Antibody for inducing immune tolerance using cell mixture having complex state, and induced lymphocyte, and cell therapeutic agent and therapeutic method using induced lymphocyte

文档序号：751310 发布日期：2021-04-02 浏览：27次中文

阅读说明：本技术 使用具有复合状态的细胞混合物诱导免疫耐受的抗体、以及所诱导的淋巴细胞、以及使用所诱导的淋巴细胞的细胞治疗剂和治疗法 (Antibody for inducing immune tolerance using cell mixture having complex state, and induced lymphocyte, and cell therapeutic agent and therapeutic method using induced lymphocyte ) 是由内田浩一郎竹田和由奥村康于 2019-06-21 设计创作，主要内容包括：本发明提供用于抗原特异性的免疫耐受或免疫抑制的药物组合物。本发明提供包含CD4阳性无反应性T细胞、及CD8阳性无反应性T细胞的药物组合物。在一些实施方式中,无反应性T细胞是通过能够抑制CD80和/或CD86与CD28的相互作用的抗体而诱导的。在特定的实施方式中,药物组合物可以还包含调节T细胞(例如,FOXP3阳性CD4阳性CD25阳性T细胞)。(The present invention provides pharmaceutical compositions for antigen-specific immune tolerance or immune suppression. The present invention provides pharmaceutical compositions comprising CD 4-positive anergic T cells, and CD 8-positive anergic T cells. In some embodiments, anergic T cells are induced by an antibody capable of inhibiting the interaction of CD80 and/or CD86 with CD 28. In particular embodiments, the pharmaceutical composition may further comprise regulatory T cells (e.g., FOXP 3-positive CD 4-positive CD 25-positive T cells).)

1. A pharmaceutical composition comprising:

CD4 positive anergic T cells, and

CD8 positive anergic T cells.

2. The pharmaceutical composition of claim 1, wherein said anergic T cells are induced by an inhibitor capable of inhibiting the interaction of CD80 and/or CD86 with CD 28.

3. The pharmaceutical composition of claim 2, wherein the inhibitory factor is selected from the group consisting of a small molecule, a protein, a nucleic acid, a lipid, a sugar, and combinations thereof.

4. The pharmaceutical composition of claim 3, wherein the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.

5. The pharmaceutical composition of claim 4, wherein the variant of the antibody is an antigen-binding fragment.

6. The pharmaceutical composition of claim 4, wherein the variant of the cell surface molecule is a fusion protein.

7. The pharmaceutical composition of any one of claims 3-6, wherein the inhibitory factor is selected from the group consisting of an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, a CD28-Ig fusion protein.

8. The pharmaceutical composition of claim 7, wherein the CTLA4-Ig fusion protein is abatacept or belief.

9. The pharmaceutical composition of any one of claims 1-8, further comprising regulatory T cells.

10. The pharmaceutical composition of any one of claims 1-9, wherein the regulatory T cells are FOXP3 positive CD4 positive CD25 positive.

11. A pharmaceutical composition comprising cells that have been induced to anergy by an inhibitor capable of inhibiting the interaction of CD80 and/or CD86 with CD28, the composition comprising CD8 positive cells, the composition further comprising at least 1 or more of FOXP3 positive cells and CD4 positive cells.

12. The pharmaceutical composition of claim 5, comprising all of FOXP3 positive cells, CD4 positive cells, and CD8 positive cells.

13. The pharmaceutical composition of claim 11 or 12, wherein the CD8 positive cells are CD44 positive.

14. The pharmaceutical composition of claim 11 or 12, wherein the CD8 positive cells are CD45RA negative and CD45RO positive.

15. The pharmaceutical composition of any one of claims 11-14, wherein the FOXP3 positive cells are CD4 positive.

16. The pharmaceutical composition of any one of claims 11-15, wherein the FOXP3 positive cells are CD25 positive.

17. The pharmaceutical composition of any one of claims 1 to 16, wherein the pharmaceutical composition is a pharmaceutical composition for antigen-specific immune tolerance or immune suppression.

18. The pharmaceutical composition of claim 3, wherein the antibody comprises an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody, or a combination thereof.

19. The pharmaceutical composition of any one of claims 2 to 18, wherein the cell is a cell induced by,

in the step, the inhibitor, cells derived from a sample, and an antigen derived from the sample, an antigen not derived from the sample, or a substance containing the antigen are mixed.

20. A pharmaceutical composition for treating or preventing a disease, disorder or condition of a subject arising from an antigen derived from or not derived from the subject, comprising the composition of any one of claims 1 to 19.

21. The pharmaceutical composition of claim 20, wherein the disease, disorder or condition is selected from the group consisting of transplant immune rejection, allergy, autoimmune disease, graft-versus-host disease, immune rejection caused by transplantation of iPS cells or ES cells and cells, tissues or organs derived from these cells.

22. The pharmaceutical composition of claim 21, wherein the transplant immune rejection response is produced by transplanting kidney, liver, heart, skin, lung, pancreas, esophagus, stomach, small intestine, large intestine, nerve, blood cells including cells of the immune system, bone, cartilage, blood vessel, cornea, eyeball, or bone marrow.

23. The pharmaceutical composition of any one of claims 20 to 22, wherein the antigen-containing substance is a cell.

24. A method of manufacturing a medicament comprising cells, the method comprising the steps of:

(A) mixing an inhibitor capable of inhibiting the interaction between CD80 and/or CD86 and CD28, cells derived from a sample, and an antigen derived from the sample, an antigen not derived from the sample, or a substance containing the antigen;

(B) confirming that the cell product obtained by the mixing contains CD 8-positive cells; and

25. The method of claim 24, wherein the inhibitor is selected from the group consisting of a small molecule, a protein, a nucleic acid, a lipid, a carbohydrate, and combinations thereof.

26. The method of claim 25, wherein the protein is an antibody or variant thereof, or is a cell surface molecule or variant thereof.

27. The method of claim 26, wherein the variant of the antibody is an antigen-binding fragment.

28. The method of claim 26, wherein the variant of the cell surface molecule is a fusion protein.

29. The method of any one of claims 25 to 28, wherein the inhibitory factor is selected from the group consisting of an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, a CD28-Ig fusion protein.

30. The method of claim 29, wherein the CTLA4-Ig fusion protein is abatacept or belazept.

31. The method of claim 17, wherein the presence of CD8 positive cells and the presence of at least 1 of FOXP3 positive cells and CD4 positive cells in the cell product indicates that the cell product can be used as a medicament.

32. The method according to any one of claims 24 to 31, wherein the drug is used for treating or preventing a disease, disorder or condition of the subject arising from an antigen derived from or not derived from the subject.

33. The method of any one of claims 24 to 32, wherein the step (B) comprises detecting CD8 by an anti-CD 8 antibody; the process (C) includes detecting at least 1 of FOXP3 and CD4 by at least 1 of an anti-FOXP 3 antibody and an anti-CD 4 antibody.

34. The method of claim 33, wherein the detecting is performed by FACS.

35. A method of managing the quality of a drug containing cells for treating or preventing a disease, disorder or condition of a specimen due to an antigen expressed by cells derived from the specimen or an antigen not derived from the specimen, comprising the steps of:

(A) confirming that the cells contain CD8 positive cells; and

(B) confirming that the cell comprises at least 1 cell selected from the group consisting of FOXP 3-positive cells and CD 4-positive cells.

36. The method of claim 35, wherein said procedure (a) comprises detecting CD8 by an anti-CD 8 antibody; the process (B) comprises detecting at least 1 of FOXP3 and CD4 by at least 1 of an anti-FOXP 3 antibody and an anti-CD 4 antibody.

37. The method of claim 36, wherein the detecting is performed by FACS, western blot, or PCR.

38. A composition comprising an inhibitor, which is an inhibitor capable of inhibiting the interaction of CD80 and/or CD86 with CD28, for use in the manufacture of a pharmaceutical composition according to any one of claims 1 to 23.

39. The composition of claim 38, wherein the inhibitor is selected from the group consisting of a small molecule, a protein, a nucleic acid, a lipid, a sugar, and combinations thereof.

40. The composition of claim 39, wherein the protein is an antibody or variant thereof, or is a cell surface molecule or variant thereof.

41. The composition of claim 40, wherein the variant of the antibody is an antigen-binding fragment.

42. The composition of claim 40, wherein the variant of the cell surface molecule is a fusion protein.

43. The composition of any one of claims 39-42, wherein the inhibitory factor is selected from the group consisting of an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, a CD28-Ig fusion protein.

44. The composition of claim 43, wherein the CTLA4-Ig fusion protein is acalep or belief.

45. A kit for use in the manufacture of a medicament comprising a mixture of cells, the kit comprising:

(A) an inhibitor capable of inhibiting the interaction of CD80 and/or CD86 with CD 28;

(B) means for detecting CD 8; and

46. The kit of claim 45, wherein the inhibitory factor is selected from the group consisting of a small molecule, a protein, a nucleic acid, a lipid, a carbohydrate, and combinations thereof.

47. The kit of claim 46, wherein the protein is an antibody or variant thereof, or is a cell surface molecule or variant thereof.

48. The method of claim 47, wherein the variant of the antibody is an antigen-binding fragment.

49. The method of claim 47, wherein the variant of the cell surface molecule is a fusion protein.

50. The kit or method of any one of claims 46 to 49, wherein the inhibitory factor is selected from the group consisting of an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, a CD28-Ig fusion protein.

51. The kit or method of claim 51, wherein the CTLA4-Ig fusion protein is abacavir or belief.

52. The kit of any one of claims 45 to 52, wherein the presence of CD 8-positive cells and the presence of at least 1 of FOXP 3-positive cells and CD 4-positive cells in the cell mixture indicates that the cell mixture is capable of being used as a medicament.

53. The kit according to any one of claims 45 to 53, wherein the drug is used for treating or preventing a disease, disorder or condition of the subject caused by an antigen derived from or not derived from the subject.

54. A kit for managing the quality of a cell-containing drug for treating or preventing a disease, disorder or condition of a subject arising from an antigen expressed by cells derived from the subject or an antigen not derived from the subject, comprising:

(A) means for detecting CD 8; and

(B) a unit for detecting at least 1 of FOXP3 and CD 4.

55. The kit of any one of claims 45 to 55, wherein the means for detecting CD8 comprises an anti-CD 8 antibody and the means for detecting at least 1 of FOXP3 and CD4 comprises at least 1 of an anti-FOXP 3 antibody and an anti-CD 4 antibody.

56. The kit of claim 56, wherein the detection is by FACS, Western blot or PCR.

Technical Field

The present invention relates to a novel technology relating to immune tolerance. More specifically, the present invention relates to a pharmaceutical composition comprising an anergic T cell, the manufacture of the pharmaceutical composition, and the quality management of the pharmaceutical composition.

Background

Liver transplantation has been widely used as a final therapy for patients with advanced liver failure. There are over 20000 cases in japan and over 500 cases in japan each year.

Transplantation is one of the major treatment methods of choice for advanced kidney, heart, liver, pancreas organ failure, and although significant advances have been made in recent years in the treatment of transplant rejection, most transplants are eventually rejected without immunosuppressive regimens. With current immunosuppressive regimens that rely on continuous drug therapy, the drug inhibits not only a significant response to transplantation, but all immune responses, thus making organ transplant patients more susceptible to increased susceptibility to infectious diseases, cancer.

Although regenerative medicine has also attracted attention, even if induced pluripotent stem cells (iPS cells) or the like are used, immune rejection may occur as long as the cells are not autologous cells at all, and thus a technique of immune tolerance has attracted attention.

As a technique for inducing immune tolerance, there is a technique of inducing antigen-specific immune unresponsiveness (anergy) of T cells. Specifically, it is reported that: a technique of directly administering an antibody that inhibits the interaction of CD80/CD86 on antigen presenting cells and CD28 on non-activated (naive) T cells to an organ transplant patient, thereby inducing donor antigen-specific anergy in vivo (patent document 1); a technique of co-culturing recipient cells and donor cells irradiated with radiation in the presence of the same antibody to induce donor antigen-specific non-reactive cells in vitro and return the cells to the recipient (patent documents 2, 3, and non-patent documents 1 to 3).

Non-patent document 1 reports: in the technique of inducing donor antigen-specific anergic cells in vitro and returning them to the recipient, even if CD 8-positive cells were removed, immunosuppression was hardly affected.

Documents of the prior art

Patent document

Patent document 1: japanese Kohyo publication No. 2002-504120

Patent document 2: japanese Kokai publication No. 2007-131598

Patent document 3: japanese examined patent publication No. 2016-520081

Non-patent document

Non-patent document 1: satoru Todo et al, hepatology, 64(vol.2), 632-

Non-patent document 2: teraoka S, Koyama I, Bashuda H, Uchida K, Tonsho M, et al (2017) J vector Res 2(1) p1-8

Non-patent document 3: bashuda H et al, j.clin.invest.115: 1896-1902(2005).

Disclosure of Invention

Means for solving the problems

The inventor and the like discover for the first time that: even if T cell anergy is induced by using an inhibitor such as an antibody that inhibits the interaction between CD80/CD86 and CD28, the ability to induce immune tolerance is significantly reduced in the absence of CD8 positive cells in the anergy-induced T cell mixture compared to the presence of CD8 positive cells. The inventors found that CD8 positive cells play an important role in inducing immune tolerance.

Accordingly, the present invention provides the following.

