から選択される、上記項目のいずれかに記載の細胞。
(C17)前記タンパク質が、抗体もしくはその改変体、または細胞表面分子もしくはその改変体である、上記項目のいずれかに記載の細胞。
(C18)前記抗体の改変体が、抗原結合断片である、上記項目のいずれかに記載の細胞。
(C19)前記細胞表面分子の改変体が、融合タンパク質である、上記項目のいずれかに記載の細胞。
(C20)前記阻害因子は、抗CD80抗体、抗CD86抗体、CD80およびCD86に対する二重特異性抗体、抗CD28抗体もしくはこれらの抗原結合断片、CTLA4-Ig融合タンパク質、CD28-Ig融合タンパク質からなる群より選択される、上記項目のいずれかに記載の細胞。
(C21)前記CTLA4-Ig融合タンパク質は、アバタセプトまたはベラタセプトである、上記項目のいずれかに記載の細胞。
(C22)被験体のアレルギーを治療または予防するための、CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、アレルギーの原因となっている抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞の使用。
(C23)前記アレルギーの原因となっている抗原は、食品、花粉、薬品、および金属から選択される、上記項目のいずれかに記載の細胞。
(C24)被験体において、iPS細胞またはES細胞またはそれらに由来する細胞、組織もしくは臓器によって生じる免疫拒絶反応を抑制するまたは予防するための、CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、該iPS細胞またはES細胞に由来する抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞の使用。
(C25)被験体に由来しない抗原によって生じる疾患、障害または状態を処置または予防するための、CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、被験体由来の細胞と、該被験体由来の抗原あるいは該験検体に由来しない抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞。
(C26)前記阻害因子が、低分子、タンパク質、核酸、脂質、糖、およびこれらの組み合わせからなる群から選択される、上記項目のいずれかに記載の細胞。
(C27)前記タンパク質が、抗体もしくはその改変体、または細胞表面分子もしくはその改変体である、上記項目のいずれかに記載の細胞。
(C28)前記抗体の改変体が、抗原結合断片である、上記項目のいずれかに記載の細胞。
(C29)前記細胞表面分子の改変体が、融合タンパク質である、上記項目のいずれかに記載の細胞。
(C30)前記阻害因子は、抗CD80抗体、抗CD86抗体、CD80およびCD86に対する二重特異性抗体、抗CD28抗体もしくはこれらの抗原結合断片、CTLA4-Ig融合タンパク質、CD28-Ig融合タンパク質からなる群より選択される、上記項目のいずれかに記載の細胞。
(C31)前記CTLA4-Ig融合タンパク質は、アバタセプトまたはベラタセプトである、上記項目のいずれかに記載の細胞。
(C32)前記疾患、障害または状態は、移植免疫拒絶反応、アレルギー、自己免疫疾患、移植片対宿主病、およびiPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応からなる群より選択される、上記項目のいずれかに記載の細胞。
(C33)前記疾患、障害または状態はアレルギーを含む、上記項目のいずれかに記載の細胞。
(C34)前記疾患、障害または状態は、iPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応を含む、上記項目のいずれかに記載の細胞。
本開示は以下をも提供する。
(D1)被験体において、ドナーに対する恒久的な免疫寛容(感染性免疫寛容)を惹起するための組成物であって、該組成物は、CD80および/またはCD86と、CD28との相互作用を阻害することができる抗体と、該被験体由来の細胞と、該ドナーに由来する抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞を含む、組成物。
(D2)前記免疫寛容は、前記被験体におけるCD8陽性T細胞において免疫寛容が惹起されている、上記項目に記載の組成物。
(D3)被験体において、疾患、障害または状態を予防または治療する方法であって、該方法は、1) CD80および/またはCD86と、CD28との相互作用を阻害することができる抗体と、該被験体由来の細胞と、該被験体由来の抗原あるいは該被験体に由来しない抗原または該抗原の含有物とを混合することによってアナジーが誘導された細胞を含む製剤を該被験体に投与する工程と、2) 該被験体のT細胞のアナジー状態を確認し、アナジー状態が確認できる場合はさらなる処置をせず、アナジー状態が確認できない場合、該細胞を含む製剤を再度投与する工程とを含む、方法。
(D3A)被験体において、疾患、障害または状態を予防または治療するための医薬であって、該医薬は、CD80および/またはCD86と、CD28との相互作用を阻害することができる抗体と、該被験体由来の細胞と、該被験体由来の抗原あるいは該被験体に由来しない抗原または該抗原の含有物とを混合することによってアナジーが誘導された細胞(例えば、T細胞)を含み、ここで、該医薬は、該医薬を投与した後に、該被験体のT細胞のアナジー状態を確認し、アナジー状態が確認できる場合はさらなる処置をせず、アナジー状態が確認できない場合、該細胞を含む医薬を再度投与されることを特徴とする、医薬。
(D4)前記製剤は、CD4陽性アナジー細胞、CD8陽性アナジー細胞、またはそれらの組合せを含む、上記項目のいずれか一項に記載の方法、医薬または組成物。
(D5)前記製剤は、CD8陽性アナジー細胞を含む、上記項目のいずれか一項に記載の方法、医薬または組成物。
(D6)前記アナジー状態の確認は、前記細胞を含む製剤が消失することを確認する工程を含む、上記項目のいずれか一項に記載の方法、医薬または組成物。
(D7)前記疾患、障害または状態は、移植免疫拒絶反応、アレルギー、自己免疫疾患、移植片対宿主病、およびiPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応からなる群より選択される、上記項目のいずれか一項に記載の方法、医薬または組成物。
(D8)前記疾患、障害または状態はアレルギーを含む、上記項目のいずれか一項に記載の方法、医薬または組成物。
(D9)前記疾患、障害または状態は、iPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応を含む、上記項目のいずれか一項に記載の方法、医薬または組成物。
(D10)被験体のアレルギーを治療または予防するための組成物であって、該組成物は、CD80および/またはCD86と、CD28との相互作用を阻害することができる抗体と、該被験体由来の細胞と、アレルギーの原因となっている抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞を含む、組成物。
(D11)前記アレルギーの原因となっている抗原は、食品、花粉、薬品、および金属から選択される、上記項目のいずれか一項に記載の組成物。
(D11A)項目D1~D9のいずれか1つまたは複数に記載の特徴をさらに含む、項目D10またはD11に記載の組成物。
(D12)被験体において、iPS細胞またはES細胞またはそれらに由来する細胞、組織もしくは臓器によって生じる免疫拒絶反応を抑制するまたは予防するための組成物であって、該組成物は、CD80および/またはCD86と、CD28との相互作用を阻害することができる抗体と、該被験体由来の細胞と、該iPS細胞またはES細胞に由来する抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞
を含む、組成物。
(D12A)項目D1~D11のいずれか1つまたは複数に記載の特徴をさらに含む、項目D12に記載の組成物。
(D13)制御性T細胞ではない、アナジーが誘導されたT細胞。
(D13A)項目D1~D11、D11A、D12またはD12Aのいずれか1つまたは複数に記載の特徴をさらに含む、項目D13に記載の組成物。
(D14)制御性T細胞ではない、被験体に対してアナジーが誘導されたT細胞を製造する方法であって、A)CD80および/またはCD86と、CD28との相互作用を阻害することができる抗体と、該被験体由来の細胞と、該被験体に由来しない抗原または該抗原の含有物とを混合する工程と、B)該混合物を培養してT細胞を得る工程と、C)必要に応じて、該被験体に由来しない抗原または該抗原の含有物で該T細胞を刺激し、該T細胞が反応しないことを確認する工程とを包含する、方法。
(D15)前記T細胞は、CD4陽性細胞および/またはCD8陽性細胞を含む、上記項目のいずれか一項に記載のT細胞または上記項目のいずれか一項に記載の方法。
(D15A)項目D1~D11、D11A、D12、D12AまたはD13のいずれか1つまたは複数に記載の特徴をさらに含む、上記項目のいずれか一項に記載の方法。