(1) A pharmaceutical composition comprising CD 4-positive anergic T cells, and CD 8-positive anergic T cells.

(2) The pharmaceutical composition according to the preceding item, wherein the aforementioned anergic T cells are induced by an inhibitor capable of inhibiting the interaction of CD80 and/or CD86 with CD 28.

(3) The pharmaceutical composition according to any of the preceding items, wherein the aforementioned inhibitory factor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars and combinations thereof.

(4) The pharmaceutical composition of any of the above items, wherein the aforementioned protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.

(5) The pharmaceutical composition of any of the preceding items, wherein the variant of the aforementioned antibody is an antigen-binding fragment.

(6) The pharmaceutical composition of any of the preceding items, wherein the variant of the aforementioned cell surface molecule is a fusion protein.

(7) The pharmaceutical composition of any of the preceding items, wherein the aforementioned inhibitory factor is selected from the group consisting of an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, a CD28-Ig fusion protein.

(8) The pharmaceutical composition according to any of the preceding items, wherein the CTLA4-Ig fusion protein is abatacept or belicept.

(9) The pharmaceutical composition according to any one of items 1 to 8, further comprising regulatory T cells.

(10) The pharmaceutical composition according to any of the preceding items, wherein the aforementioned regulatory T cells are FOXP3 positive CD4 positive CD25 positive.

(11) A pharmaceutical composition comprising cells that have been induced to anergy by an inhibitor capable of inhibiting the interaction of CD80 and/or CD86 with CD28, the composition comprising CD8 positive cells, the composition further comprising at least 1 or more of FOXP3 positive cells and CD4 positive cells.

(12) The pharmaceutical composition of any one of the above items, comprising all of FOXP 3-positive cells, CD 4-positive cells, and CD 8-positive cells.

(13) The pharmaceutical composition of any of the above items, wherein the aforementioned CD8 positive cells are CD44 positive.

(14) The pharmaceutical composition of any of the above items, wherein the aforementioned CD8 positive cells are CD45RA negative and CD45RO positive.

(15) The pharmaceutical composition of any of the above items, wherein the FOXP3 positive cells are CD4 positive.

(16) The pharmaceutical composition of any of the above items, wherein the FOXP3 positive cells are CD25 positive.

(17) The pharmaceutical composition according to any one of the preceding items, wherein the pharmaceutical composition is a pharmaceutical composition for antigen-specific immune tolerance or immune suppression.

(18) The pharmaceutical composition of any of the above items, wherein the aforementioned antibodies comprise an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody, or a combination thereof.

(19) The pharmaceutical composition according to any one of the above items, wherein the cells are induced by mixing the inhibitor, cells derived from a sample, and an antigen derived from the sample, an antigen not derived from the sample, or a substance containing the antigen.

(20) A pharmaceutical composition for treating or preventing a disease, disorder or condition of a subject caused by an antigen derived from or not derived from the subject, comprising the composition of any one of the above items.

(21) The pharmaceutical composition according to any of the preceding items, wherein the aforementioned disease, disorder or condition is selected from the group consisting of transplant immune rejection, allergy, autoimmune disease, graft-versus-host disease, immune rejection caused by transplantation of iPS cells or ES cells and cells, tissues or organs derived from these cells.

(22) The pharmaceutical composition according to any of the preceding items, wherein the transplant immune rejection is produced by transplanting kidney, liver, heart, skin, lung, pancreas, esophagus, stomach, small intestine, large intestine, nerve, blood cells including cells of the immune system, bone, cartilage, blood vessel, cornea, eyeball, or bone marrow.

(23) The pharmaceutical composition according to any of the preceding items, wherein the antigen-containing substance is a cell.

(24) A method of manufacturing a medicament comprising cells, the method comprising the steps of:

(B) confirming that the cell product obtained by the mixing contains CD 8-positive cells; and

(25) The method according to any one of the preceding items, wherein the aforementioned inhibitory factor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars and combinations thereof.

(26) The method according to any one of the preceding items, wherein the aforementioned protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.

(27) The method of any one of the preceding items, wherein the variant of the aforementioned antibody is an antigen-binding fragment.

(28) The method of any one of the preceding items, wherein the variant of the cell surface molecule is a fusion protein.

(29) The method according to any of the preceding items, wherein the aforementioned inhibitor is selected from the group consisting of an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, a CD28-Ig fusion protein.

(30) The method of any one of the preceding items, wherein the CTLA4-Ig fusion protein is abatacept or belicept.

(31) The method of any one of the preceding items, wherein the presence of CD 8-positive cells and the presence of at least 1 of FOXP 3-positive cells and CD 4-positive cells in the aforementioned cell product indicates that the aforementioned cell product can be used as a medicament.

(32) The method according to any one of the preceding items, wherein the drug is used for treating or preventing a disease, disorder or condition of the subject caused by an antigen derived from or not derived from the subject.

(33) The method according to any one of the preceding items, wherein the step (B) comprises detecting CD8 by an anti-CD 8 antibody; the aforementioned process (C) includes detecting at least 1 of FOXP3 and CD4 by at least 1 of the anti-FOXP 3 antibody and the anti-CD 4 antibody.

(34) The method according to any of the preceding items, wherein the detection is performed by FACS.

(35) A method of managing the quality of a drug containing cells for treating or preventing a disease, disorder or condition of a specimen due to an antigen expressed by cells derived from the specimen or an antigen not derived from the specimen, comprising the steps of: (A) confirming that the cells contain CD8 positive cells; and (B) confirming that the cells contain at least 1 cell selected from the group consisting of FOXP 3-positive cells and CD 4-positive cells.

(36) The method according to any one of the preceding items, wherein the aforementioned step (a) comprises detecting CD8 by an anti-CD 8 antibody; the aforementioned process (B) includes detecting at least 1 of FOXP3 and CD4 by at least 1 of the anti-FOXP 3 antibody and the anti-CD 4 antibody.

(37) The method according to any of the preceding items, wherein the detection is performed by FACS, western blot or PCR.

(38) A composition comprising an inhibitor, which inhibitor is an inhibitor capable of inhibiting the interaction of CD80 and/or CD86 with CD28, for use in the manufacture of a pharmaceutical composition according to any of the preceding items.

(39) The composition of any of the preceding items, wherein the aforementioned inhibitory factor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars, and combinations thereof.

(40) The composition of any one of the preceding items, wherein the aforementioned protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.

(41) The composition of any one of the preceding items, wherein the variant of the aforementioned antibody is an antigen-binding fragment.

(42) The composition of any one of the preceding items, wherein the variant of the aforementioned cell surface molecule is a fusion protein.

(43) The composition of any one of the preceding items, wherein the aforementioned inhibitor is selected from the group consisting of an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, a CD28-Ig fusion protein.

(44) The composition of any one of the preceding items, wherein the CTLA4-Ig fusion protein is abatacept or belicept.

(45) A kit for use in the manufacture of a medicament comprising a mixture of cells, the kit comprising: (A) an inhibitor capable of inhibiting the interaction of CD80 and/or CD86 with CD 28; (B) means for detecting CD 8; and (C) a means for detecting at least 1 of FOXP3 and CD 4.

(46) The kit according to any of the preceding items, wherein the aforementioned inhibitor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars and combinations thereof.

(47) The kit according to any one of the preceding items, wherein the aforementioned protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.

(48) The method of any one of the preceding items, wherein the variant of the aforementioned antibody is an antigen-binding fragment.

(49) The method of any one of the preceding items, wherein the variant of the cell surface molecule is a fusion protein.

(50) The kit or method of any one of the above items, wherein the aforementioned inhibitor is selected from the group consisting of an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, a CD28-Ig fusion protein.

(52) The kit or method of any one of the preceding items, wherein the CTLA4-Ig fusion protein is abatacept or belicept.

(53) The kit according to any one of the preceding items, wherein the presence of CD8 positive cells and the presence of at least 1 of FOXP3 positive cells and CD4 positive cells in the aforementioned cell mixture indicates that the aforementioned cell mixture can be used as a medicament.

(54) The kit according to any one of the above items, wherein the aforementioned drug is used for treatment or prevention of a disease, disorder or condition of the specimen due to an antigen derived from or not derived from the aforementioned specimen.

(55) A kit for managing the quality of a cell-containing drug for treating or preventing a disease, disorder or condition of a subject arising from an antigen expressed by cells derived from the subject or an antigen not derived from the subject, comprising: (A) means for detecting CD 8; and (B) means for detecting at least 1 of FOXP3 and CD 4.

(56) The kit according to any one of the preceding items, wherein the aforementioned means for detecting CD8 comprises an anti-CD 8 antibody and the means for detecting at least 1 of FOXP3 and CD4 comprises at least 1 of an anti-FOXP 3 antibody and an anti-CD 4 antibody.

(57) The kit according to any one of the preceding items, wherein the detection is performed by FACS, western blot or PCR.

(B1) A pharmaceutical composition for use with CD 4-positive anergic cells to induce immune tolerance comprising CD 8-positive anergic T cells.

(B2) The pharmaceutical composition of clause B1, further comprising 1 or more of the features set forth in any of the preceding clauses 1-57 or other clauses.

(C1) A pharmaceutical composition for use with CD 8-positive anergic cells to induce immune tolerance comprising CD 4-positive anergic T cells.

(C2) The pharmaceutical composition of clause B1, further comprising 1 or more of the features set forth in any of the preceding clauses 1-57 or other clauses.

(D1) A method for treating or preventing a disease, disorder or condition in a subject caused by an antigen derived from or not derived from the subject, the method comprising the steps of: administering a CD 4-positive non-reactive T cell and a CD 8-positive non-reactive T cell to the specimen in an effective amount.

(D2) The method according to the above item, wherein the aforementioned anergic T cells are induced by an antibody capable of inhibiting the interaction of CD80 and/or CD86 with CD 28.

(D3) The method according to any one of the preceding items, wherein the aforementioned inhibitory factor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars and combinations thereof.

(D4) The method according to any one of the preceding items, wherein the aforementioned protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.

(D5) The method of any one of the preceding items, wherein the variant of the aforementioned antibody is an antigen-binding fragment.

(D6) The method of any one of the preceding items, wherein the variant of the cell surface molecule is a fusion protein.

(D7) The method according to any of the preceding items, wherein the aforementioned inhibitor is selected from the group consisting of an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, a CD28-Ig fusion protein.

(D8) The method of any one of the preceding items, wherein the CTLA4-Ig fusion protein is abatacept or belicept.

(D9) The pharmaceutical composition according to any one of items 1 to 8, which further comprises a step of administering regulatory T cells.

(D10) The method of any one of the above items, wherein the aforementioned regulatory T cells are FOXP3 positive CD4 positive CD25 positive.

(D11) A method for treating or preventing a disease, disorder or condition in a subject caused by an antigen derived from or not derived from the subject, the method comprising the steps of: and administering to the subject an effective amount of cells induced to anergy by an inhibitor capable of inhibiting the interaction between CD80 and/or CD86 and CD28, wherein the anergy-induced cells comprise CD 8-positive cells, and the anergy-induced cells further comprise at least 1 or more of FOXP 3-positive cells and CD 4-positive cells.

(D12) The method of any one of the above items, comprising all of FOXP 3-positive cells, CD 4-positive cells, and CD 8-positive cells.

(D13) The method of any one of the above items, wherein the aforementioned CD 8-positive cells are CD 44-positive.

(D14) The method of any one of the above items, wherein the aforementioned CD 8-positive cells are CD45 RA-negative and CD45 RO-positive.

(D15) The method of any one of the above items, wherein the FOXP3 positive cells are CD4 positive.

(D16) The method of any one of the above items, wherein the FOXP3 positive cells are CD25 positive.

(D17) The method of any of the preceding items, wherein the induction of anergy in the cells induces antigen-specific immune tolerance or immunosuppression.

(D18) The method of any one of the above items, wherein the aforementioned antibody comprises an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody, or a combination thereof.

(D19) The method according to any one of the above items, wherein the cells are cells induced by mixing the inhibitor, cells derived from a sample, and an antigen derived from the sample, an antigen not derived from the sample, or a substance containing the antigen.

(D20) A method for treating or preventing a disease, disorder or condition in a subject arising from an antigen derived from or not derived from the subject, the method comprising a composition as defined in any one of the preceding items.

(D21) The method according to any of the preceding claims, wherein the aforementioned disease, disorder or condition is selected from the group consisting of transplant immune rejection, allergy, autoimmune disease, graft-versus-host disease, immune rejection caused by transplantation of iPS cells or ES cells and cells, tissues or organs derived from these cells.

(D22) The method according to any one of the preceding items, wherein the transplant immune rejection is produced by transplanting kidney, liver, heart, skin, lung, pancreas, esophagus, stomach, small intestine, large intestine, nerve, blood cells including cells of the immune system, bone, cartilage, blood vessel, cornea, eyeball, or bone marrow.

(D23) The method according to any one of the preceding items, wherein the antigen-containing substance is a cell.

(E1) Use of a CD 4-positive anergic T cell and a CD 8-positive anergic T cell in the manufacture of a medicament for treating or preventing a disease, disorder or condition in a subject arising from an antigen derived from or not derived from the subject.

(E2) Use according to the preceding item, wherein the aforementioned anergic T cells are induced by antibodies capable of inhibiting the interaction of CD80 and/or CD86 with CD 28.

(E3) The use according to any of the preceding items, wherein the aforementioned inhibitor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars and combinations thereof.

(E4) The use according to any one of the preceding items, wherein the aforementioned protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.

(E5) The use according to any one of the preceding items, wherein the variant of the aforementioned antibody is an antigen-binding fragment.

(E6) Use according to any one of the preceding items, wherein the variant of the aforementioned cell surface molecule is a fusion protein.

(E7) The use according to any of the preceding items, wherein the aforementioned inhibitor is selected from the group consisting of an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, a CD28-Ig fusion protein.

(E8) The use according to any of the preceding items, wherein the CTLA4-Ig fusion protein is abatacept or belatacept.

(E9) The use according to any one of items 1 to 8, wherein the aforementioned CD 4-positive and CD 8-positive anergic T cells further comprise regulatory T cells.

(E10) The use according to any one of the preceding items, wherein the aforesaid regulatory T cells are FOXP3 positive CD4 positive CD25 positive.

(E11) Use of anergy-induced cells for the manufacture of a medicament for treating or preventing a disease, disorder or condition in a subject arising from an antigen derived from or not derived from the subject, the cells being anergy-induced by an inhibitor capable of inhibiting the interaction of CD80 and/or CD86 with CD28, the anergy-induced cells comprising CD8 positive cells, the anergy-induced cells comprising at least 1 or more of FOXP3 positive cells and CD4 positive cells.

(E12) The use according to any one of the preceding items, wherein the aforementioned anergy-induced cells comprise all of FOXP 3-positive cells, CD 4-positive cells and CD 8-positive cells.

(E13) The use according to any one of the preceding items, wherein the aforementioned CD 8-positive cells are CD 44-positive.

(E14) The use according to any one of the preceding items, wherein the aforementioned CD8 positive cells are CD45RA negative and CD45RO positive.

(E15) The use according to any one of the preceding items, wherein the FOXP3 positive cells are CD4 positive.

(E16) The use according to any one of the preceding items, wherein the FOXP3 positive cells are CD25 positive.

(E17) The use according to any of the preceding items, wherein the induction of anergy in the cells induces antigen-specific immune tolerance or immunosuppression.

(E18) The use of any one of the preceding items, wherein the aforementioned antibody comprises an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody, or a combination thereof.

(E19) The use according to any one of the above items, wherein the anergy-induced cells are cells induced by mixing the inhibitor, cells derived from a sample, and an antigen derived from the sample, an antigen not derived from the sample, or a substance containing the antigen.

(E20) Use of a composition comprising any of the above items for treating or preventing a disease, disorder or condition in a subject arising from an antigen derived from or not derived from the subject.

(E21) Use according to any of the preceding items, wherein the aforementioned disease, disorder or condition is selected from the group consisting of transplant immune rejection, allergy, autoimmune disease, graft-versus-host disease, immune rejection caused by transplantation of iPS cells or ES cells and cells, tissues or organs derived from these cells.

(E22) The use according to any of the preceding items, wherein the transplant immune rejection is produced by transplanting kidney, liver, heart, skin, lung, pancreas, esophagus, stomach, small intestine, large intestine, nerve, blood cells including cells of the immune system, bone, cartilage, blood vessel, cornea, eyeball, or bone marrow.

(E23) The use according to any one of the preceding items, wherein the antigen-containing substance is a cell.

(F1) A cell mixture of CD 4-positive anergic T cells and CD 8-positive anergic T cells for use in treating or preventing a disease, disorder or condition in a subject arising from an antigen derived from or not derived from the subject.

(F2) The cell mixture according to the above item, wherein the aforementioned anergic T cells are induced by an antibody capable of inhibiting the interaction of CD80 and/or CD86 with CD 28.

(F3) The cell mixture according to any of the preceding items, wherein the aforementioned inhibitory factor is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars and combinations thereof.

(F4) A cell mixture according to any of the preceding items, wherein the aforementioned protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.

(F5) The cell mixture according to any of the preceding items, wherein the variant of the aforementioned antibody is an antigen-binding fragment.

(F6) The cell mixture according to any of the preceding items, wherein the variant of the aforementioned cell surface molecule is a fusion protein.

(F7) The cell mixture according to any of the preceding items, wherein the aforementioned inhibitory factor is selected from the group consisting of an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, a CD28-Ig fusion protein.

(F8) The cell mixture of any of the preceding items, wherein the CTLA4-Ig fusion protein is abatacept or belicept.

(F9) The cell mixture according to any one of items 1 to 8, further comprising regulatory T cells.

(F10) The cell mixture according to any of the preceding items, wherein the aforementioned regulatory T cells are FOXP3 positive CD4 positive CD25 positive.

(F11) An anergy-induced cell induced anergy by an inhibitor capable of inhibiting the interaction of CD80 and/or CD86 with CD28 for use in treating or preventing a disease, disorder or condition of a subject due to an antigen derived from or not derived from the subject, the anergy-induced cell comprising a CD 8-positive cell, the anergy-induced cell comprising at least 1 or more of a FOXP 3-positive cell and a CD 4-positive cell.

(F12) The anergic-induced cell of any of the above items, comprising all of a FOXP 3-positive cell, a CD 4-positive cell, and a CD 8-positive cell.

(F13) The anergic cell according to any of the above items, wherein the CD 8-positive cell is CD 44-positive.

(F14) The anergic-induced cell according to any of the above items, wherein the aforementioned CD 8-positive cell is CD45 RA-negative and CD45 RO-positive.

(F15) The anergic cell according to any of the preceding items, wherein the FOXP 3-positive cell is CD 4-positive.

(F16) The use according to any one of the preceding items, wherein the FOXP3 positive cells are CD25 positive.

(F17) The anergic-induced cell according to any of the above items, which induces antigen-specific immune tolerance or immune suppression.

(F18) The anergic cell of any of the preceding items, wherein the aforementioned antibody comprises an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody, or a combination thereof.

(F19) The anergy-induced cell according to any one of the above items, wherein the anergy-induced cell is a cell induced by mixing the inhibitor, a cell derived from a sample, and an antigen derived from the sample, an antigen not derived from the sample, or a substance containing the antigen.

(F20) An anergic-induced cell for treating or preventing a disease, disorder or condition in a subject caused by an antigen derived from or not derived from the subject, comprising the composition of any one of the preceding items.

(F21) The anergic cell according to any of the preceding items, wherein the aforementioned disease, disorder or condition is selected from the group consisting of transplant immune rejection, allergy, autoimmune disease, graft versus host disease, immune rejection caused by transplantation of iPS cells or ES cells and cells, tissues or organs derived from these cells.

(F22) The cell inducing anergy according to any one of the above items, wherein the transplant immune rejection is generated by transplanting kidney, liver, heart, skin, lung, pancreas, esophagus, stomach, small intestine, large intestine, nerve, blood cell including immune system cell, bone, cartilage, blood vessel, cornea, eyeball, or bone marrow.

(F23) The cell in which anergy is induced according to any one of the above items, wherein the antigen-containing substance is a cell.

The present invention also provides the following solutions.

(G1) A pharmaceutical composition comprising: CD4 positive anergic T cells, and

CD8 positive anergic T cells.

(G2) The composition according to the preceding item, wherein the aforementioned anergic T cells are induced by antibodies capable of inhibiting the interaction of CD80 and/or CD86 with CD 28.

(G3) The pharmaceutical composition of any one of the above items, further comprising regulatory T cells.

(G4) The composition of any one of the preceding items, wherein the aforementioned regulatory T cells are FOXP3 positive CD4 positive CD25 positive.

(G5) A composition which is a pharmaceutical composition comprising cells that have been induced to anergy by an antibody capable of inhibiting the interaction of CD80 and/or CD86 with CD28, the composition comprising CD8 positive cells, the composition further comprising at least 1 or more of FOXP3 positive cells and CD4 positive cells.

(G6) The composition of any one of the above items, wherein all of FOXP 3-positive cells, CD 4-positive cells, and CD 8-positive cells are comprised.

(G7) The composition of any one of the preceding items, wherein the aforementioned CD 8-positive cells are CD 44-positive.

(G8) The composition of any one of the preceding items, wherein the FOXP3 positive cells are CD4 positive.

(G9) The composition of any one of the preceding items, wherein the FOXP3 positive cells are CD25 positive.

(G10) The composition according to any one of the preceding items, wherein the aforementioned pharmaceutical composition is for antigen-specific immune tolerance or immune suppression.

(G11) The composition of any one of the preceding items, wherein the aforementioned antibody comprises an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody, or a combination thereof.

(G12) The composition according to any one of the above items, wherein the cells are induced by mixing the antibody, cells derived from a sample, and an antigen derived from the sample, an antigen not derived from the sample, or a substance containing the antigen.

(G13) A pharmaceutical composition for treating or preventing a disease, disorder or condition in a subject caused by an antigen derived from or not derived from the subject, comprising the composition of any one of items G1 to G12.

(G14) The composition according to any of the preceding claims, wherein the aforementioned disease, disorder or condition is selected from the group consisting of transplant immune rejection, allergy, autoimmune disease, graft-versus-host disease, immune rejection caused by transplantation of iPS cells or ES cells and cells, tissues or organs derived from these cells.

(G15) The composition according to item G14, wherein the transplant immune rejection response is produced by transplanting kidney, liver, heart, skin, lung, pancreas, esophagus, stomach, small intestine, large intestine, nerve, blood cells including cells of the immune system, bone, cartilage, blood vessel, cornea, eyeball, or bone marrow.

(G16) The composition according to any one of the preceding items, wherein the antigen-containing substance is a cell.

(G17) A method for inhibiting the production of a cell-containing drug, the method comprising the steps of:

(A) mixing an antibody capable of inhibiting the interaction between CD80 and/or CD86 and CD28, cells derived from a sample, and an antigen derived from the sample, an antigen not derived from the sample, or a substance containing the antigen;

(B) confirming that the cell product obtained by the mixing contains CD 8-positive cells; and

(G18) The method of any one of the preceding items, wherein the presence of CD 8-positive cells and the presence of at least 1 of FOXP 3-positive cells and CD 4-positive cells in the aforementioned cell product indicates that the aforementioned cell product can be used as a medicament.

(G19) The method according to any one of the preceding items, wherein the drug is used for treating or preventing a disease, disorder or condition of the subject caused by an antigen derived from or not derived from the subject.

(G20) The method according to any one of the preceding items, wherein the step (B) comprises detecting CD8 by an anti-CD 8 antibody; the aforementioned process (C) includes detecting at least 1 of FOXP3 and CD4 by at least 1 of the anti-FOXP 3 antibody and the anti-CD 4 antibody.

(G21) The method according to any of the preceding items, wherein the detection is performed by FACS.

(G22) A method of managing the quality of a drug containing cells for treating or preventing a disease, disorder or condition of a specimen due to an antigen expressed by cells derived from the specimen or an antigen not derived from the specimen, comprising the steps of:

(A) confirming that the cells contain CD8 positive cells; and

(B) confirming that the cell comprises at least 1 cell selected from the group consisting of FOXP 3-positive cells and CD 4-positive cells.

(G23) The method according to item G22, wherein the aforementioned procedure (a) comprises detecting CD8 by anti-CD 8 antibody and the aforementioned procedure (B) comprises detecting at least 1 of FOXP3 and CD4 by at least 1 of anti-FOXP 3 antibody and anti-CD 4 antibody.

(G24) The method according to any of the preceding items, wherein the detection is performed by FACS, western blot or PCR.

(G25) A kit for use in the manufacture of a medicament comprising a mixture of cells, the kit comprising:

(A) an antibody capable of inhibiting the interaction of CD80 and/or CD86 with CD 28;

(B) means for detecting CD 8; and

(G25A) a kit for managing the quality of a medicament comprising a mixture of cells, the kit comprising:

(A) means for detecting CD 8; and

(B) a unit for detecting at least 1 of FOXP3 and CD 4.

(G26) The kit according to any one of the preceding items, wherein the presence of CD8 positive cells and the presence of at least 1 of FOXP3 positive cells and CD4 positive cells in the aforementioned cell mixture indicates that the aforementioned cell mixture can be used as a medicament.

(G27) The kit according to any one of the above items, wherein the aforementioned drug is used for treatment or prevention of a disease, disorder or condition of the specimen due to an antigen derived from or not derived from the aforementioned specimen.

(item G27A) the kit of any one of the above items, wherein the drug is treated or prevented by immune tolerance.

(G28) A kit for managing the quality of a cell-containing medicament for treating or preventing a disease, disorder or condition of a subject arising from an antigen expressed by cells derived from the subject or an antigen not derived from the subject, the kit comprising:

(A) means for detecting CD 8; and

(B) a unit for detecting at least 1 of FOXP3 and CD 4.

(G29) The kit according to any one of the preceding items, wherein the aforementioned means for detecting CD8 comprises an anti-CD 8 antibody and the means for detecting at least 1 of FOXP3 and CD4 comprises at least 1 of an anti-FOXP 3 antibody and an anti-CD 4 antibody.

(G30) The kit according to any one of the preceding items, wherein the detection is performed by FACS, western blot or PCR.

In the present invention, one or more of the above-mentioned features may be provided intentionally in further combination in addition to the combination explicitly shown. Further embodiments and advantages of the present invention will be appreciated to those skilled in the art upon reading and understanding the following detailed description, as needed.