(D16)特定の抗原に対する、被験体内のCD8陽性T細胞の無反応または低反応を誘導するための組成物。
(D16A)項目D1~D1、D11A、D12、D12A、D13、D14、D15またはD15Aのいずれか1つまたは複数に記載の特徴をさらに含む、上記項目のいずれか一項に記載の組成物。
(D17)CD80および/またはCD86と、CD28との相互作用を阻害することができる抗体と、被験体由来の細胞と、該被験体由来の抗原あるいは該験検体に由来しない抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞を含む、該被験体に由来しない抗原によって生じる疾患、障害または状態を処置または予防するための医薬。
(D18)前記疾患、障害または状態は、移植免疫拒絶反応、アレルギー、自己免疫疾患、移植片対宿主病、およびiPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応からなる群より選択される、上記項目のいずれか一項に記載の医薬。
(D19)前記疾患、障害または状態はアレルギーを含む、上記項目のいずれか一項に記載の医薬。
(D20)前記疾患、障害または状態は、iPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応を含む、上記項目のいずれか一項に記載の医薬。
(D20A)項目D1~D11、D11A、D12、D12A、D13、D14、D15、D15A、D16、D16A、D17~D19のいずれか1つまたは複数に記載の特徴をさらに含む、上記項目のいずれか一項に記載の医薬。
本明細書において「約」とは、本明細書で使用される場合、後に続く数値の±10%を意味する。
以下に好ましい実施形態の説明を記載するが、この実施形態は本開示の例示であり、本開示の範囲はそのような好ましい実施形態に限定されないことが理解されるべきである。当業者はまた、以下のような好ましい実施例を参考にして、本開示の範囲内にある改変、変更などを容易に行うことができることが理解されるべきである。これらの実施形態について、当業者は適宜、本明細書中の記載を参酌して、任意の実施形態を組み合わせ得る。また、本開示の以下の実施形態は単独でも使用されあるいはそれらを組み合わせて使用することができることが理解される。
本発明者らは、CD80/CD86とCD28との相互作用を阻害する阻害因子によりアナジーが誘導された細胞を含む細胞製剤を臓器移植患者(レシピエント)に投与して、免疫寛容を誘導する技術において、予想外にも、細胞製剤に由来する細胞がレシピエントから無くなった(検出されなくなった)後も免疫寛容が続くこと(感染性免疫寛容)を見出した。
別の局面において、本開示は、被験体において、疾患、障害または状態を予防または治療する方法であって、該方法は、(1)CD80および/またはCD86と、CD28との相互作用を阻害することができる抗体等の阻害因子と、該被験体由来の細胞と、該被験体由来の抗原あるいは該被験体に由来しない抗原または該抗原の含有物とを混合することによってアナジーが誘導された細胞を含む製剤を該被験体に投与する工程と、(2)該被験体のT細胞のアナジー状態を確認し、アナジー状態が確認できる場合はさらなる処置をせず、アナジー状態が確認できない場合、該細胞を含む製剤を再度投与する工程とを含む、方法を提供する。
1つの局面において、本開示は、本開示の医薬を用いてアレルギーおよび/または自己免疫疾患を治療または予防する方法およびそれに用いる医薬、組成物、細胞混合物を提供する。アレルギーおよび自己免疫疾患に関しては、患者の末梢血から得たマクロファージを慣例的な方法により抗原提示能の高い樹状細胞(マクロファージ由来樹状細胞)へと分化させ、放射線(γ線)照射後のこの細胞にアレルギーや自己免疫疾患における過剰反応の原因となっている抗原を提示させ、同じ患者末梢血から得たT細胞群と、抗CD80抗体および/または抗CD86抗体あるいはCTLA4-Ig融合タンパク質などの阻害因子の存在下で1~2週間共培養し、アレルギーや自己免疫疾患の原因となっている抗原に特異的なアナジー細胞を得る。このアナジー細胞を患者に投与することで、アレルギーや自己免疫疾患の原因となっている抗原に特異的な免疫寛容を誘導し、アレルギーおよび自己免疫疾患の予防および治療に用いる。予防的療法であるか治療であるか、さらに症状の強弱等の諸条件により、投与回数は複数回となることもある。
1つの局面において、本開示は、本開示の医薬を用いて移植片対宿主病を治療または予防する方法およびそれに用いる医薬、組成物、細胞混合物を提供する。移植片対宿主病においては、移植免疫拒絶反応に対する治療とは対照的に、移植片を提供するドナーのPBMCまたはT細胞等の移植片対宿主病の原因となりうる細胞を、放射線(γ線)照射した宿主由来のPBMCまたはそれ以外の細胞と抗CD80抗体および/または抗CD86抗体あるいはCTLA4-Ig融合タンパク質などの阻害因子の存在下で1~2週間共培養し、宿主に特異的なアナジー細胞を得る。このアナジー細胞を宿主に投与することで、移植片対宿主病の原因となっている移植片による宿主への反応を抑制し(免疫寛容を誘導し)移植片対宿主病を予防および治療する。予防的療法であるか治療であるか、さらに移植する組織やその大きさ、症状の強弱等の諸条件により、投与回数は複数回となることもある。
1つの局面の局面において、本開示は、本開示の医薬を用いてiPS細胞またはES細胞を用いた予防または治療において、iPS細胞またはES細胞およびそれらの細胞に由来する細胞、組織または臓器の移植によって引き起こされる免疫拒絶反応またはその他の副作用を治療または予防する方法およびそれに用いる医薬、組成物、細胞混合物を提供する。iPS細胞またはES細胞を用いた治療への応用では、iPS細胞またはES細胞およびそれらの細胞に由来する細胞、組織または臓器の移植によって引き起こされる免疫拒絶反応が代表的に治療対象として例示される。
1つの局面において、本開示は、本開示の医薬を用いてアレルギーを治療または予防する方法およびそれに用いる医薬、組成物、細胞混合物を提供する。本発明者らは、CD80/CD86とCD28との相互作用を阻害する抗体等の阻害因子によりアナジーが誘導された細胞を含む製剤が、アレルギーに対する免疫寛容を惹起することができることを実証した。したがって、別の局面の局面において、本開示は、被験体のアレルギーを治療または予防するための組成物であって、該組成物は、CD80および/またはCD86と、CD28との相互作用を阻害することができる抗体等の阻害因子と、該被験体由来の細胞と、アレルギーの原因となっている抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞を含む、組成物を提供する。
1つの局面の局面において、本開示は、iPS細胞等によって生じる免疫拒絶反応の抑制または予防のための方法を提供し、その抑制または予防のための、医薬、組成物、細胞混合物を提供する。本発明者らは、CD80/CD86とCD28との相互作用を阻害する抗体等の阻害因子によりアナジーが誘導された細胞を含む製剤が、iPS細胞等またはそれらに由来する細胞、組織または臓器によって生じる免疫拒絶反応に対する免疫寛容を惹起することができることを実証した。したがって、別の局面の局面において、本開示は、被験体において、iPS細胞またはES細胞またはそれらに由来する細胞、組織もしくは臓器によって生じる免疫拒絶反応を抑制するまたは予防するための組成物であって、該組成物は、CD80および/またはCD86と、CD28との相互作用を阻害することができる抗体等の阻害因子と、該被験体由来の細胞と、該iPS細胞またはES細胞に由来する抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞を含む、組成物を提供する。
本発明者らは、移植後後期(例えば、移植後80日以降)には、CD8陽性細胞の反応性が失われ、CD4陽性T細胞を除去しても免疫寛容が誘導されることを実証した。したがって、本開示の一つの局面において、制御性T細胞ではない、アナジーを誘導する活性を有するT細胞が提供される。さらなる態様において、本開示は、制御性T細胞ではない、被験体に対してアナジーを誘導する活性を有するT細胞を製造する方法であって、(A)CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、該被験体に由来しない抗原または該抗原の含有物とを混合する工程と、(B)該混合物を培養してT細胞を得る工程と、(C)必要に応じて、該被験体に由来しない抗原または該抗原の含有物で該T細胞を刺激し、該T細胞が反応しないことを確認する工程とを包含する、方法を提供する。
別の局面において、本開示は、CD80および/またはCD86と、CD28との相互作用を阻害することができる抗体等の阻害因子と、被験体由来の細胞と、該被験体由来の抗原あるいは該験検体に由来しない抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞を含む、該被験体に由来しない抗原によって生じる疾患、障害または状態を処置または予防するための医薬を提供する。