ADVANTAGEOUS EFFECTS OF INVENTION

The composition of the present invention comprises CD 4-positive anergic T cells and CD 8-positive anergic T cells, and has higher immune tolerance induction ability than those who do not comprise CD 8-positive anergic T cells. In addition, in the production of a drug for inducing immune tolerance, the quality of the drug can be controlled using CD 8-positive cells as an index.

Drawings

FIG. 1 is a graph showing the results of a loss of function test (loss of function assay) for identifying components required for inducing immune tolerance. (i) Production of anergic cells: spleens were collected from C57BL6 (hereinafter abbreviated as B6) mice and BALB/C mice to obtain spleen cells (lymphocytes). Then, BALB/c spleen cells as stimulators were irradiated with 30Gy of radiation (γ rays), and then the ratio of 1: 1 was mixed with B6-derived spleen cells, and anti-CD 80 antibody and anti-CD 86 antibody were added so that the respective final concentrations were 10. mu.g/mL, in a suitable volume of culture medium at 37 ℃ with 5% CO₂The incubation was started in the incubator. Removing the culture medium by centrifugation on the 7 th day after the start of culturing, and culturingThe culture medium containing BALB/c-derived radiation-irradiated spleen cells and anti-CD 80 antibody/anti-CD 86 antibody was added again under the same conditions as the start of the culture. Cells were recovered on day 14 of culture to obtain non-reactive cells. (ii) Screening of each cell phenotype: after the PE fluorescently labeled anti-CD 8 antibody was reacted with the obtained non-reactive cells, the cells were screened for CD8 positive and CD8 negative by an automatic magnetic cell separation apparatus (auto-MACS) using anti-PE magnetic beads. In addition, cells were screened for CD19 positive and CD19 negative by the same procedure using the PE fluorescence labeled CD19 antibody. Furthermore, in order to identify regulatory T cells (regT cells) expressing FOXP3 by expression of cell surface antigens, B6-derived mice genetically modified so that FOXP3 and human CD2 were expressed simultaneously by responder cells were used, and auto-MACS using PE fluorescent-labeled anti-human CD2 antibody and anti-PE magnetic beads was used to screen human CD2 positive cells (FOXP 3-expressing regT cells) and human CD2 negative cells for immune response suppression ability test. (iii) Immune response inhibition test: freshly collected splenocytes from B6 mice were used as effectors, and freshly collected splenocytes from BALB/c mice were used as stimulators, and the cells were prepared in a volume of 200 μ L in each well of a 96-well plate in a manner of 1: 1 ratio of 1 Mixed culture containing cells (number of cells per well 1X 10)⁵One). To the mixed culture, total non-reactive cells without any selection (FIG. 1a), a cell population containing only non-reactive CD 8-positive cells or a remaining cell population from which non-reactive CD 8-positive cells were removed (FIG. 1B), a cell population containing only regT cells or a remaining cell population from which regT cells were removed (FIG. 1c), a cell population containing only CD 19-positive cells (B cells) or a remaining cell population from which B cells were removed (FIG. 1d) were added so that the ratio of the number of splenocytes responding to B6 to the mixed culture was 1, 1/2, 1/4, 1/8 or 1/16, and 5% CO was added at 37 ℃₂Culturing in a thermostat. Adding on day 4 after the start of culture³H-Thymidine, at day 5 of the beginning of culture (addition)³After 16 to 20 hours of H-thymidine) recovery of cultured cells, and measurement thereof by means of a scintillation counter³H-thymidine uptake. In each figure, "initial" (naive) is the effector-only well and "allogenic" (allo) is the effective-only responsePores of the applicator and stimulus, will be "initial³The average value of the H-thymidine uptake was 1, and a graph was prepared.

FIG. 2 is a graph showing the results of an experiment obtained based on the inhibitory function of CD80/86 blockade. In the same manner as in example 1, non-reactive cells were obtained by treating splenocytes obtained from a wild-type B6 mouse and a B6-derived mouse genetically modified to express FoxP3 and human CD2 simultaneously with irradiation of BALB/c-derived radiation and anti-CD 80 antibody/anti-CD 86 antibody. Non-stimulated primary splenocytes obtained from wild type B6 mice and B6-derived mice genetically engineered to express both FoxP3 and human CD2 were reacted with PE fluorescently labeled anti-mouse CD8 antibody or PE fluorescently labeled anti-human CD2 antibody, respectively, along with the non-reactive cells. Then, the cells were screened for CD8 positive and CD8 negative, or for human CD2 positive and human CD2 negative by auto-MACS using anti-PE magnetic beads. The selected cells were added to the mixed culture system on a 96-well plate described in example 1, and their immunosuppressive ability was examined. In each figure, "initial" (i more accurately denotes Trema (2 points above). as in this specification) is the only effector aperture and "allogeneic" is the only effector and stimulator aperture, the "initial" aperture³The average value of the H-thymidine uptake was 1, and a graph was prepared.

Fig. 3 shows the results of confirming whether or not purified cells after induction of anergy have tolerance-inducing ability after transplantation by an experiment using mice. In the same manner as in example 1, splenocytes of a wild-type B6 mouse and a B6-derived mouse genetically modified to express both FoxP3 and human CD2 were irradiated with BALB/c-derived radiation and anti-CD 80 antibody/anti-CD 86 antibody to obtain non-reactive cells. From the obtained anergic cells (total anergic cells), CD 8-positive cells, CD 4-positive cells, or human CD 2-positive cells were selected in the same manner as in example 1. For wild type B6 mice, the hearts of BALB/c mice were transplanted 3 days after 2Gy irradiation (gamma ray), and the initial B administration was performed from the tail vein immediately after the transplantation (or on the same day as the heart transplantation)Rejection of the heart was observed with splenocytes or resulting anergy cells. Each group was tested with 5 or more. Fig. 3a shows: in the case of non-transplanted non-reactive cells or administration of 2X 10⁶4 x 10 pieces⁶Or 6 x 10⁶Cumulative survival of heart-transplanted mice at total anergic cells. Figure 3b shows the effect of radiation irradiation on recipient mice. The recipient mice were given irradiation with non-radiation plus total non-reactive cells 5X 10⁶2.5Gy radiation + Primary B6 splenocytes 4X 10 irradiation of recipient mice⁶2.5Gy radiation irradiation + Total non-reactive cells 4X 10 was applied to one or more recipient mice⁶Individual treatment followed the survival of the mice thereafter. Fig. 3c and 3d show the survival of heart-transplanted mice when given various purified cell populations.

Figure 4 shows the capacity of anergic cells derived from human PBMCs. (i) Production of anergic cells: mononuclear Cells (PBMCs) were isolated from human peripheral blood of 4 volunteers (2 persons as stimuli and 2 persons as effectors), and the stimuli PBMCs were irradiated with radiation (γ -rays) 30Gy at a rate of 1: 1 were mixed with responding PBMCs. To the mixed PBMC, anti-human CD80 antibody and anti-human CD86 antibody were added so that the final concentrations were 10. mu.g/mL, respectively, and the mixture was cultured in a suitable volume of culture medium at 37 ℃ with 5% CO₂The incubation was started in the incubator. After removing the culture solution by centrifugation on the 7 th day after the start of the culture, a culture solution containing radiation irradiation stimuli PBMC and anti-CD 80 antibody/anti-CD 86 antibody was added under the same conditions as at the start of the culture. Cells were recovered on the 14 th day of culture, and the culture broth was washed away by centrifugation to obtain non-reactive cells. (ii) Screening of each cell phenotype: after staining the obtained non-reactive cells with PE fluorescence-labeled mouse anti-human CD25 antibody, FITC fluorescence-labeled mouse anti-human CD4 antibody, and APC fluorescence-labeled mouse anti-human CD8 antibody, each cell was purified using a JSAN cell sorter (Bay bioscience co., Ltd.). (iii) Immune response inhibition test: freshly collected PBMCs from the same volunteer were used as effector, stimulator and prepared in a volume of 200 μ Ι _, in each well of a 96-well plate in a 1: 1 ratio of mixed cultures containing cells (number of cells per well 2X 10)⁵One). Total non-reactive cells were added at 37 ℃ with 5% CO in such a way that the ratio of the number of PBMC-responding cells to the mixed culture was 1/2, 1/4, 1/8 or 1/16₂Incubations were performed in an incubator (FIG. 4 a). In FIG. 4b, the cell population purified from CD 25-positive cells (CD25+), the cell population purified from CD 4-positive CD 25-positive regT cells (CD4+ CD25+), or the cell population added with purified CD 4-positive CD 25-positive regT cells and purified CD 8-positive cells (CD25+/CD8+) were added to the mixed culture, and the ratio of the number of responsive PBMCs was 1/2, respectively. In fig. 4c, the immunosuppressive ability was compared by adding a cell population purified from CD 4-positive cells, a cell population purified from CD 4-negative cells, or total non-reactive cells to the mixed culture, and setting the ratio of the number of cells responding to PBMCs to 1/2, 1/4, 1/8, or 1/16. In each figure, "initial" is the only responsive well, and "allogenic" is the only effective effector and stimulus well, and will be "initial"³The average value of the H-thymidine uptake was 1, and a graph was prepared.

Fig. 5 is a graph showing antigen specificity of immune suppression based on anergic cells. This figure shows the survival of the heart after injection of B6 mouse-derived anergic cells into B6 mice after stimulation with Balb/C mouse splenocytes in the presence of anti-CD 80/86 antibody. Whereas the heart of the transplanted BALB/c mouse was 100% inhibited rejection of the heart and survived after 100 days, the heart of the CBA mouse, a foreign (third party) was rapidly rejection after transplantation and was shown to be 100% rejected at about 50 days.

Fig. 6 shows the case where the non-responsive cells bind to the donor (stimulus) cells more rapidly than the initial cells, thereby inhibiting the reaction and proliferation of the initial cells. In the same manner as in example 1, the splenocytes obtained from B6 mice were irradiated with BALB/c-derived radiation and anti-CD 80 antibody/anti-CD 86 antibody to obtain non-reactive cells. In this experiment, splenocytes newly obtained from B6 mice genetically modified in such a manner as to stably express fluorescent pigment GFP were used as effectors. In a 12-well plate, in each case contain1×10⁶The above-mentioned non-reactive cells were added to a 4ml mixed culture system of the above-mentioned B6-responsive splenocytes and stimulus (donor) BALB/c splenocytes at a ratio of 1/2 to B6-responsive splenocytes at 37 ℃ with 5% CO₂Culturing in a thermostat. The well plate was observed from 1 day to 3 days after the start of the culture, and photographs were taken.

Fig. 7 shows the case where cells exerting immunosuppressive ability (anergy-inducing ability) among anergy cells were positive for CD 44. In the same manner as in example 1, the splenocytes obtained from B6 mice were irradiated with BALB/c-derived radiation and anti-CD 80 antibody/anti-CD 86 antibody to obtain non-reactive cells. After staining the non-reactive cells with PE fluorescence-labeled anti-mouse CD8 antibody, PE fluorescence-labeled anti-mouse CD4 antibody, and APC fluorescence-labeled anti-mouse CD44 antibody, a cell population from which CD 8-positive CD 44-negative cells or CD 4-positive CD 44-negative cells were removed was prepared using a JSAN cell sorter, and the cell population or total non-reactive cells were added to the mixed culture system so that the ratio to B6-responsive splenocytes was 1/2, 1/4, 1/8, or 1/16 (fig. 7 a). Further, a cell population obtained by purifying CD 8-positive CD 44-positive cells or CD 4-positive CD 44-positive cells was prepared using a JSAN cell sorter, and added to the mixed culture system so that the ratio to the splenocytes responding to B6 was 1/2, 1/4, or 1/8 (fig. 7B). Fig. 7c shows the results of investigating the phenotype of non-reactive cells by FACS.

Detailed Description

The best mode is shown below and the present invention will be explained. Throughout this specification, the singular expression should be understood to include the plural concept unless it is specifically stated otherwise. Thus, articles in the singular (e.g., articles "a," "an," "the," etc. in the english case) should be understood to also include the plural concepts unless specifically stated otherwise. In addition, unless otherwise specified, terms used in the present specification should be understood to be used in the meaning generally used in the field. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(definition of terms)

In the present specification, "about" means ± 10% of the value continuing thereafter in the case of use in the present specification.

In the present specification, "immune tolerance" means: it does not show a specific immune response to a specific antigen or a state in which a specific immune response is suppressed. Immune tolerance can be defined as a state in which immune cells (particularly T cells) do not exhibit a specific immune response to a particular antigen, or the specific immune response is suppressed; and a state in which the human does not show a specific immune response to the specific antigen or the specific immune response is suppressed. It has been noticed that immunological rejection can be treated or allergy can be treated by inducing immunological tolerance. In the present specification, "non-reactive" means: a state in which co-stimulation is not input when an antigen is presented by antigen-presenting cells, and thus no response occurs even when the antigen is next stimulated under the condition of co-stimulation. Thus, in the present specification, "non-reactive cells" means: an immunologically tolerizing (non-immunocompromised) cell, an "anergic T cell" is an immunologically tolerizing (non-immunocompromised) T cell that, in addition to not being activated, includes T cells that are non-reactive when they encounter the same antigen again. In the present specification, "immune tolerance-inducing PBMC (or T cells)" and "anergic PBMC (or T cells)" have the same meaning. Whether or not the cells are non-reactive cells can be confirmed by confirming the cells are positive for CD44, for example, but the present invention is not limited thereto.

In the present specification, the "subject" includes domesticated animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., non-human primates such as humans and monkeys), rabbits, and rodents (e.g., mice and rats). In a particular embodiment, the subject is a human.

In this specification, "reagent", "agent" or "factor" (in english, each equivalent to an agent) may be used interchangeably in a broad sense, and may be any substance or other element (e.g., light, radiant energy, heat, electricity, etc.) as long as the intended purpose is achieved. Examples of such substances include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including, for example, cDNA, DNA such as genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, information transfer substances, other small organic molecule compounds, molecules chemically synthesized by combinatorial synthesis, small molecules useful as drugs (for example, small molecule ligands, etc.), and complex molecules thereof.

In the present specification, "inhibitor" means: all types of small molecules, proteins, nucleic acids, lipids, sugars, etc., that are capable of inhibiting a particular effect (e.g., interaction, signaling, etc.). While not wishing to be bound by a particular theory, in the present invention, T cell anergy is induced by blocking the cell surface interaction of CD80 and/or CD86 with CD28, inhibiting the CD28 costimulatory signal. In the present invention, the inhibitor for blocking the interaction of CD80 and/or CD86 with CD28 is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars and combinations thereof. In one embodiment, the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof. In another embodiment, the variant of the above antibody is an antigen binding fragment. In another embodiment, the variant of the cell surface molecule is a fusion protein. In another embodiment, the inhibitor is selected from the group consisting of an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, and a CD28-Ig fusion protein. In another embodiment, the CTLA4-Ig fusion protein is abatacept or belatacept. In addition, it is also conceivable that the above-described interaction may be indirectly inhibited (for example, an inhibitor of a signal upstream or downstream of signal transmission).

In the present specification, the term "antibody" in a broad sense means an antibody capable of binding to a specific antigenA molecule or population thereof to which an epitope specifically binds. An "antibody" in the broad sense of the present specification may be a full-length antibody (i.e., an antibody having an Fc portion) or an antibody lacking an Fc portion. An antibody lacking an Fc portion may be any antibody as long as it can bind to a target antigen, and examples of such an antibody include Fab antibody and F (ab')₂Antibodies, Fab' antibodies, Fv antibodies, scFv antibodies, and the like, but are not limited thereto. The antibody may be any type of antibody, i.e., may be an immunoglobulin known in the art. In exemplary embodiments, the antibody is of the isotype IgA, IgD, IgE, IgG, or IgM class. In exemplary embodiments, an antibody described herein comprises 1 or more alpha, delta, epsilon, gamma, and/or mu heavy chains. In exemplary embodiments, the antibodies described herein comprise 1 or more kappa or light chains. In an exemplary form, the antibody is an IgG antibody, which is of 4 human subclasses: 1 of IgG1, IgG2, IgG3, and IgG 4. In addition, as antibodies envisaged to be used in the present invention, there may be mentioned: antibodies of camelid origin (e.g., VHH antibodies), antibodies of shark origin (e.g., single-chain antibodies), peptibodies (peptibodies), nanobodies (nanobodies, single-domain antibodies), minibodies (minibodies), multispecific antibodies (e.g., bispecific antibodies, diabodies (diabodies), triabodies (triabodies), tetrabodies (tetrabodies), tandem bivalent antibodies-scFV, tandem trivalent antibodies-scFV), and the like are well known in the art. See, for example, Kortt et al, Biomol eng.200118: pages 95-108, (2001) and Todorovska et al, J Immunol methods.248: pages 47-66, (2001), etc. In addition, the antibody includes a modified antibody or an unmodified antibody. The modified antibody may be one obtained by binding an antibody to various molecules such as polyethylene glycol. The modified antibody can be obtained by chemically modifying an antibody by a known method. For various artificial antibodies and methods for modifying/transforming antibodies, reference is also made to Biochemical (2016) volume 88, pages 3-380.

"antibody" in the narrow sense of the present specification 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 "variant of antibodyBody ". The "antibody" in the narrow sense of the present specification may be a full-length antibody (i.e., an antibody having an Fc portion), and the "variant of an antibody" in the present specification may be a variant lacking the Fc portion of the above-mentioned antibody. Accordingly, an antibody defined narrowly in the specification may also be referred to as a full-length antibody, and a variant of an antibody may also be referred to as a variant of a full-length antibody. The variant lacking the Fc portion may be any variant as long as it can bind to the target antigen, and examples of such variants include Fab antibody and F (ab')₂Antibodies, Fab' antibodies, Fv antibodies, scFv antibodies, and the like, but are not limited thereto. In addition, variants of the antibody include antibody modifications or antibody non-modifications. The modified antibody may be one obtained by binding an antibody to various molecules such as polyethylene glycol. The modified antibody can be obtained by chemically modifying an antibody by a known method.

In one embodiment of the present invention, for example, in order to induce the production of an antigen-specific polyclonal antibody, the "polyclonal antibody" can be produced by administering an immunogen containing a target antigen to mammals (e.g., rats, mice, rabbits, cows, monkeys, etc.), birds, etc. In the administration of the immunogen, 1 or more kinds of immunizing agents and, if necessary, adjuvants may be injected. Adjuvants may also be used to enhance the immune response and may include Freund's adjuvant (complete or incomplete), mineral gels (aluminum hydroxide, etc.), or surface active substances (lysolecithin, etc.), etc. Immunization protocols are well known in the art, and may be carried out by any method that induces an immune response depending on the host organism selected (protein laboratory Manual, sheep soil Co., Ltd. (2003): 86-91.).

In one embodiment of the present invention, "monoclonal antibody" includes the following cases: each antibody comprising the population is substantially the same antibody corresponding to a single epitope, except for a small number of antibodies having mutations that occur naturally. Alternatively, each antibody comprising the population may be substantially identical except for a small number of antibodies having naturally occurring mutations. Monoclonal antibodies are highly specific and differ from conventional polyclonal antibodies, which typically comprise different antibodies corresponding to different epitopes and/or from conventional polyclonal antibodies, which typically comprise different antibodies corresponding to the same epitope. In addition to their specificity, monoclonal antibodies are useful in that they can be synthesized by hybridoma culture that is not contaminated with other immunoglobulins. The description of "monoclonal" may show that this feature is essentially obtained from a homogeneous population of antibodies, but does not imply that the antibodies must be produced using a particular method. For example, monoclonal antibodies can be produced by conjugation with "Kohler G, Milstein c., nature.1975aug 7; 256(5517): 495-497 by the same method as that of the hybridoma disclosed in "above. Alternatively, the monoclonal antibody can be produced by the same method as the recombinant method described in U.S. Pat. No. 4816567. Alternatively, monoclonal antibodies similar to those described in "Clackson et al, nature.1991aug 15; 352(6336): 624-; 222(3): 581-597 ", from phage antibody library. Alternatively, the protein can be prepared by the following methods of "protein laboratory manual, sheep soil society (2003): 92-96. ".

In one embodiment of the present invention, a "chimeric antibody" is constructed by, for example, connecting a variable region of an antibody between heterogeneous organisms to a constant region of the antibody, and can be constructed by gene recombination techniques. Mouse-human chimeric antibodies can be obtained, for example, using the antibody sequences of "Roguska et al, Proc Natl Acad Sci U S A.1994Feb1; 91(3): 969 and 973. The basic methods for producing a mouse-human chimeric antibody are, for example: the mouse leader sequence and variable region sequences present in the cloned cDNA are ligated to sequences encoding human antibody constant regions already present in the mammalian cell expression vector. Alternatively, 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, followed by ligation to a mammalian cell expression vector. The fragment of the human antibody constant region may be any fragment of the H chain constant region of a human antibody and the L chain constant region of a human antibody, and for example, for the human H chain, there are listed: c γ 1, C γ 2, C γ 3 or C γ 4, for the L chain, there may be mentioned: c λ or C κ.

In one embodiment of the present invention, a "humanized antibody" is, for example, an antibody having 1 or more CDRs derived from a non-human species and a Framework Region (FR) derived from a human immunoglobulin, and further a constant region derived from a human immunoglobulin, and binding to a desired antigen. Humanization of antibodies can be performed using various methods known in the art (Almagro et al, Front biosci.2008Jan 1; 13: 1619-1633.). Examples thereof include: CDR mapping (Ozaki et al, blood.1999Jun 1; 93 (11): 3922-. To modify (preferably improve) antigen binding, amino acid residues in the human FR region may be substituted for the corresponding residues from the CDR donor antibody. The FR replacement may be carried out by methods well known in the art (Riechmann et al, Nature.1988Mar 24; 332 (6162): 323-327.). For example, FR residues important for antigen binding can be identified by modeling the interaction of CDRs with FR residues. Alternatively, aberrant FR residues at a particular position can be identified by sequence comparison.

In one embodiment of the present invention, a "human antibody" is, for example, an antibody comprising regions comprising the variable and constant regions of the heavy chain and the variable and constant regions of the light chain constituting the antibody derived from a gene encoding a human immunoglobulin. The main production methods include: transgenic mouse method and phage display method for human antibody. In the transgenic mouse method for human antibody production, when a functional human Ig gene is introduced into a mouse from which an endogenous Ig gene has been deleted, a human antibody having diversified antigen-binding ability is produced instead of a mouse antibody. When the mouse is further immunized, a human monoclonal antibody can be obtained by the conventional hybridoma method. For example, "Lonberg et al, Int Rev immunol.1995; 13(1): 65-93. ". Typically, the phage display method is a system in which a foreign gene that does not lose the infectivity of phage is expressed as a fusion protein at the N-terminal side of the coat protein (g3p, g10p, etc.) of a fibrous phage such as M13 and T7, which are one of Escherichia coli viruses. For example, "Vaughan et al, Nat biotechnol.1996mar; 14(3): 309 and 314.

In the present specification, "cells derived from a sample" means: a cell obtained from a sample to which the composition of the present invention has been administered or a cell of a cell obtained from the sample. In the present specification, the "antigen derived from a sample" means: an antigen produced by the specimen itself that generates an immune response, for example, an antigen produced by the specimen itself that causes an autoimmune disease in a specimen having an autoimmune disease. In the present specification, the phrase "antigen not derived from a sample" means: a foreign antigen capable of generating an immune response. In the present specification, "antigen-containing material" that is not derived from a sample "refers to any substance or collection of substances that contains an antigen that is not derived from a sample, and examples thereof include cells, cell groups, tissues, and the like that express an antigen that is not derived from a sample.

In the present specification, "transplant immune rejection" means: in a test subject that has received a transplant of an organ, tissue or cell, the immune system of the test subject attacks, damages or destroys the transplanted organ, tissue or cell.

In the present specification, "allergy" means: a state in which an excessive immune response occurs against an antigen not derived from a specimen. Antigens which cause allergy and are not derived from the sample are also called allergens, and examples thereof include: mite antigen, protein 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 dander antigen, insect antigen, pollen antigen, dog dander antigen, fungal antigen, bacterial antigen, latex, hapten, metal, and the like, but is not limited thereto.

In the present specification, "autoimmune disease" means: any disease in which the immune system exerts an unwanted immune response against its own cells, tissues or organs. Examples of autoimmune diseases include: rheumatoid arthritis, multiple sclerosis, type 1 diabetes, inflammatory bowel disease (e.g., crohn's disease or ulcerative colitis), systemic lupus erythematosus, psoriasis, scleroderma, autoimmune thyroid disease, alopecia areata, graves ' disease, guillain-barre syndrome, celiac disease, sjogren's syndrome, rheumatic fever, gastritis, autoimmune atrophic gastritis, autoimmune hepatitis, pancreatitis, oophoritis, seminal inflammation, uveitis, lens-induced uveitis, myasthenia gravis, primary myxedema, pernicious anemia, autoimmune hemolytic anemia, addison's disease, scleroderma, goodpasture's syndrome, nephritis (e.g., glomerulonephritis), psoriasis, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, Wegener's granulomatosis and polymyositis, but is not limited to these.

In the present specification, "graft versus host disease" means: the transplanted organ, tissue or cells attack, damage or destroy the cells, tissue or organ of the subject to be transplanted by an immune response.

In the present specification, the "immune rejection reaction caused by transplantation of iPS cells or ES cells or cells, tissues or organs derived from these cells" means: an immune rejection reaction due to an antigen possessed by the iPS cell or the ES cell, or an antigen possessed by a cell, tissue, or organ derived from the iPS cell or the ES cell.

(preferred embodiment)

The following describes preferred embodiments, which are examples of the present invention, and it should be understood that the scope of the present invention is not limited to such preferred embodiments. It is to be understood that those skilled in the art can easily make changes, modifications, etc. within the scope of the present invention with reference to the following preferred embodiments. With respect to these embodiments, a person skilled in the art can appropriately combine any of the embodiments with reference to the description in the present specification. In addition, it can be understood that: the following embodiments of the present invention may be used alone or in combination of these.

(drugs and treatments and preventions)

The present inventors and others confirmed that: even if T cell anergy is induced using an inhibitor that inhibits the interaction of CD80/CD86 with CD28, the ability to induce immune tolerance is reduced in the absence of CD8 positive cells in a mixture comprising T cells that induce anergy. This result is unexpected, contrary to non-patent document 4, which reports that even removal of CD8 positive cells may hardly affect immunosuppression.