1.生物由来原料およびその対応状況
一つの実施形態では、アナジーT細胞の製造工程において、表1に記載の生物由来原料基準に適合した生物由来原料を使用する。
ベラタセプト(例えば、Bristol-Myers Squibb、New York、NYから入手可能)
2.細胞製剤の製法
一つの実施形態では、細胞製剤は以下のように製造することができる。以下に例示する各種数値等は、代表例であり、当業者は適宜変更して細胞製剤の製造を行うことができる。
1) 投与19日前頃に、医療機関においてドナーにアフェレーシスを実施し、ドナーアフェレーシス産物を30Gyの放射線を照射し、細胞増殖能を無くした後に、細胞加工を実施する細胞培養加工施設へ発送する。
2) ドナーアフェレーシス産物受領後、細胞培養加工施設において、ドナー単核球を密度勾配遠心法により分離、回収後、2つに分けて凍結し、-80±10℃で保管する。
3) 投与14日前頃に、医療機関においてレシピエントにアフェレーシスを実施し、レシピエントアフェレーシス産物を、細胞加工を実施する細胞培養加工施設へ発送する。
4) レシピエントアフェレーシス産物受領後、細胞培養加工施設において、レシピエント単核球を密度勾配遠心法により分離、回収し、解凍したドナー単核球ならびに抗CD80抗体および抗CD86抗体またはCTLA4-Ig融合タンパク質などの阻害因子と共培養する。
5) 投与7日前頃に培地交換を行う。7日間培養した中間製品を回収し、解凍したドナー単核球ならびに抗CD80抗体および抗CD86抗体またはCTLA4-Ig融合タンパク質などの阻害因子と共培養する。
6) 投与当日に細胞加工物を密度勾配遠心法により回収後に、洗浄し、生理食塩液に充填する。
7) 医療機関に発送し、医療機関でレシピエントに投与する。
3.工程内管理試験
一つの実施形態では、製造工程内において、表2に記載した工程内管理試験を実施することができる。以下に例示する各種数値等や手順は、代表例であり、当業者は適宜変更して工程内管理試験を実施することができる。
4.規格試験、特性解析試験
一つの実施形態では、最終製品を用いて表3に記載した規格試験を行うことができる。表3に例示する手順は、代表例であり、当業者は適宜変更して規格試験、特性解析試験を実施することができる。例えば、その改変例としては、実施例11に例示した規格を挙げることができる。誘導性抑制性T細胞投与時には結果が判明しない場合、工程内管理試験の結果を参考に、治験製品の出荷を判断することができる。
最終製品は、一例をあげると、下記表に記載の構成物で構成される。これらの規格もまた、当業者は適宜変更して組成を変更して再生医療等製品を構成することができる。
一つの実施形態において、臓器移植後14日後に一回投与する。当業者は、投与方法やその期間等について、本明細書に記載される技術的事項を適宜参酌して必要に応じて変更を行って具体的に実施することができる。
以下に、自己由来制御性T細胞の典型的な製造方法を説明する。
一つの実施形態では、事前の確認は以下のように行うことができる。以下に例示する各種数値、試薬、手順等は、代表例であり、当業者は適宜変更して事前の確認の製造を行うことができる。
一つの実施形態では、ドナーリンパ球の分離は以下のように製造することができる。以下に例示する各種数値、試薬や手順等は、代表例であり、当業者は適宜変更してドナーリンパ球の分離を行うことができる。
・アフェレーシスでドナーリンパ球を回収バッグに採取し、その回収バッグを放射線照射する。
・前述の放射線照射済み末梢血単核球を、適量のFicoll-Paque PREMIUM(GE Healthcare #17-5442-02)またはLymphocyte separation Solution(ナカライテスク#20828)等(例えば、20mL)が入った遠心管に入れ、860Gで20分間、22℃で遠心分離(遠心分離機のアクセルをslow、ブレーキをslowに設定する)する。
・上清を捨て、リンパ球層を含む細胞浮遊液を、別の遠心管(例えば、50mL遠心管2本)に移す。
・細胞浮遊液の入った遠心管に、生理食塩液を追加し(例えば、全液量が50mLになるまで適量)、シリンジ(例えば、18G注射針をつけた50mLシリンジ)またはピペットで吸引排出を繰り返して、よく混和させる。
・500Gで10分間、22℃で遠心分離(遠心分離機のアクセルをfast、ブレーキをfastに設定してもよい)する。
・上清を捨て、再度生理食塩液を追加し(例えば、全液量が50mLになるまで適量)、細胞ペレットを、ピペットで吸引排出を繰り返して、よく混和させる。
・500Gで5分間、22℃で遠心分離(遠心分離機のアクセルをfast、ブレーキをfastに設定してもよい)する。
・上清を捨てる。
・予めドナーより採取した血漿を含むALyS505N-0培養液(細胞科学研究所 (CSTI)1020P10))を、細胞ペレットに加え(例えば、全液量が31mLになるまで適量)、
ピペットで吸引排出を繰り返して、よく混和させる。
・シリンジ(例えば、18G注射針をつけた1mLシリンジ)またはピペットで、適量(例えば、0.3mL)抜き取り、細胞数および生細胞数を確認する。
一つの実施形態では、ドナーリンパ球の凍結保存は以下のように行うことができる。以下に例示する各種数値、試薬や手順等は、代表例であり、当業者は適宜変更してドナーリンパ球の凍結保存を行うことができる。
・凍結バッグ(例えば、フローズバッグF-050 25mL凍結バッグ(株)ニプロ89-101)
を無菌下で開封し、ラベルに必要事項(日付、製造番号、ドナー名)を記入する。
・シリンジ(例えば、18G注射針をつけた30mLシリンジ)で、細胞浮遊液を取り、凍結バッグに入れる。
・ACD液((株)テルモTP-A05ACD、例えば、細胞浮遊液15mLに対して2mL)を、
細胞浮遊液が入った凍結バッグに加え、4℃で冷却した保冷剤に挟んで、10分間程度冷やす。
・シリンジ(例えば、18G注射針をつけた20mLシリンジ)を用いて、4℃で冷却したCP-1(極東製薬工業(株) 551-27202-4細胞凍害保護液CP-1)例えば、8.5mL)を、凍結バッグに1分半程度の時間をかけて加える。この際、凍結バッグをゆっくり攪拌する。
・シリンジを用いて、凍結バッグおよびそのポート内の空気を全て抜き取る。
・チューブシーラーを使用して凍結バッグをシールし、まず4℃で約5~10分冷却し、その後-80℃の冷凍庫で保管する。
一つの実施形態では、ドナーリンパ球の解凍は以下のように行うことができる。以下に例示する各種数値、試薬や手順等は、代表例であり、当業者は適宜変更してドナーリンパ球の解凍を行うことができる。
・保存されたドナー細胞の凍結バッグを、例えば、37℃恒温槽で解凍する。以降の操作は、好ましくは無菌下で行う。
・シリンジ(例えば、18G注射針をつけた50mLシリンジ)を用いて、解凍された凍結バッグから細胞浮遊液を抜き取り、遠心管(例えば、50mL遠心管2本に12.5mLずつ)に移す。
・細胞浮遊液の入った遠心管に、例えば5%アルブミン液(日本製薬(株) 123146364 献血アルブミン5%静注12.5g/250mL)(例えば、細胞浮遊液12.5mLに対して37.5mL)を追加し、よく混和させる。その後、約5分間静置する。
・例えば、600Gで10分間、22℃で遠心分離(例えば、好ましくは、遠心分離機のアクセルをfast、ブレーキをslowに設定する)する。
・上清を静かに捨て、細胞ペレットに、洗浄用のアルブミン加生理食塩液(例えば、5%アルブミン液25mLと生理食塩液19mLから作製する)等の適宜の液を加えて懸濁する。
・例えば、600Gで10分間、22℃で遠心分離(例えば、好ましくは、遠心分離機のアクセルをfast、ブレーキをslowに設定する)する。
・上清を静かに捨て、細胞ペレットに、ALyS505N培養液(例えば、50mL遠心管に対して10mL)を加えて懸濁する。
・ALyS505N-0培養液またはそれと同等の液が入った培養バッグ(例えば、(株)ニプロ87598 ニプロメディウムALyS505NB10)に、抗ヒトCD80抗体(例えば、m2D10.4;Cat.No.16-0809-85、eBioscience社)と抗ヒトCD86抗体(例えば、IT2.2;Cat.No.16-0869-85、eBioscience社)をそれぞれ例えば最終濃度10μg/mLで加え(またはCTLA4-Ig融合タンパク質(例えば、ベラタセプト)などの阻害因子を加え)、この培養バッグに、上記の細胞浮遊液をシリンジ(例えば、18G注射針をつけた20mLシリンジ)で注入して加える。一例では、培養バッグ中の総液量は、約840mLである。
一つの実施形態では、患者リンパ球の分離は以下のように行うことができる。以下に例示する各種数値、試薬や手順等は、代表例であり、当業者は適宜変更して患者リンパ球の分離を行うことができる。
・患者より採取した血漿は、恒温槽中、例えば、56℃で30分間加温し、非働化しておく。直ちに使用しないものは、凍結保存する。
・患者より採取した末梢血を、適量の適切な媒体、例えばFicoll-Paque(例えば、20mL)が入った遠心管に入れ、例えば、860Gで20分間、22℃で遠心分離(例えば、好ましくは、遠心分離機のアクセルをslow、ブレーキをslowに設定する)する。