In one embodiment, the present invention provides a pharmaceutical composition comprising CD 4-positive anergic T cells, and CD 8-positive anergic T cells. Such pharmaceutical compositions may be used for antigen-specific immune tolerance or immunosuppression. The anergic T cells may be induced by an inhibitor capable of inhibiting the interaction of CD80 and/or CD86 with CD 28. Such inhibitors are selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars, and combinations thereof. In one embodiment, the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof. In another embodiment, the variant of the above antibody is an antigen binding fragment. In another embodiment, the variant of the cell surface molecule is a fusion protein. In another embodiment, the inhibitor is selected from the group consisting of an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, and a CD28-Ig fusion protein. In another embodiment, the CTLA4-Ig fusion protein is abatacept or belatacept.

In some embodiments, CD80 and/or CD86 is expressed by an antigen presenting cell and CD28 is expressed by a T cell.

In some embodiments, the pharmaceutical composition of the invention may further comprise regulatory T cells, such as FOXP 3-positive CD 4-positive CD 25-positive T cells. Such regulatory T cells are not directly involved in "immune tolerance" that does not exhibit a specific immune response to a particular antigen, nor are essential components, but in order to be able to suppress an immune response, in a preferred embodiment, the composition of the invention may further comprise regulatory T cells.

In another aspect of the present invention, the present invention provides a composition which is a pharmaceutical composition comprising cells induced anergy by an inhibitor capable of inhibiting the interaction of CD80 and/or CD86 with CD28, the composition comprising CD8 positive cells, the composition further comprising at least 1 or more of FOXP3 positive cells and CD4 positive cells. Such pharmaceutical compositions may be used for antigen-specific immune tolerance or immunosuppression. In particular embodiments, the compositions of the invention may further comprise all of FOXP3 positive cells, CD4 positive cells, and CD8 positive cells.

The inventor proves that: anergic CD 8-positive cells and CD 4-positive cells (also referred to as CD 8-positive anergic cells and CD 4-positive anergic cells) were induced to contain a large number of CD 44-positive cells. Thus, in some embodiments, CD 8-positive cells and/or CD 4-positive cells may be CD 44-positive. In addition, in order to confirm the production of non-reactive cells, CD45RA/CD45RO may be used in addition to CD 44. For example, in one protocol, CD 8-positive cells and/or CD 4-positive cells are CD45 RA-negative and CD45 RO-positive.

In some embodiments, FOXP 3-positive cells may be CD 4-positive and/or CD 25-positive. The composition of the invention preferably further comprises regulatory T cells which are FOXP3 positive and may further be CD4 positive and/or CD25 positive.

In some embodiments, the inhibitor capable of inhibiting the interaction of CD80 and/or CD86 with CD28 is selected from the group consisting of small molecules, proteins, nucleic acids, lipids, sugars, and combinations thereof. In one embodiment, the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof. In another embodiment, the variant of the above antibody is an antigen binding fragment. In another embodiment, the variant of the cell surface molecule is a fusion protein. In another embodiment, the inhibitor is selected from the group consisting of an anti-CD 80 antibody, an anti-CD 86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD 28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, and a CD28-Ig fusion protein. In another embodiment, the CTLA4-Ig fusion protein is abatacept or belatacept. In some embodiments, CD80 and/or CD86 is expressed by an antigen presenting cell and CD28 is expressed by a T cell. In particular embodiments, the inhibitory factor capable of inhibiting the interaction of CD80 and/or CD86 with CD28 may be an anti-CD 80 antibody and/or an anti-CD 86 antibody or CTLA4-Ig fusion protein. As the inhibitor to be used in the present invention, CTLA4-Ig fusion protein can be mentioned as mentioned above. Binding to CD80/CD86 on antigen presenting cells, CTLA4-Ig fusion protein competes with CD28 as a costimulatory receptor on T cells, and as a result, acts to inhibit activation of T cells. In the present invention, as the CTLA4-Ig fusion protein, it is assumed that there are Abiradept (oracia (registered trademark)), Belacidept (belatadept), and Maxy-4. Belacicept (belatacept) contains 2 amino acid substitutions (L104E and A29Y) that significantly increase binding affinity to CD80 and CD86 (see Davies JK et al, Cell transfer. (2012); 21 (9): 2047-61, Adams AB et al, J Immunol. (2016) (197 (6): 2045-50)). Further, as an inhibitor which can be expected to have the same effect as that of CTLA4-Ig fusion protein, CD28-Ig fusion protein (Peach RJ et al, J Exp Med. (1994)180 (6): 2049 to 2058) can be mentioned. The inhibitor of the present invention may be used in the form of a nucleic acid. To cite an example, it is also conceivable: the nucleic acid encoding the CTLA4-Ig fusion protein is introduced into cells by an adenovirus vector or the like for expression. See, for example, Jin YZ et al, Transplant Proc, (2003); 35(8): 3156-9.

In some embodiments, the non-reactive cells may be cells induced by mixing an inhibitor such as an antibody, cells derived from a sample, and an antigen derived from the sample, an antigen not derived from the sample, or a substance containing the antigen. The antigen-containing substance may be a cell, and irradiation with radiation may be performed to prevent proliferation and activation of the cell.

In a particular embodiment, a pharmaceutical composition for treating or preventing a disease, disorder or condition of a subject arising from an antigen derived from or not derived from the subject may be provided, comprising a composition of the invention. Examples of the disease, disorder or condition of the subject caused by the antigen derived from the subject or the antigen not derived from the subject include, but are not limited to, diseases, disorders or conditions requiring immune tolerance such as transplant immune rejection, allergy, autoimmune disease, graft-versus-host disease, and immune rejection caused by transplantation of iPS cells or ES cells and cells, tissues or organs derived from these cells.

In some embodiments, the transplant immune rejection response is characterized by the transplantation of kidney, liver, heart, skin, lung, pancreas, esophagus, stomach, small intestine, large intestine, nerve, blood cells including cells of the immune system, bone, cartilage, blood vessels, cornea, eyeball, or bone marrow.

In the embodiment of the present invention in which the subject disease or the like is transplant immune rejection, the non-reactive cells can be induced by mixing an inhibitor such as an antibody, recipient-derived cells (PBMC or splenocytes), and a donor-derived antigen or a donor-derived antigen-containing substance. The antigen-containing material derived from a donor may be PBMCs, splenocytes, or cells derived from transplanted organs, etc.

In the embodiment of the present invention in which the disease or the like to be treated is an allergy, the anergic cell can be induced by mixing an inhibitor such as an antibody, a cell derived from a sample (PBMC or splenocyte), and an antigen that causes an allergy and is not derived from a sample.

In the embodiment where the target disease or the like to which the present invention is directed is an autoimmune disease, the non-reactive cells can be induced by mixing an inhibitor such as an antibody, cells derived from a sample (PBMC or splenocytes), and an antigen derived from the sample that causes the autoimmune disease.

In the embodiment where the subject disease or the like to which the present invention is directed is graft-versus-host disease, the anergic cell can be induced by mixing an inhibitor such as an antibody, PBMC or spleen cells of a donor to which a graft is applied, and an antigen derived from a recipient or a substance containing the antigen. The antigen-containing substance derived from the recipient may be PBMCs, splenocytes, cells around the site of the transplanted organ, or cells derived therefrom.

In the embodiment where the subject disease or the like to which the present invention is directed is immune rejection reaction caused by transplantation of iPS cells or ES cells and cells, tissues or organs derived from these cells, the non-reactive cells can be induced by mixing an inhibitor such as an antibody, cells derived from a sample (PBMC or splenocytes), and cells used in transplantation differentiated from iPS cells or ES cells.

Examples of treatments for diseases and the like according to the present invention are shown below, but the present invention is not limited to the following.

(allergy and autoimmune diseases)

In allergic reactions and autoimmune diseases, macrophages obtained from peripheral blood of a patient are differentiated into dendritic cells (macrophage-derived dendritic cells) having high antigen presenting ability by a conventional method, and the cells after irradiation with radiation (γ rays) present antigens causing allergic reactions and autoimmune diseases, and are cultured together with T cell populations obtained from peripheral blood of the same patient for 1 to 2 weeks in the presence of an appropriate inhibitor such as anti-CD 80 antibody and/or anti-CD 86 antibody or CTLA4-Ig fusion protein, to obtain antigen-specific non-reactive cells causing allergic reactions and autoimmune diseases. By administering the anergic cells to a patient, specific immune tolerance against an antigen causing allergy or autoimmune disease is induced, and the prevention and treatment of allergy or autoimmune disease are provided. The administration frequency may be multiple times depending on various conditions such as prophylactic treatment, therapeutic treatment, and severity of symptoms.

(graft versus host disease)

In contrast to treatment for graft-versus-host disease, cells that can cause graft-versus-host disease, such as PBMC or T cells from a donor who provides a graft, host-derived PBMC that has been irradiated with radiation (γ rays), or other cells are co-cultured for 1 to 2 weeks in the presence of an appropriate inhibitor, such as anti-CD 80 antibody and/or anti-CD 86 antibody or CTLA4-Ig fusion protein, to obtain host-specific non-reactive cells. By administering the anergic cell to a host, the reaction to the host (induction of immune tolerance) caused by a graft that is a cause of graft-versus-host disease is suppressed, thereby preventing and treating graft-versus-host disease. The administration frequency may be multiple times depending on various conditions such as prophylactic or therapeutic treatment, transplanted tissue, size thereof, severity of symptoms, and the like.

(immunological rejection reaction caused by transplantation of iPS cells or ES cells and cells, tissues or organs derived from these cells)

For application to therapy using iPS cells or ES cells, cells or dendritic cells used for transplantation differentiated from iPS cells or ES cells are irradiated with radiation (γ rays), and the cells and PBMCs or T cell population of a patient to be transplanted are co-cultured for 1 to 2 weeks in the presence of an appropriate inhibitor such as an anti-CD 80 antibody and/or an anti-CD 86 antibody or CTLA4-Ig fusion protein, to obtain non-reactive cells specific to cells differentiated from iPS cells or ES cells. By administering the anergic cells to a host, thereby inducing immune tolerance specific to cells, tissues, and organs derived from transplantation of iPS cells or ES cells, rejection reactions to them are prevented and treated. The number of administration may be multiple times depending on whether prophylactic therapy or treatment is performed, and further, various conditions such as transplanted tissue, size thereof, and severity of symptoms.

(method for producing drug)

In another aspect of the present invention, the present invention provides a method for producing a cell-containing drug, the method comprising the steps of: (A) mixing an inhibitor such as an antibody capable of inhibiting the interaction between CD80 and/or CD86 and CD28, cells derived from a sample, and an antigen derived from the sample, an antigen not derived from the sample, or a substance containing the antigen; (B) confirming that the cell product obtained by the mixing contains CD 8-positive cells; and (C) confirming that the cell product contains at least 1 cell out of FOXP3 positive and CD4 positive cells. The inventors of the present invention found that: the inclusion of CD8 positive cells in a mixture of anergic T cells is important for exerting sufficient immunosuppressive capacity. Therefore, in the manufacture of a medicament comprising T cells in which anergy is induced, it is important to ensure that CD8 positive cells are contained. The thus-produced medicament can be used for treating or preventing a disease, disorder or condition in a subject which is caused by an antigen derived from or not derived from the subject. Any inhibitor can be used as long as it produces an inhibitor such as CD8 positive cells.

In some embodiments, the presence of CD 8-positive cells and the presence of at least 1 of FOXP 3-positive cells and CD 4-positive cells in the cell product indicates that the cell product is capable of being used as a medicament.

In the step (B), CD8 can be detected using an anti-CD 8 antibody in order to confirm that CD 8-positive cells are contained. In the step (C), in order to confirm at least 1 cell including FOXP 3-positive and CD 4-positive, at least 1 of FOXP3 and CD4 can be detected using at least 1 of the anti-FOXP 3 antibody and the anti-CD 4 antibody. Specific methods for detection include, but are not limited to, flow cytometry (FACS), western blotting, and RNA detection. Preferably, the specific method used for detection is FACS. The detection of RNA can be carried out by a method known in the art, such as PCR. Although FOXP3 is expressed intracellularly, it can be stained intracellularly by performing an osmotic treatment after fixing the cells, and detection can be similarly performed by a method such as FACS. Suitable commercially available products for use in this method include eBioscience^TMHuman Regulatory T Cell Staining Kit#3(cat#88-8995-40)、eBioscience^TM Mouse Regulatory T Cell Staining Kit#1(cat#88-8111-40)、eBioscience^TMHuman/Non-Human primer Regulation T Cell Staining Kit #1(cat #88-4999-40) and eBioscience^TMHuman Regulatory T Cell wheel Blood stabilizing Kit (cat #88-8996-40), etc.

(quality control method)

In another aspect, the invention provides a method for quality management of a cell preparation (cell-containing drug). More specifically, the present invention provides a method for managing the quality of a cell-containing drug for treating or preventing a disease, disorder or condition of a sample due to an antigen expressed by cells derived from the sample or an antigen not derived from the sample, the method comprising the steps of: (A) confirming that the cells contain CD8 positive cells; and (B) confirming that the cells contain at least 1 cell selected from the group consisting of FOXP 3-positive cells and CD 4-positive cells. In order to maintain constant quality (sufficient immune tolerance), it is important to manage whether or not CD8 positive cells are included in a drug containing cells for treating or preventing a disease, disorder or condition of a specimen caused by an antigen expressed by the cells derived from the specimen or an antigen not derived from the specimen.