・上清を捨て、リンパ球層を含む細胞浮遊液を、別の遠心管(例えば、50mL遠心管2本)に移す。
・細胞浮遊液の入った遠心管に、生理食塩液を追加し(例えば、全液量が50mLになるまで適量)、ピペットで吸引排出を繰り返して、よく混和させる。
・例えば、500Gで10分間、22℃で遠心分離(遠心分離機のアクセルをfast、ブレーキをfastに設定してもよい)する。
・上清を捨て、再度生理食塩液を追加し(例えば、全液量が50mLになるまで適量)、細胞ペレットを、ピペットで吸引排出を繰り返して、よく混和させる。
・例えば、500Gで5分間、22℃で遠心分離(遠心分離機のアクセルをfast、ブレーキをfastに設定してもよい)する。
・上清を捨て、細胞ペレットに、例えば、ALyS505N-0培養液(例えば、10mL)を加えて懸濁し、細胞浮遊液を作製する(例えば、合計20mLになるまでALyS505N-0培養液を追加する)。ここで、0.5mL程度の細胞浮遊液を抜き取り、細胞数、生細胞数および表面抗原の発現を確認する。
・「3.ドナーリンパ球の解凍」で作製したALyS505N-0培養液中ドナー細胞および抗体等の阻害因子が入った培養バッグに患者由来の非働化血漿を追加する。
・この培養バッグに、上記患者由来細胞浮遊液をシリンジ(例えば、18G注射針をつけた20mLシリンジ)で注入して加え、チューブシーラーを使用して培養バッグをシールする。一例では、培養バッグ中の総液量は、約1000mLである。
・37℃インキュベーター内で、例えば、1週間培養する。
一つの実施形態では、培地交換は以下のように行うことができる。以下に例示する各種数値、試薬や手順等は、代表例であり、当業者は適宜変更して培地交換を行うことができる。
・インキュベーターから培養バッグを取り出し、内容物を、遠心管(例えば、225mL遠心管4本)に分注する。
・例えば、600Gで10分間、22℃で遠心分離(例えば、好ましくは、遠心分離機のアクセルをfast、ブレーキをfastに設定してもよい)する。
・上清を静かに捨て、細胞ペレットに、例えば、ALyS505N-0培養液を加えて懸濁し、細胞浮遊液を作製する(例えば、合計20mLになるまでALyS505N-0培養液を追加する)。ここで、0.3mL程度の細胞浮遊液を抜き取り、細胞数および生細胞数を確認する。
・例えば、ALyS505N-0培養液が入った培養バッグに、細胞浮遊液をシリンジ(例えば、18G注射針をつけた20mLシリンジ)で注入して加える。
・抗ヒトCD80抗体(例えば、2D10.4)希釈液と抗ヒトCD86抗体(例えば、IT2.2)希釈液を、それぞれ例えば、最終濃度10μg/mLとなるように(またはCTLA4-Ig融合タンパク質(例えば、ベラタセプト)等の阻害因子を)、培養バッグにシリンジ(例えば、18G注射針をつけた20mLシリンジ)で注入して加える。
一つの実施形態では、ドナーリンパ球の解凍・抗原再刺激~二次培養開始は以下のように行うことができる。以下に例示する各種数値、試薬や手順等は、代表例であり、当業者は適宜変更してドナーリンパ球の解凍・抗原再刺激~二次培養開始を行うことができる。
・保存されたドナー細胞の凍結バッグと患者由来の非働化血漿を、例えば、37℃恒温槽で解凍する。以降の操作は、好ましくは、無菌下で行う。
・シリンジ(例えば、18G注射針をつけた50mLシリンジ)を用いて、解凍された凍結バッグからドナー細胞浮遊液を抜き取り、遠心管(例えば、50mL遠心管2本)に移す。
・ドナー細胞浮遊液の入った遠心管に、5%アルブミン液(例えば、50mL遠心管2本につき、合計で約50mL)を追加し、よく混和させる。その後、約5分間静置する。
・例えば、600Gで10分間、22℃で遠心分離(遠心分離機のアクセルをfast、ブレーキをslowに設定する)する。
・上清を静かに捨て、細胞ペレットに、洗浄用のアルブミン加生理食塩液(例えば、5%アルブミン液25mLと生理食塩液19mLから作製する)を加えて懸濁する。
・例えば、600Gで10分間、22℃で遠心分離(遠心分離機のアクセルをfast、ブレーキをslowに設定する)する。
・上清を静かに捨て、細胞ペレットに、例えば、ALyS505N-0培養液(例えば、50mL遠心管に対して10mL)を加えて懸濁する。
・「3.ドナーリンパ球の解凍」で作製したALyS505N培養液中患者細胞および抗体等の阻害因子が入った培養バッグに、解凍した患者由来の非働化血漿(例えば、10mL)をシリンジ(例えば、18G注射針をつけた20mLシリンジ)で注入して加え、さらに、この培養バッグに、上記の細胞浮遊液をシリンジ(例えば、18G注射針をつけた20mLシリンジ)で注入して加える。一例では、培養バッグ中の総液量は、約1000mLである。
・チューブシーラーを使用して培養バッグをシールする。
・37℃インキュベーター内で、例えば、1週間培養する。
一つの実施形態では、二次培養中の検査は以下のように行うことができる。以下に例示する各種数値、試薬や手順等は、代表例であり、当業者は適宜変更して二次培養中の検査を行うことができる。
・代表的に、二次培養開始から3日目(培養通算10日目)に少量の培養液を、培養バッグから抜き取り、マイコプラズマ汚染などについて検査する。
一つの実施形態では、培養リンパ球の回収・充填は以下のように行うことができる。以下に例示する各種数値、試薬や手順等は、代表例であり、当業者は適宜変更して培養リンパ球の回収・充填を行うことができる。
・例えば、二次培養開始から7日目(培養通算14日目)インキュベーターから培養バッグを取り出し、内容物を、遠心管(例えば、225mL遠心管4本)に分注する。
・例えば、600Gで10分間、22℃で遠心分離(遠心分離機のアクセルをfast、ブレーキをfastに設定してもよい)する。
・上清を静かに捨て、細胞ペレットに、生理食塩液を加えて懸濁する。
・例えば、600Gで10分間、22℃で遠心分離(例えば、好ましくは、遠心分離機のアクセルをfast、ブレーキをfastに設定してもよい)する。
・上清を静かに捨て、細胞ペレットに、生理食塩液(例えば、10mL)を加えて懸濁し、細胞浮遊液を作製する。
・細胞浮遊液を、適量のFicoll-Paque(例えば、20mL)が入った遠心管(例えば、50mL遠心管)に静かに入れて重層させる。
・例えば、860Gで20分間、22℃で遠心分離(遠心分離機のアクセルをslow、ブレーキをslowに設定する)する。
・上清を捨て、リンパ球層を含む細胞浮遊液を、別の遠心管(例えば、50mL遠心管)に移す。
・細胞浮遊液の入った遠心管に、生理食塩液を追加し(例えば、全液量が50mLになるまで適量)、シリンジ(例えば、18G注射針をつけた50mLシリンジ)で吸引排出を繰り返して、よく混和させる。
・例えば、500Gで10分間、22℃で遠心分離(遠心分離機のアクセルをfast、ブレーキをfastに設定してもよい)する。
・上清を5mL程度残して他は捨て、ピペットで吸引排出を繰り返して、よく混和させる。
・生理食塩液を追加し(例えば、全液量が50mLになるまで適量)、シリンジ(例えば、例えば、18G注射針をつけた50mLシリンジ)で吸引排出を繰り返して、よく混和させる(a)。
・例えば、500Gで5分間、22℃で遠心分離(遠心分離機のアクセルをfast、ブレーキをfastに設定してもよい)する(b)。
・上清を5mL程度残して他は捨て、ピペットで吸引排出を繰り返して、よく混和させる(c)。
・上記(a)、(b)および(c)をさらに2回繰り返す。
・最後の遠心分離後の上清を、適量(例えば、4mL)を抜き取り、無菌検査およびマイコプラズマ検査に供する。
・再度生理食塩液を加えて懸濁し、細胞浮遊液を、最終的な容器(例えば、100mL生理食塩液のボトル)に移す。適量(例えば、4mL)を抜き取り、最終的な生成物の、細胞数、生細胞数、表面抗原の発現およびエンドトキシンの含有量を確認する。
一つの実施形態では、二次包装は以下のように行うことができる。以下に例示する各種数値、試薬や手順等は、代表例であり、当業者は適宜変更して二次包装を行うことができる。
・代表的には、適切な基準(代表的には、NUHCPC-M-12-ATREG)に基づいてラベルに被験者ID、製造番号、使用期限を入力して印刷し、容器にラベルを貼付する。
・適切な基準(代表的には、NUHCPC-PMF-ATREG14)に基づいて「用法・用量・効能または効果ならびに使用上の注意または取扱い上の注意」を発行する。
・チャック付ビニール袋に試験物および「用法・用量・効能または効果ならびに使用上の注意または取扱い上の注意」を収納する。
・出荷するまで移送容器に入れてモニタリングユニット内に保管する。
本明細書において「または」は、文章中に列挙されている事項の「少なくとも1つ以上
」を採用できるときに使用される。「もしくは」も同様である。本明細書において「2つ
の値の範囲内」と明記した場合、その範囲には2つの値自体も含む。
本実施例では、免疫寛容を惹起するために投与される細胞製剤が反応すべきレスポンダー細胞と直接接触することが、抑制作用の発揮に必要であるかどうかを試験した。以下に手法を説明する。
(材料および方法)
(アナジー細胞の調製)
既に文献に記載された方法に従い図1に示す実験を行った(1-4)。概説すると、Promo cell社製Lymphocyte separation Media(Cat.No.C―44010)またはFicoll-Paque PREMIUM(GE Healthcare #17-5442-02)またはLymphocyte separation Solution(ナカライテスク#20828)等を用いて、4人のボランティア(2人をスティミュレイター、2人をレスポンダーとする)のヒト末梢血から単核細胞(PBMC)を分離し、4×106細胞/mlとなるよう2%ヒトAB型血清(プール)添加Biowest社製ALyS505N-0培地(細胞科学研究所(CSTI)1020P10)で調整した。スティミュレイターPBMCには放射線(γ線)30Gyを照射し、レスポンダーPBMCと1:1で混合した。上記にて混合したPBMCに、eBioscience社製マウス抗ヒトCD80抗体(2D10.4)(Cat.No.16-0809-85)とマウス抗ヒトCD86抗体(IT2.2)(Cat.No.16-0869-85)をそれぞれ最終濃度が10μg/mLになる様に添加し、12ウェルプレート(Corning、#3513)(1~2.5ml)、6ウェルプレート(Corning、Cat.No.3516)(3~6ml)、6cmシャーレ(Greiner CELLSTAR(登録商標)dish,Cat.No.628160)(3~6ml)、または10cmシャーレ(Corning、Cat.No.430167)(10~15ml)にて37℃、5%CO2インキュベーター中で培養を開始した(day0)。培養開始後5日目(day5)に遠心により培養液を除いた後に、放射線照射スティミュレイターPBMCと抗CD80抗体/抗CD86抗体を含む培養液を培養開始時と同じ条件で添加した。2日後(day7)に細胞を回収し遠心により培養液をウォッシュアウトし、アナジー細胞を得た。
得られたアナジー細胞またはレスポンダーPBMCを、放射線照射したスティミュレーターPMBCと1:1の比率に調整し、通称トランスウェルメンブレンと呼ばれる液性因子は透過するが、細胞は透過できない膜を有する上段のウェル中にて培養した。この下段には、新たに採取したレスポンダーPBMCおよび放射線照射スティミュレーターPBMCを各2×106細胞/mLで1:1に混合した(最終200μL/ウェルで4ウェルずつ)。これらの上段、下段のウェルを、96ウェルプレート(Corning社製、Cat.No.3381)上に組み、37℃、5%CO2インキュベーター中で培養した。
結果を図1右パネル上に示す。
本実施例では、マウスモデルを用いて、細胞製剤に由来する細胞がレシピエントから無くなってしまっても免疫寛容が続くことを実証した。以下にその手順を示す。
以下に本実施例で用いた方法および使用した材料を示す。
結果を図2および図3に示す。図2aに示すように、アナジー細胞の移入により、細胞数依存的に、移植した心臓の生着率が改善し、6×106個のアナジー細胞の移入により全てのマウスで100日以上の移植した心臓の生着が観察された、すなわち、移植した心臓に対する免疫寛容の誘導が見られた。しかし、図2bに示す様に、移入したアナジー細胞は、末梢血、リンパ節、脾臓のみならず、移植した心臓内においても、蛍光発現によっては検出されなかった。さらに図3aに示す様に、0.1%(1/1000希釈GFPマウスゲノムDNAの存在:レーン4)でも検出可能な、ゲノム遺伝子中のGFP遺伝子をPCRにより増幅するより感度の高い検出手法でも、図3bに示す様に、GFP遺伝子の発現は、移植後3日目までは末梢血、脾臓、腸間膜リンパ節、移植した心臓内のすべてにおいて確認できたものの、移植後7日目以降においては、いずれの組織においてもその遺伝子の発現は検出されなかった。このことは、アナジー細胞の移入により100日以上のドナー特異的な免疫抑制に由来する移植した心臓の生着(免疫寛容)が誘導されるものの、移入した細胞は1週間以内にレシピエント生体内で消失することを示唆している。
本実施例では、アナジー細胞が特異性を持ち、3rd party由来の抗原に対する拒絶を抑制しないことを実証した。
実施例2と同様の手法で、野生型B6マウス(H-2b)から得た脾臓細胞を、抗CD80/86抗体存在下でBALB/cマウス(H-2b)由来の脾臓細胞で刺激しアナジー細胞を得た。このアナジー細胞を、2Gyの放射線照射3日後にBALB/cマウスまたはCBAマウス(H-2k)の心臓を移植した野生型B6マウスに5×106個尾静脈から投与し、心臓の拒絶を観察した。
図4に示す様に、抗CD80/86抗体存在下でBalb/Cマウス脾臓細胞で刺激されたB6マウス由来のアナジー細胞のB6マウスへの移入は、移植されたBALB/cマウスの心臓の拒絶を100%阻害し100日後でも心臓が生着している。これに対して3rd partyであるCBAマウスの心臓は移植後に速やかに拒絶反応を引き起こし、約50日で100%拒絶される。以上から、抗CD80/86抗体存在下で抗原に反応させられたレシピエント由来のリンパ球は、刺激された抗原特異的に免疫反応の抑制能を有することが示された。
本実施例では、感染性免疫寛容が生じることを実証した。
実施例2と同様の手法でアナジー細胞を得て、図5に示す実験を行った。概説すると、蛍光色素GFPを全ての細胞が発現するよう遺伝子改変されたB6マウスから得た脾臓細胞を、抗CD80/86抗体存在下でBALB/cマウス由来の放射線照射した脾臓細胞で刺激しアナジー細胞を得た。このアナジー細胞を、2Gyの放射線照射4日後にBALB/cマウスの心臓を移植した野生型B6マウスに5×106個尾静脈から投与した。免疫寛容が誘導されたとみなされる心臓移植から100日以上経過した後に、レシピエントマウスを屠殺し、脾臓を摘出した後、赤血球を溶血し脾臓細胞を得た。このアナジー細胞から、CD8-T細胞アイソレーションキット(BioLegend 480035)、もしくはCD4-T細胞アイソレーションキット(BioLegend 480033)を用いて、レシピエントマウス由来のGFP陰性CD8陽性またはGFP陰性CD4陽性細胞を分離した。同様に、B6マウスから新たに取得した新鮮な(ナイーブ)脾臓細胞からも、CD8陽性またはCD4陽性細胞を同様に採取した。これら単離された細胞を、2Gyの放射線照射4日後にBALB/cマウスの心臓を移植した野生型B6マウスに4×106個尾静脈から投与し、心臓の拒絶を観察した。
結果を図6に示す。
本実施例では、ヒトインビトロモデルでのアロ特異的抑制性アナジー細胞の継代移入でも効果があることを実証した。
実施例1と同様の手法で、ヒト由来のアナジー細胞を得た(1-4)。概説すると、Promo cell社製Lymphocyte separation Media(Cat.No.C―44010)を用いて、4人のボランティア(2人をスティミュレイター、2人をレスポンダーとする)のヒト末梢血から単核細胞(PBMC)を分離し、4×106細胞/mLとなるよう2%ヒトAB型血清(プール)添加Biowest社製ALyS505N-0培地で調整した。スティミュレイターPBMCには放射線30Gyを照射し、レスポンダーPBMCと1:1で混合した。上記にて混合したPBMCに、eBioscience社製マウス抗ヒトCD80抗体(Cat.No.16-0809-85)とマウス抗ヒトCD86抗体(Cat.No.16-0869-85)をそれぞれ最終濃度が10μg/mLになる様に添加し、12ウェルプレート(Corning、#3513)、6ウェルプレート(Corning、Cat.No.3516)、6cmディッシュ(Greiner CELLSTAR(登録商標)dish、Cat.No.628160)または10cmディッシュ(Greiner CELLSTAR(登録商標)dish、Cat.No.664 160-013)にて37℃、5%CO2インキュベーター中で培養を開始した(day0)。培養開始後5日目(day5)に遠心により培養液を除いた後に、放射線照射スティミュレイターPBMCと抗CD80抗体/抗CD86抗体を含む培養液を培養開始時と同じ条件で添加した。その2日後(day7)に細胞を回収し遠心により培養液をウォッシュアウトし、アナジー細胞(1st)を得て、この細胞を蛍光色素CFSEでラベルした。
各混合培養系において培養後2日目に、CFSE陰性のレスポンダーCD4陽性細胞をソーティングにより単離し、IL-2、IL-10の発現mRNAレベルを定量的なリアルタイムPCR(qPCR)(TaqManTM)法にて解析した。
図7に示されるこの実験では、図8aの模式図に示す様な反応がin vitroで起きていると推定され、この機構により、アナジー細胞存在下で反応したナイーブ細胞に免疫抑制能が付与されると考えられる。
本実施例では、マウスにおいて、移植後後期(80日以降)には、CD8陽性細胞の反応性が失われ、CD4陽性T細胞を除去しても免疫寛容が誘導されることを確認した。
以下に、本実施例での手順および使用した材料を説明する。