In the step (a), CD8 can be detected using an anti-CD 8 antibody in order to confirm that CD 8-positive cells are included. In the step (B), in order to confirm that the cells include at least 1 of FOXP 3-positive and CD 4-positive cells, at least 1 of FOXP3 and CD4 can be detected using at least 1 of the anti-FOXP 3 antibody and the anti-CD 4 antibody. Specific means for detection include, but are not limited to, FACS, Western blotting, PCR, and the like. Although FOXP3 is expressed intracellularly, it can be stained intracellularly by performing an osmotic treatment after fixing the cells, and detection can be similarly performed by a method such as FACS.

A typical example of the method for producing the cell preparation and managing the quality of the cell preparation of the present invention is shown below.

(production and quality control of cell preparation containing non-reactive T cells)

1. Raw material of biological origin and corresponding situation thereof

In one embodiment, in the process for producing an anergic T cell, a biogenic raw material conforming to the biogenic raw material standard shown in table 1 is used.

Administration of the anergic T cells may be performed after performing organ transplantation (e.g., liver transplantation) from a donor to a recipient. Donor-derived monocytes as a material for autologous non-reactive T cells are contained in a donor organ (e.g., liver) in a state in which viruses are not removed, and the donor organ (e.g., liver) is transplanted into a recipient in a state in which the donor-derived monocytes are contained. Therefore, it is considered that the donor-derived monocytes used as a material for the autoreactive T cells do not belong to a biologically-derived material.

[ Table 1]

Table 1 list of biogenic materials

Belacicept (belatacept) (e.g., available from Bristol-Myers Squibb, New York, NY)

2. Method for preparing cell preparation

In one embodiment, the cell preparation can be made as follows. Various numerical values and the like shown below are typical examples, and those skilled in the art can produce cell preparations with appropriate modifications.

1) Approximately 19 days before administration, the donor was subjected to blood component separation in a medical institution, and the product of the blood component separation from the donor was irradiated with 30Gy of radiation and, after losing the cell growth ability, was transported to a cell culture processing facility where cell processing was performed.

2) After receiving the separated product of donor blood components, donor monocytes were separated and recovered by density gradient centrifugation in a cell culture processing facility, and then frozen in 2 portions and stored at-80. + -. 10 ℃.

3) Approximately 14 days prior to administration, the recipient is subjected to blood component separation in a medical institution, and the product of the blood component separation from the recipient is transported to a cell culture processing facility where cell processing is performed.

4) After receiving the product of the separation of the recipient blood components, the recipient monocytes are separated and recovered by density gradient centrifugation at a cell culture processing facility, and co-cultured with the thawed donor monocytes and an inhibitor such as anti-CD 80 antibody, anti-CD 86 antibody or CTLA4-Ig fusion protein.

5) Medium exchange was performed around 7 days prior to administration. The intermediate product cultured for 7 days was recovered and co-cultured with thawed donor monocytes and inhibitors such as anti-CD 80 antibody, anti-CD 86 antibody or CTLA4-Ig fusion protein.

6) On the day of administration, the processed cells were collected by density gradient centrifugation, washed, and then filled in physiological saline.

7) Delivered to a medical facility where it is administered to a recipient.

(representative example of production and quality test flow)

3. In-process management test

In one embodiment, the production process may be subjected to the in-process management test shown in table 2. Various numerical values and steps shown below are representative examples, and those skilled in the art can perform the in-process management test with appropriate modifications.

[ Table 2]

TABLE 2 in-Process management test List

4. Standard test and characteristic analysis test

In one embodiment, the final article may be used to perform the standard tests set forth in table 3. The steps shown in table 3 are representative examples, and those skilled in the art can perform standard tests and property analysis tests with appropriate modifications. For example, as a modification thereof, the standard exemplified in example 8 may be cited. When the result is unclear when the induced suppressive T cells are administered, the shipment of the clinical test product can be judged by referring to the result of the in-process management test.

In addition, the characteristic analysis test described in table 3 can be performed using the cells and the final product in the production process.

[ Table 3]

TABLE 3 Final product Standard test List

As described in the present specification, the clinical test product standard test may include appearance, cell number, viable cell rate, cell surface markers (CD3, CD4, CD8, CD25, CD44, CD45RA/CD45RO), production process-derived impurities (donor-derived cells, medium components, anti-CD 80 antibody, anti-CD 86 antibody, cell cryoprotectant components, gravity separation fluid components), virus negative test, sterility test, mycoplasma negative test, and endotoxin. Examples of the pharmacodynamic test include a cytokine production test or a tritium uptake test based on Mixed Lymphocyte Reaction (MLR) using cultured cells. The reference value of the cellular phenotype is, for example, a typical reference value of the CD 3-positive cell ratio, which is 50% or more of the reference values in the table, or may be, 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 may be a numerical value therebetween (may be set on a scale of 1%, 0.5%, or the like). The percentage of CD 8-positive CD 44-positive cells among CD 3-positive cells may be typically 10% or more of the reference value in the table, 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, or the like. The ratio of CD 4-positive CD 44-positive cells in CD 3-positive cells may not be set, 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, or the like may be setA reference value. The ratio of CD 8-positive CD45 RA-negative cells in CD 3-positive cells may not be set, 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, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, or the like may be set as a reference value. The ratio of CD 8-positive CD45 RA-negative CD45 RO-positive cells in CD 3-positive cells may not be set, 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, or the like may be set as a reference value. The ratio of CD 4-positive CD45 RA-negative CD45 RO-positive cells in CD 3-positive cells may not be set, 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, or the like may be set as a reference value. The percentage of CD 4-positive CD25 cells in CD 3-positive cells may be typically 5% or more of the reference value in the table, 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, or the like. The number of cells is typically 1X 10⁹More than one as a reference, or may be, for example, 1 × 10⁸More than one, 5 × 10⁸More than one, 1 × 10⁹More than one, 2 × 10⁹More than one, 3 × 10⁹More than one, etc. The living cell rate may be typically 70% or more, or may be 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or the like.

(composition of the final product)

For example, the final product was composed of the components shown in the following table. Those skilled in the art can also modify the composition of these criteria to construct products for regenerative medicine and the like by appropriate modification.

[ Table 3-1]

List of table composition

Composition of matter	Composition of
		Autologous non-reactive cells	1×10⁹More than one
Physiological saline	100mL
		Human serum albumin	1％

The standard test and the characteristic analysis test can be specifically carried out by those skilled in the art by appropriately referring to the technical matters described in the present specification and by making changes as necessary.

5. Method for administering regenerative medicine or the like

In one embodiment, the administration is performed once 14 days after the organ transplant. The administration method, the period thereof, and the like can be specifically carried out by those skilled in the art by making appropriate changes as needed with reference to the technical matters described in the present specification.

(kit)

In another aspect, the invention provides a kit for making a cell preparation. In yet another aspect, the present invention provides a kit for quality management of a cell preparation. In detail, the present invention provides a kit for the manufacture of a medicament comprising a mixture of cells, the kit comprising: (A) inhibitors such as antibodies capable of inhibiting the interaction of CD80 and/or CD86 with CD 28; (B) means for detecting CD 8; and (C) a means for detecting at least 1 of FOXP3 and CD 4.

In some embodiments, the presence of CD 8-positive cells and the presence of at least 1 of FOXP 3-positive cells and CD 4-positive cells in the cell mixture indicates that the cell mixture is capable of being used as a medicament.

The medicament comprising a cell mixture manufactured using the kit of the present invention comprises: the anergic CD 8-positive T cells, and at least 1 of the FOXP 3-positive cells and the CD 4-positive cells, which induce anergy, can be used for treating or preventing a disease, disorder or condition in a subject due to an antigen derived from or not derived from the subject.

In another aspect, there is provided a kit for managing the quality of a cell-containing drug for treating or preventing a disease, disorder or condition of a subject due to an antigen expressed by cells derived from the subject or an antigen not derived from the subject, the kit comprising: (A) means for detecting CD 8; and (B) a means for detecting at least 1 of FOXP3 and CD 4. In some embodiments, the means for detecting CD8 comprises an anti-CD 8 antibody, and the means for detecting at least 1 of FOXP3 and CD4 comprises at least 1 of an anti-FOXP 3 antibody and an anti-CD 4 antibody. Detection can be performed by, for example, FACS, western blot, PCR, or the like.

(autologous regulatory T cell production step)

A typical method for producing autologous regulatory T cells will be described below.

Prior confirmation

In one embodiment, the prior confirmation may be performed as follows. Various numerical values, reagents, steps, and the like, which are exemplified below, are representative examples, and those skilled in the art can carry out the production confirmed in advance with appropriate modifications.

Infectious diseases of donors and patients were screened, and all of HBs antigen, HCV antibody, HIV-1/2, and HTLV-1 antibody were confirmed to be negative for the donors.

1. Representative examples of isolation (under aseptic conditions) of donor lymphocytes

In one embodiment, isolation of donor lymphocytes can be made in the following manner. The following examples of various values, reagents, procedures, etc. are representative examples, and those skilled in the art can isolate donor lymphocytes by appropriate modification.

Collecting donor lymphocytes in a collection bag by blood component separation, and irradiating the collection bag with radiation.

The peripheral blood mononuclear cells irradiated with the radiation ray are put into a centrifuge tube containing an appropriate amount of Ficoll-Paque PREMIUM (GE Healthcare #17-5442-02), Lymphocyte separation Solution (Nacalai Tesque #20828), or the like (for example, 20mL), and centrifuged at 22 ℃ for 20 minutes at 860G (the accelerator of the centrifuge is set to slow, and the stopper is set to slow).

Discard the supernatant and transfer the cell suspension containing the lymphocyte layer to another centrifuge tube (e.g., 2 in a 50mL centrifuge tube).

The cell suspension is added with physiological saline (for example, an appropriate amount of the total liquid amount to 50mL) to a centrifuge tube containing the cell suspension, and the mixture is thoroughly mixed by repeatedly sucking and discharging the mixture with a syringe (for example, a 50mL syringe with an 18G syringe needle) or a pipette.

The centrifugation was carried out at 22 ℃ for 10 minutes at 500G (the accelerator and brake of the centrifuge may be set to high).

The supernatant is discarded, physiological saline (for example, an appropriate amount of total liquid volume to 50mL) is added again, and the cell pellet is repeatedly aspirated and discharged with a pipette, and is mixed thoroughly.

The centrifugation was carried out at 22 ℃ for 5 minutes at 500G (the accelerator and brake of the centrifuge may be set to high).

Discard the supernatant.

The culture solution of ALyS505N-0 (cell science research institute (CSTI)1020P10)) containing the plasma collected from the donor in advance was added to the cell pellet (for example, an appropriate amount of the total solution amount to 31 mL), and the mixture was repeatedly aspirated and discharged with a pipette to mix the solution well.

The number of cells and the number of viable cells were confirmed by taking out an appropriate amount (e.g., 0.3mL) with a syringe (e.g., 1mL syringe with 18G injection needle) or pipette.

2. Cryopreservation of donor lymphocytes (performed under sterile conditions)

In one embodiment, cryopreservation of donor lymphocytes can be performed as follows. Various numerical values, reagents, procedures, and the like, which are exemplified below, are representative examples, and those skilled in the art can perform cryopreservation of donor lymphocytes as appropriate.

Aseptically unsealing a freezer bag (e.g., Frozen bag F-05025 mL freezer bag Nipro Corporation 89-101), and filling the label with the necessary items (date, manufacturing number, donor name).

The cell suspension is removed with a syringe (e.g., a 30mL syringe with an 18G needle) and placed in a freezer bag.

ACD solution (Terumo Corporation TP-A05ACD, for example, 2mL relative to 15mL of cell suspension) was added to a freezing bag containing the cell suspension, and the bag was cooled for about 10 minutes in a cold-keeping agent cooled at 4 ℃.

CP-1 (cell cryoprotectant CP-1 from Jidong pharmaceutical industries, 551-. At this point, the freezer bag was slowly stirred.

Using a syringe, evacuate all air in the freezing bag and its port.

The frozen bag is sealed by a plastic tube sealer, first cooled at 4 ℃ for about 5 to 10 minutes, and then stored in an ice house at-80 ℃.

3. Thawing of donor lymphocytes (performed aseptically)

In one embodiment, thawing of the donor lymphocytes can be performed as follows. The various numerical values, reagents, procedures, and the like, which are exemplified below, are representative examples, and those skilled in the art can thaw donor lymphocytes with appropriate modifications.

The frozen bags of preserved donor cells are thawed, for example, in a 37 ℃ incubator. The subsequent operations are preferably carried out under aseptic conditions.

Using a syringe (e.g., a 50mL syringe with an 18G needle), the cell suspension is removed from the thawed cryobag and transferred to a centrifuge tube (e.g., 250mL centrifuge tubes each 12.5 mL).

For example, 5% albumin solution (Nihon Pharmaceutical co., Ltd, 123146364) was added to a centrifuge tube containing the cell suspension, and 12.5g/250mL of the blood albumin solution was injected 5% intravenously (e.g., 37.5mL for 12.5mL of the cell suspension), followed by thorough mixing. Then, it was left to stand for about 5 minutes.

For example, the centrifugation is performed at 600G at 22 ℃ for 10 minutes (for example, it is preferable to set the accelerator and the brake of the centrifuge to be fast and slow).

The supernatant is discarded gently, and an appropriate liquid such as albumin-containing physiological saline for washing (for example, 25mL of 5% albumin solution or 19mL of physiological saline) is added to the cell pellet to suspend the cell pellet.