結果を図9に示す。抗CD4抗体によるregT細胞を含むCD4陽性細胞の除去を、心移植後早期(3~24日)または中期(25~42日)に行った場合、移植した心臓の拒絶が起きるマウスが認められた。それに対して、後期(80~100日)においてregT細胞を含むCD4陽性細胞を除去した場合は、全てのマウスで移植した心臓の拒絶は見られなかった。すなわち、約42日後まではregT細胞を含むCD4陽性T細胞が免疫寛容に必要であり、これを枯渇させるとCD8陽性T細胞による移植した心臓の拒絶が誘導される。しかし、80日以降にはregT細胞を含むCD4陽性T細胞の必要性が無くなり、CD8陽性細胞の反応性自体が失われることが示唆された。すなわち、細胞治療を受けたレシピエントには、最終的には(80日以降には)免疫抑制性の細胞の存在なしでも免疫寛容が誘導されていることを示すものである。
本実施例では、ドナーに対するCD8陽性T細胞の反応を確認した。
実施例2と同様の手法で、野生型B6マウスから得た脾臓細胞を、抗CD80/86抗体存在下、BALB/c細胞で刺激しアナジー細胞を得た。このアナジー細胞を、2Gyの放射線照射3日後にBALB/cマウスの心臓を移植した野生型B6マウスに5×106個尾静脈から投与した。拒絶反応が見られずに移植した心臓が生着して100日以上経過した免疫寛容誘導マウスとナイーブマウスから脾臓細胞を得て、CFSE cell proliferation kit(Invitrogen C34554)を用いてCSFEでラベルした(図10a)。CSFEでラベルしたこの細胞を、BALB/c由来の放射線照射脾臓細胞と12ウェルプレート(Corning社製、Cat.No.3513)もしくは24ウェルプレート(corningCat.No3526)にて2×106細胞/mLで1:1で混合し、培養を行い、培養5日目のCD8陽性T細胞のCSFEの発現をフローサイトメーター(BD verse)により調べた(図10b)。
結果を図10に示す。
1.本開示のアナジー細胞は、レシピエントのナイーブ細胞に接触することにより免疫抑制能を発揮する。
2.移入した抑制性アナジーT細胞は検出不能になる。
3.移入した抑制性アナジー細胞によりレシピエント生体内で新たな免疫抑制能を持った細胞が誘導される。
4.最終的にドナー抗原特異的CD8陽性クローンの反応性は顕著に低下する。これは、クローンアナジーまたはクローン除去が誘導されている可能性を示している。
本実施例では、アレルギー性肺炎モデル(喘息)において免疫寛容が生じるか実証した。
図11に示す様に、既に文献に記載されたアレルギー性肺炎(喘息)モデルを用いて実験を行った(8)。
OVA(Sigma-Aldrich #O1641またはMBLライフサイエンス TS-5001-P等)100μgと抗マウスCD80/86抗体それぞれ250μgを同時に腹腔内投与して、OVAに感作させたB6マウスを5日後に屠殺し、脾臓を摘出し、溶血により脾臓細胞を得た。この脾臓細胞を、4×106細胞/mLとなるように10%FCSを含むRPMI1640培地にて調整し、ここにOVAを100μg/mL、CD80/86抗体をそれぞれ最終濃度10μg/mLとなる様に添加した。この脾臓細胞懸濁物を、37℃、5%CO2インキュベーター中で7日間培養して、OVA特異的アナジー細胞を得た。
アレルギー性肺炎(喘息)モデルとして、OVA100μgとアラムアジュバントを含むエマルジョンを0日と7日に腹腔内投与してOVAに感作させたマウスに、14日、17日、20日にOVA140μgを含む70μLのPBSを点鼻投与するモデルを使用した。誘導したアナジー細胞を14日目に1×107個尾静脈から投与し、24日目に気管支洗浄液(BAL:Bronchoalveolar Lavage)を1mlの生食水を用いた気管洗浄により回収し、そこに浸出した白血球数(CD45陽性細胞)と好酸球数(CD45陽性CD11b陽性SiglecF陽性細胞のCD45陽性細胞中の比率として算出)を測定した。加えて、BAL中のIL-4をELISAにより測定した(eBioscience #88-704-88)。
結果を図12に示す。図12aは気管支浸出液(BAL)中に含まれる白血球の数を示しており、図12bはBAL中に含まれる好酸球の数を示している。アナジー細胞の移入により、浸出する白血球数および好酸球が減っている。さらに図12cに示される様に、BAL中に含まれるアレルギー反応に関係するとされているIL-4も減少している。このことから、OVAによるアレルギー性肺炎(喘息)モデルにおいて、抗CD80/86抗体とOVAとの共培養で誘導されたアナジー細胞の移入が免疫寛容を誘導し、アレルギー反応を減弱することが示された。
本実施例では、食品アレルギーモデルにおける免疫寛容惹起を調べた。
図13に示すように、既に文献に記載された食品アレルギーモデルを用いて実験を行った(9)。概説すると、OVA100μgとアラムアジュバント(Thermo Fisher#77161)を含むエマルジョンを0日目と14日目に腹腔内投与してOVAに感作したマウスに、28日目から2~3日毎に7回、OVA50mgを経口投与して誘導する食物アレルギーモデルに、実施例8と同様の方法で得たアナジー細胞を14日目に5×106個尾静脈から投与した。36日後にマウスを屠殺した後、腸管を採取し、ビオチン標識抗FoxP3抗体(eBioscience #13-5773-829)とAlexa594標識ストレプトアビジン(Molecular Probes #S11227)とFITC標識抗CD4抗体(RM4-5, Molecular Probes #553047)を用いた免疫染色により腸管に存在するregT細胞を、HE染色により腸管に存在する好酸球(エオジン強染色細胞)を染色した。CD4陽性Foxp3陽性細胞密度は、蛍光顕微鏡Axioplan 2 imaging(Zeiss)で、CCDカメラAxioCam HRc(Zeiss)にて蛍光免疫染色画像を取得後、画像解析ソフトKS400(Zeiss)を用いて、測定対象領域面積の測定と同領域に存在する陽性細胞を計数し陽性細胞数/mm2を算出した。好酸球密度は、光学顕微鏡Axioskop 2 plus(Zeiss)で、CCDカメラAxioCam MRc(Zeiss)にてHE(ヘマトキシリンエオジン)染色画像を取得後、画像解析ソフトKS400(Zeiss)を用いて、測定対象領域面積の測定と同領域に存在するエオジン強陽性好酸球を計数し陽性細胞数/mm2を算出した
(結果)
結果を図14および15に示す。図14には典型的なFoxP3/CD4の免疫染色の結果を示す。アレルギー発症マウスが、マウス#11、#12(各腸管の異なる部位の画像3枚)、アナジー細胞移入マウスがマウス#21、#22(各腸管の異なる部位の画像3枚)、無処置の正常マウスがマウス#31、#32(各1枚)である。図15aには各マウスの典型的なHE染色の写真1枚ずつ示す。図14で示された画像の解析結果を数値化し、1mm2当たりのregT細胞の個数をグラフにしたものが図15bであり、図15aで示された画像の解析結果を数値化し、1mm2当たりの好酸球の個数をグラフにしたものが図15cである。
本実施例では、iPS細胞およびそれから分化した細胞・組織を用いた移植実験において生じた深刻な免疫拒絶反応を、本開示のアナジー細胞を用いて治癒し得るかどうかを実証する実験を行った。以下のように免疫寛容を惹起させた。以下説明する。
(iPS細胞からのニューロン分化~免疫寛容惹起実験)
既に文献(10)に記載された方法に従い、iPS細胞を神経細胞に分化させた。この神経細胞に放射線(γ線)30Gyを照射してスティミュレイター細胞として用いた。レスポンダー細胞には、MHCの異なるボランティアのヒト末梢血から得た単核細胞(PBMC)を用い、スティミュレイター細胞とレスポンダー細胞とが各々2×106細胞/mLとなるように2%ヒトAB型血清(プール)添加Biowest社製ALyS505N-0培地にて調整し、実施例1と同様に抗ヒトCD80抗体と抗ヒトCD86抗体各10μg/mL存在下で、12ウェルプレート(Corning社製、Cat.No.3513)にて37℃、5%CO2インキュベーター中で7日間培養した。7日後に細胞を回収し遠心により培養液をウォッシュアウトし、アナジー細胞を得た。
iPS細胞由来神経細胞(スティミュレイター細胞)との共培養で得られたアナジー細胞を、新たに採取した同一ボランティアのPBMCとの比率が1/2から1/16になるように希釈した後に、同一iPS細胞由来の放射線(γ線)30Gyを照射した神経細胞と、新たに採取した同一ボランティアのPBMCとを約1:1の細胞数で含む96ウェルプレート(Corning社製、Cat.No.3799)での混合培養系(各2×105細胞/200μL/ウェルで4ウェルずつ)に添加し、37℃、5%CO2インキュベーター中で培養した。培養開始4日目に3H-チミジン(10μL)を添加し、培養開始5日目(3H-チミジン添加の16~20時間後)にCell Harvester(Molecular Devices)により培養細胞を回収し、3H-チミジン取込み量をシンチレーションカウンターで測定した。
図16に示す様に、iPS細胞から分化した神経細胞は、MHCの一致しないボランティアのPBMCを刺激し増殖を誘導する。このiPS細胞から分化した神経細胞に反応して起きるリンパ球の増殖反応を、抗CD80/86抗体とiPS細胞から分化した神経細胞との共培養で誘導されたアナジー細胞はin vitroにおいて抑制することが示された。これはiPS細胞由来の細胞や組織を用いた移植において生じる免疫拒絶反応を本開示が減弱させ得ることが実証したものである。
本実施例では、様々な阻害因子を用いても免疫寛容を惹起することができることを示す。
基本的には、実施例1に記載の方法および既に文献に記載された方法に従い実験を行う(1~3)。レシピエントPBMCおよびドナーPBMCには、それぞれヒト末梢血から新鮮に分離したものを使用するか、または-80℃で凍結保存したものを急速解凍して使用し、これらの細胞はともに、自己血漿または10%非働化ウシ胎児血清(FCS)(SIGMA # 172012-500ML Lot 11D257またはbiosera #FB-1380/500 Lot.015BS482)を含むRPMI1640培地(Sigma;R8758-500MK)にて4×106細胞/mLに調整する。ドナーPBMCには、20Gy放射線を照射しておく。これらのレシピエントPBMCとドナーPBMCとを、1:1で混合し、この混合物に、阻害因子(例えば、抗CD80抗体/抗CD86抗体では、それぞれ最終濃度10μg/ml、ベラタセプト(またはアバタセプト)では、最終濃度10μg/ml~40μg/ml)を添加する。培養は、6cmシャーレ(Greiner CELLSTAR(登録商標)dish,Cat.No.628160)(培養体積3~6mL)または10cmシャーレ(Corning、Cat.No.430167)(培養体積10~15mL)にて37℃、5%CO2インキュベーター中で7日間行う(培養開始時の細胞密度は、4×106細胞/mL)。
培養開始から通算して14日目に遠心により誘導細胞を回収し、基本的には既に文献に記載された方法に従いリンパ球混合試験を行った(3)。細胞懸濁物を、37℃、5%CO2インキュベーターで共培養する。共培養開始4日目に3H-チミジン(10μl)を添加し、共培養開始5日目(3H-チミジン添加の16~20時間後)に培養液中の3H-チミジンを除去し、3H-チミジン取込み量を測定し、免疫反応抑制能を確認することができる。
細胞製剤の製法については、上述の実施例1~10における記載を参照のこと。実施例に準じて製造された細胞製剤について、以下のように品質管理を行う。
生理食塩液中に懸濁させたアナジー細胞を、外観について目視で調べる。品質規格に合致する懸濁液は、微黄白色~淡黄色の細胞からなるはずである。
アナジー細胞を、各表現型についてフローサイトメトリーで調べるために、例えば以下の抗体を使用し、多重染色により解析する:
CD3:FITC蛍光標識抗ヒトCD3抗体(UCHT1;eBioscience #11-0038-42)またはPacific Blue蛍光標識抗ヒトCD3抗体(UCHT1;Invitrogen #CD0328)
CD4:PE蛍光標識抗ヒトCD4抗体(RPA-T4;eBioscience #25-0049-42)
CD8:APC蛍光標識抗ヒトCD8抗体(RPA-T8;eBioscience #17-0088-42)
CD25:PerCP蛍光標識抗ヒトCD25抗体 (MEM-181;eBioscience #A15802)
CD44:PE-Cy7蛍光標識抗ヒトCD44抗体(IM7;eBioscience #25-0441-82)
CD45:Brilliant Violet蛍光標識抗ヒトCD45抗体(HI30;BioLegend #304032)
CD45RA:FITC蛍光標識抗CD45RA抗体(ALB11;Beckman Coulter A07786)またはPE蛍光標識抗CD45RA抗体(ALB11;Beckman Coulter IM1834U)
CD45RO:ECD蛍光標識抗CD45RO抗体(UCHL1;Beckman Coulter IM2712U)またはPE蛍光標識抗CD45RO抗体(UCHL1;Beckman Coulter A07787)またはAPC蛍光標識抗CD45RO抗体(UCHL1;Bay biosciences 20-0457)
(手順)
CD3陽性細胞比率、生細胞中のCD45陽性細胞比率、CD3陽性細胞中のCD8陽性CD44陽性細胞比率、CD3陽性細胞中のCD4陽性CD44陽性細胞比率、CD3陽性細胞中のCD8陽性CD45RA陰性細胞比率、CD3陽性細胞中のCD8陽性CD45RA陰性CD45RO陽性細胞比率、CD3陽性細胞中のCD4陽性CD45RA陰性CD45RO陽性細胞比率およびCD3陽性細胞中のCD4陽性CD25陽性細胞比率
生理食塩液中に懸濁させたアナジー細胞を、上記抗体に反応させた後に、Zombie NIR Fixable Viability Kit(BioLgened #423106)を用いて死細胞を染色する。この多重蛍光染色された細胞をFACS Verse (BD Bioscience社)において、全生細胞中のCD3陽性細胞の比率を決定する。
基本的には、日本薬局方または該当する各国の薬局方の記載に準拠して試験を行う。以下に、例示的な実施形態を述べる。
アナジー細胞浮遊液を軽く遠心分離し、その上清を、無菌試験に供する。日本薬局方における代表的な無菌試験の一つである直接法では、上清を、ソイビーン・カゼイン・ダイジェスト培地または液状チオグリコール酸培地に接種し、それぞれ30~35℃または20~25℃で14日間以上培養する。その後、培養物を培養期間中に数回観察する。別の代表的な無菌試験であるメンブランフィルター法では、上清を無菌希釈液(例えば、1g/Lの肉製又はカゼイン製ペプトン溶液(pH7.1±0.2))で希釈し、希釈した上清をメンブランフィルター上に移し、濾過する。その後そのメンブランフィルターを、上述の2種類の培地にそれぞれ入れ、14日間以上培養する。品質規格に合致する製品では、培養期間中及び最終日に、培地に肉眼的な微生物の増殖は存在しない。
アナジー細胞浮遊液を、適宜生理食塩液で希釈し、pH6.0~8.0に調製する。その後、ライセート試薬と混合し、ライセート試液のゲル形成を指標として(ゲル化法)、またはライセート試液のゲル化過程における濁度変化を指標として(比濁法)もしくは合成基質の加水分解による発色を指標として(比色法)、試料中のエンドトキシン濃度を定量的に求める。必要に応じて、ライセート試薬の表示感度を確認する予備試験を行う。品質規格に合致する製品では、エンドトキシン濃度は、0.25EU/mL未満にならなければならない。
培養液、またはアナジー細胞浮遊液を軽く遠心分離し、その上清を、マイコプラズマ否定試験に供する。日本薬局方における代表的なマイコプラズマ否定試験の一つである培養法では、試料を、寒天平板培地に接種し、5~10%の炭酸ガスを含む窒素ガス中で、適切な湿度のもと35~37℃で14日間以上培養する、あるいは、試料を、液体培地を入れた容器に接種し、35~37℃で培養し、液体培地の色調変化を観察したときか、または培養開始から一定期間ごとに液体培養物からアリコートを取り、新たな寒天平板培地に播いて、培養を続ける。その後、全ての寒天平板培地を対象に7日目と14日目に倍率100倍以上の顕微鏡でマイコプラズマの集落の有無を調べる。別の代表的なマイコプラズマ否定試験である指標細胞を用いたDNA染色法では、典型的には、指標細胞であるVero細胞と指定のマイコプラズマ菌株を用いる。この方法では、カバーグラスを沈めた培養ディッシュなどに指標細胞を接種し、5%炭酸ガスを含む空気中、35~38℃で一日増殖させる。その後試料(培養液または上清)を添加して、同様の条件で3~6日間培養を続ける。カバーグラス上の培養細胞を固定後、ビスベンズイミド等の染色剤によりDNA蛍光染色し、蛍光顕微鏡(倍率400~600倍またはそれ以上の倍率)で検鏡し、陰性(非接種)対照及びマイコプラズマ陽性対照と比較しながら、細胞核を囲むように微小な核外蛍光斑点を持つ細胞が0.5%以上存在した場合には、マイコプラズマ陽性と判断する。品質規格に合致する製品では、マイコプラズマ陰性であるべきである。
生理食塩液中に懸濁させたアナジー細胞を、血球計算盤を用いて顕微鏡下で、または自動セルカウンターにより細胞数を測定する。品質規格に合致する、投与に適切なアナジー細胞の数は、1×108~30×108個(例えば、100mL生理食塩液中)であり、この範囲を下回る場合には、適宜細胞を追加すべきである。
生理食塩液中に懸濁させたアナジー細胞を、0.3~0.5%トリパンブルー染色液(例えば、カタログ#35525-02、ナカライテスク)と混合し、血球計算盤を用いて顕微鏡下で、または自動セルカウンターにより生細胞数を測定する。品質規格に合致する製品では、70%以上の細胞が生細胞であるべきである。
以下の参考文献は、実施例等において基本技術として参照したものであり、これらの文献が本開示に対して先行技術を構成することを認めるものではない。これらの内容は参考として援用される。
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10. Matsumoto T, Fujimori K, Andoh-Noda T, Ando T, Kuzumaki N, Toyoshima M, et al. Functional Neurons Generated from T Cell-Derived Induced Pluripotent Stem Cells for Neurological Disease Modeling. Stem cell reports. 2016;6(3):422-35.
(注記)
以上のように、本開示の好ましい実施形態を用いて本開示を例示してきたが、本開示は、特許請求の範囲によってのみその範囲が解釈されるべきであることが理解される。本明細書において引用した特許、特許出願及び他の文献は、その内容自体が具体的に本明細書に記載されているのと同様にその内容が本明細書に対する参考として援用されるべきであることが理解される。本出願は、日本国特許出願特願2018-119003(2018年6月22日出願)に対して優先権主張の利益を主張するものであり、当該出願の内容は本願において、その内容が(全部であり得る)参考として援用されることが理解される。また、日本国特許出願特願2018-118996および特願2018-119001(いずれも2018年6月22日出願)ならびにそれらに対して優先権主張する国際出願の内容は、その一部または全文が、本明細書において参考として援用される。
Claims (44)
- 被験体において、ドナーに対する恒久的な免疫寛容(感染性免疫寛容)を惹起するための組成物であって、該組成物は、
CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、該ドナーに由来する抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞
を含む、組成物。 - 前記免疫寛容は、前記被験体におけるCD8陽性T細胞において免疫寛容が惹起されている、請求項1に記載の組成物。
- 前記阻害因子が、低分子、タンパク質、核酸、脂質、糖、およびこれらの組み合わせからなる群から選択される、請求項2に記載の組成物。
- 前記タンパク質が、抗体もしくはその改変体、または細胞表面分子もしくはその改変体である、請求項2に記載の組成物。
- 前記抗体の改変体が、抗原結合断片である、請求項4に記載の組成物。
- 前記細胞表面分子の改変体が、融合タンパク質である、請求項4に記載の組成物。
- 前記阻害因子は、抗CD80抗体、抗CD86抗体、CD80およびCD86に対する二重特異性抗体、抗CD28抗体もしくはこれらの抗原結合断片、CTLA4-Ig融合タンパク質、CD28-Ig融合タンパク質からなる群より選択される、請求項3~6のいずれか一項に記載の組成物。
- 前記CTLA4-Ig融合タンパク質は、アバタセプトまたはベラタセプトである、請求項7に記載の組成物。
- 被験体において、疾患、障害または状態を予防または治療する方法であって、該方法は、
1) CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、該被験体由来の抗原あるいは該被験体に由来しない抗原または該抗原の含有物とを混合することによってアナジーが誘導された細胞を含む製剤を該被験体に投与する工程と、
2) 該被験体のT細胞のアナジー状態を確認し、アナジー状態が確認できる場合はさらなる処置をせず、アナジー状態が確認できない場合、該細胞を含む製剤を再度投与する工程と
を含む、方法。 - 前記製剤は、CD4陽性アナジー細胞、CD8陽性アナジー細胞、またはそれらの組合せを含む、請求項9に記載の方法。
- 前記製剤は、CD8陽性アナジー細胞を含む、請求項10に記載の方法。
- 前記アナジー状態の確認は、前記細胞を含む製剤が消失することを確認する工程を含む、請求項9に記載の方法。
- 前記疾患、障害または状態は、移植免疫拒絶反応、アレルギー、自己免疫疾患、移植片対宿主病、およびiPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応からなる群より選択される、請求項9に記載の方法。
- 前記疾患、障害または状態はアレルギーを含む、請求項9に記載の方法。
- 前記疾患、障害または状態は、iPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応を含む、請求項9に記載の方法。
- 前記阻害因子が、低分子、タンパク質、核酸、脂質、糖、およびこれらの組み合わせからなる群から選択される、請求項15に記載の方法。
- 前記タンパク質が、抗体もしくはその改変体、または細胞表面分子もしくはその改変体である、請求項16に記載の方法。
- 前記抗体の改変体が、抗原結合断片である、請求項17に記載の方法。
- 前記細胞表面分子の改変体が、融合タンパク質である、請求項17に記載の方法。
- 前記阻害因子は、抗CD80抗体、抗CD86抗体、CD80およびCD86に対する二重特異性抗体、抗CD28抗体もしくはこれらの抗原結合断片、CTLA4-Ig融合タンパク質、CD28-Ig融合タンパク質からなる群より選択される、請求項16~19のいずれか一項に記載の方法。
- 前記CTLA4-Ig融合タンパク質は、アバタセプトまたはベラタセプトである、請求項20に記載の方法。
- 被験体のアレルギーを治療または予防するための組成物であって、該組成物は、
CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、アレルギーの原因となっている抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞
を含む、組成物。 - 前記アレルギーの原因となっている抗原は、食品、花粉、薬品、および金属から選択される、請求項22に記載の組成物。
- 被験体において、iPS細胞またはES細胞またはそれらに由来する細胞、組織もしくは臓器によって生じる免疫拒絶反応を抑制するまたは予防するための組成物であって、該組成物は、
CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、該iPS細胞またはES細胞に由来する抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞
を含む、組成物。 - 制御性T細胞ではない、アナジーが誘導されたT細胞。
- 制御性T細胞ではない、被験体に対してアナジーが誘導されたT細胞を製造する方法であって、
A)CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、該被験体由来の細胞と、該被験体に由来しない抗原または該抗原の含有物とを混合する工程と、
B)該混合物を培養してT細胞を得る工程と、
C)必要に応じて、該被験体に由来しない抗原または該抗原の含有物で該T細胞を刺激し、該T細胞が反応しないことを確認する工程と
を包含する、方法。 - 前記阻害因子が、低分子、タンパク質、核酸、脂質、糖、およびこれらの組み合わせからなる群から選択される、請求項26に記載の方法。
- 前記タンパク質が、抗体もしくはその改変体、または細胞表面分子もしくはその改変体である、請求項27に記載の方法。
- 前記抗体の改変体が、抗原結合断片である、請求項28に記載の方法。
- 前記細胞表面分子の改変体が、融合タンパク質である、請求項28に記載の方法。
- 前記阻害因子は、抗CD80抗体、抗CD86抗体、CD80およびCD86に対する二重特異性抗体、抗CD28抗体もしくはこれらの抗原結合断片、CTLA4-Ig融合タンパク質、CD28-Ig融合タンパク質からなる群より選択される、請求項27~30のいずれか一項に記載の方法。
- 前記CTLA4-Ig融合タンパク質は、アバタセプトまたはベラタセプトである、請求項31に記載の方法。
- 前記T細胞は、CD4陽性細胞および/またはCD8陽性細胞を含む、請求項25に記載のT細胞または請求項26に記載の方法。
- 特定の抗原に対する、被験体内のCD8陽性T細胞の無反応または低反応を誘導するための組成物。
- CD80および/またはCD86と、CD28との相互作用を阻害することができる阻害因子と、被験体由来の細胞と、該被験体由来の抗原あるいは該験検体に由来しない抗原または該抗原の含有物とを混合することによって免疫寛容が誘導された細胞を含む、該被験体に由来しない抗原によって生じる疾患、障害または状態を処置または予防するための医薬。
- 前記阻害因子が、低分子、タンパク質、核酸、脂質、糖、およびこれらの組み合わせからなる群から選択される、請求項35に記載の医薬。
- 前記タンパク質が、抗体もしくはその改変体、または細胞表面分子もしくはその改変体である、請求項36に記載の医薬。
- 前記抗体の改変体が、抗原結合断片である、請求項37に記載の医薬。
- 前記細胞表面分子の改変体が、融合タンパク質である、請求項37に記載の医薬。
- 前記阻害因子は、抗CD80抗体、抗CD86抗体、CD80およびCD86に対する二重特異性抗体、抗CD28抗体もしくはこれらの抗原結合断片、CTLA4-Ig融合タンパク質、CD28-Ig融合タンパク質からなる群より選択される、請求項36~39のいずれか一項に記載の医薬。
- 前記CTLA4-Ig融合タンパク質は、アバタセプトまたはベラタセプトである、請求項40に記載の医薬。
- 前記疾患、障害または状態は、移植免疫拒絶反応、アレルギー、自己免疫疾患、移植片対宿主病、およびiPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応からなる群より選択される、請求項36に記載の医薬。
- 前記疾患、障害または状態はアレルギーを含む、請求項36に記載の医薬。
- 前記疾患、障害または状態は、iPS細胞もしくはES細胞またはそれらに由来する細胞、組織もしくは臓器の移植によって引き起こされる免疫拒絶反応を含む、請求項36に記載の医薬。
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