For example, the centrifugation is performed at 600G at 22 ℃ for 10 minutes (for example, it is preferable to set the accelerator and the brake of the centrifuge to be fast and slow).

The supernatant was gently discarded, and the cell pellet was suspended by adding ALyS505N culture medium (e.g., 10mL relative to a 50mL centrifuge tube).

In a culture bag (for example, Nipro Corporation 87598Nipro medium ALyS505NB10) containing ALyS505N-0 culture solution or a liquid equivalent thereto, an anti-human CD80 antibody (for example, m2D10.4; Cat. No.16-0809-85, eBioscience) and an anti-human CD86 antibody (for example, IT 2.2; Cat. No.16-0869-85, eBioscience) were added at a final concentration of, for example, 10. mu.g/mL, respectively (or an equivalent inhibitor such as CTLA4-Ig fusion protein (for example, Beilacipt) was added), and the above cell suspension was injected into the culture bag by a syringe (for example, a 20mL syringe with an 18G needle). In one example, the total volume of fluid in the bag is about 840 mL.

4. Isolation of patient lymphocytes-initiation of one culture (performed aseptically)

In one embodiment, isolation of the patient's lymphocytes can be performed as follows. The following examples of various values, reagents, procedures, etc. are representative examples, and those skilled in the art can isolate lymphocytes from a patient by appropriate modification.

For plasma collected from the patient, the inactivation is carried out beforehand in a thermostatic bath, for example heated at 56 ℃ for 30 minutes. Will not be directly stored by the user.

Peripheral blood collected from the patient is put into a centrifuge tube containing an appropriate amount of a suitable medium such as Ficoll-Paque (e.g., 20mL), and centrifuged at 22 ℃ for 20 minutes at 860G, for example (for example, it is preferable to set the accelerator and brake of the centrifuge to slow).

Discard the supernatant and transfer the cell suspension containing the lymphocyte layer to another centrifuge tube (e.g., 2 in a 50mL centrifuge tube).

The cell suspension is added with physiological saline (for example, an appropriate amount of the total liquid amount to 50mL) to a centrifuge tube, and the mixture is thoroughly mixed by repeatedly sucking and discharging the mixture with a pipette.

For example, the centrifugation is performed at 22 ℃ for 10 minutes at 500G (the accelerator and brake of the centrifuge may be set to be fast).

For example, the centrifugation is performed at 22 ℃ for 5 minutes at 500G (the accelerator and brake of the centrifuge may be set to be fast).

The supernatant is discarded, and for example, ALyS505N-0 culture medium (for example, 10mL) is added to the cell pellet to suspend the cell pellet, thereby preparing a cell suspension (for example, ALyS505N-0 culture medium is added to 20mL in total). Here, about 0.5mL of the cell suspension was taken out, and the number of cells, the number of viable cells, and the expression of surface antigen were confirmed.

Patient-derived inactivated plasma was added to the culture bag containing the donor cells and the inhibitor such as antibody in the ALyS505N-0 culture solution prepared in "3. thawing of donor lymphocytes".

The patient-derived cell suspension is injected and added to the culture bag with a syringe (e.g., a 20mL syringe with an 18G injection needle), and the culture bag is sealed with a plastic tube sealer. In one example, the total volume of fluid in the bag is about 1000 mL.

The cells are cultured in an incubator at 37 ℃ for 1 week, for example.

5-1 Medium exchange (e.g., week 1, preferably done aseptically)

In one embodiment, medium exchange may be performed as follows. The following examples of various values, reagents, steps, etc. are representative examples, and those skilled in the art can perform medium exchange with appropriate modifications.

The culture bag is taken out of the incubator, and the contents are dispensed into a centrifuge tube (for example, 4 pieces of 225mL centrifuge tube).

For example, the centrifugation is performed at 600G at 22 ℃ for 10 minutes (for example, it is preferable that the accelerator and the brake of the centrifuge are set to be fast).

The supernatant is discarded gently, and for example, ALyS505N-0 culture medium is added to the cell pellet to suspend the cell pellet, thereby preparing a cell suspension (for example, ALyS505N-0 culture medium is added up to 20mL in total). Here, about 0.3mL of the cell suspension was taken out, and the number of cells and the number of viable cells were confirmed.

For example, the addition is carried out by injecting the cell suspension into a culture bag containing the ALyS505N-0 culture solution with a syringe (e.g., a 20mL syringe with an 18G injection needle).

Each of the anti-human CD80 antibody (e.g., 2D10.4) diluent and the anti-human CD86 antibody (e.g., IT2.2) diluent is injected into the culture bag with a syringe (e.g., 20mL syringe with 18G needle) so that the final concentration becomes 10 μ G/mL (or an inhibitor such as CTLA4-Ig fusion protein (e.g., belatacept) can be used).

5-2 thawing/antigen restimulation of Donor lymphocytes to the beginning of Secondary culture (e.g., week 1, performed under sterile conditions)

In one embodiment, the thawing/antigen restimulation to initiation of secondary culture of donor lymphocytes can be performed as follows. The following examples of various values, reagents, procedures, etc. are representative examples, and those skilled in the art can perform thawing of donor lymphocytes, antigen restimulation, and initiation of secondary culture, as appropriate.

Thawing the frozen bags of preserved donor cells and patient-derived inactivated plasma in, for example, a 37 ℃ incubator. The subsequent operations are preferably carried out under aseptic conditions.

Using a syringe (e.g., a 50mL syringe with an 18G needle), the donor cell suspension is removed from the thawed cryobag and transferred to a centrifuge tube (e.g., 2 in a 50mL centrifuge tube).

A5% albumin solution (for example, about 50mL in total for 250mL centrifuge tubes) was added to the centrifuge tube containing the donor cell suspension, and the mixture was thoroughly mixed. Then, it was left to stand for about 5 minutes.

For example, the centrifugation is performed at 600G at 22 ℃ for 10 minutes (accelerator of the centrifuge is set to fast, brake is set to slow).

The supernatant was discarded gently, and albumin-containing physiological saline for washing (for example, 25mL of 5% albumin solution and 19mL of physiological saline) was added to the cell pellet to suspend the cell pellet.

For example, the centrifugation is performed at 600G at 22 ℃ for 10 minutes (accelerator of the centrifuge is set to fast, brake is set to slow).

The supernatant is gently discarded, and, for example, ALyS505N-0 culture medium (e.g., 10mL relative to a 50mL centrifuge tube) is added to the cell pellet and suspended.

The culture bag containing the patient cells and the inhibitor such as the antibody in the ALyS505N culture solution prepared in "3. thawing of donor lymphocytes" was filled with the thawed inactivated plasma (for example, 10mL) derived from the patient by injecting it with a syringe (for example, a 20mL syringe with an 18G injection needle), and the cell suspension was injected into the culture bag with a syringe (for example, a 20mL syringe with an 18G injection needle). In one example, the total volume of fluid in the bag is about 1000 mL.

The bags were sealed using a plastic tube sealer.

The cells are cultured in an incubator at 37 ℃ for 1 week, for example.

6. Detection in Secondary culture (cultured cell removal test)

In one embodiment, the detection in the secondary culture can be performed in the following manner. The following examples of various numerical values, reagents, procedures, etc. are representative examples, and those skilled in the art can perform detection in secondary culture with appropriate modifications.

Typically, a small amount of culture solution is taken out from the culture bag on the 3 rd day from the start of the secondary culture (10 th day in total of the culture), and mycoplasma contamination and the like are detected.

7. Recovery/filling of cultured lymphocytes (performed under sterile conditions)

In one embodiment, the recovery/filling of cultured lymphocytes may be performed as follows. The following examples of various numerical values, reagents, steps, etc. are typical examples, and those skilled in the art can collect and fill cultured lymphocytes by appropriate modification.

For example, the culture bag is taken out from the incubator from the 7 th day (total 14 th day of culture) after the start of the secondary culture, and the contents are dispensed into a centrifuge tube (for example, 4 roots in a 225mL centrifuge tube).

For example, the centrifugation is performed at 600G at 22 ℃ for 10 minutes (the accelerator and brake of the centrifuge may be set to be fast).

The supernatant was discarded gently, and the cell pellet was suspended by adding physiological saline.

For example, the centrifugation is performed at 600G at 22 ℃ for 10 minutes (for example, it is preferable that the accelerator and the brake of the centrifuge are set to be fast).

The supernatant is discarded gently, and physiological saline (e.g., 10mL) is added to the cell pellet to suspend the cell pellet, thereby preparing a cell suspension.

The cell suspension is gently added to a centrifuge tube (e.g., a 50mL centrifuge tube) containing an appropriate amount of Ficoll-Paque (e.g., 20mL) for stacking.

For example, centrifugation is performed at 22 ℃ for 20 minutes at 860G (the accelerator and brake of the centrifuge are set to slow).

Discard the supernatant and transfer the cell suspension containing the lymphocyte layer to another centrifuge tube (e.g., a 50mL centrifuge tube).

The cell suspension is added with physiological saline (for example, an appropriate amount of the total liquid amount to 50mL) to a centrifuge tube containing the cell suspension, and the mixture is thoroughly mixed by repeatedly aspirating and discharging the mixture with a syringe (for example, a 50mL syringe with an 18G injection needle).

For example, the centrifugation is performed at 22 ℃ for 10 minutes at 500G (the accelerator and brake of the centrifuge may be set to be fast).

The supernatant was left at about 5mL, and the remainder was discarded, and the mixture was thoroughly mixed by repeatedly aspirating and discharging with a pipette.

Adding physiological saline (for example, an appropriate amount of total liquid volume to 50mL), and repeating aspiration and discharge with a syringe (for example, a 50mL syringe with an 18G injection needle) to mix them thoroughly (a).

For example, centrifugation is performed at 22 ℃ for 5 minutes at 500G (the accelerator and brake of the centrifuge may be set to be fast) (b).

The supernatant was left at about 5mL, and the remainder was discarded, and the mixture was repeatedly aspirated and discharged by a pipette (c).

Further repeating the above (a), (b) and (c) 2 times.

The supernatant after the final centrifugation is removed in an appropriate amount (e.g., 4mL) for sterility testing and mycoplasma testing.

The cell suspension is suspended again by adding physiological saline, and the cell suspension is transferred to a final container (for example, a bottle of 100mL physiological saline). An appropriate amount (e.g., 4mL) was taken out, and the cell number, viable cell number, expression of surface antigen and endotoxin content of the final product were confirmed.

8. Secondary package

In one embodiment, secondary packaging may be performed as follows. The following examples of various values, reagents, steps, etc. are representative examples, and those skilled in the art can perform secondary packaging with appropriate modifications.

Typically, the label is attached to the container by inputting the printed subject ID, the product number, and the service life on the label based on an appropriate standard (typically, NUHCPC-M-12-ATREG).

Make "usage/dosage/efficacy or effect and attention on usage or attention on operation" based on a suitable reference (typically NUHCPC-PMF-ATREG 14).

The test article and "usage/dose/efficacy or effect and attention on use or attention on operation" are contained in a plastic bag with a clip.

The container is placed in the transfer container and stored in the monitoring unit until shipment.

(remarks)

In the present specification, "or" is used when "at least 1 or more of the items listed in the text can be adopted. The same applies to "or". In the present specification, the case where "within a range of 2 values" is explicitly described, the range also includes 2 values themselves.

All references cited in the present specification, such as scientific documents, patents, and patent applications, are incorporated by reference in their entirety into the present specification to the same extent as the contents of each of the individual references are specifically described.

The foregoing description shows preferred embodiments for ease of understanding the invention. The present invention will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is defined only by the claims.

Examples

The present invention will be described more specifically below based on examples. However, the present invention is not limited to these examples. It is noted that throughout this specification, all documents cited are directly incorporated into this application by reference.

Example 1 loss of function test

In this example, a loss-of-function test was performed to identify components required to induce immune tolerance. The following description is made.

(materials and methods)

(Generation of non-reactive cells)

The experiments (1-3) were carried out according to methods already described in the literature. Briefly, spleen was extracted from C57BL6 (hereinafter referred to as B6) and BALB/C mice (CLEA Japan, Inc., CHARLES RIVER LABORATORIES JAPAN, INC., etc.) and hemolyzed to obtain spleen cells (lymphocytes), which were then converted into 4X 10 cells⁶The individual cells/ML were conditioned with RPMI1640 medium (Sigma; R8758-500MK) containing 10% inactivated Fetal Calf Serum (FCS) (SIGMA #172012-500ML Lot 11D257 or biosera # FB-1380/500 Lot.015BS482). After 30Gy of radiation (γ -rays) was applied to BALB/c spleen cells as stimulators, the ratio of 1: 1 and B6 spleen cells were mixed, and hamster anti-mouse CD80 antibody (16-10A1) (Cat. No.16-0801-82) manufactured by eBioscience and rat anti-mouse CD86 antibody (GL1) (Cat. No.14-0862-82) were added to the mixture so that the final concentration of each was 10. mu.g/mL, and the mixture was placed in 12-well plates (Corning, #3513) (1-2.5mL), 6-well plates (Corning, Cat. No. 16) (3-6 mL), 6-cm dishes (Greiner CELLSTAR (registered trademark) dish, Cat. No.628160) (3-6 mL) or 10-cm dishes (Corning, Cat. No. 430) (10-15 mL) at 37 ℃ with 5% CO₂The culture was carried out in an incubator for 14 days. After the culture solution was removed by centrifugation on the 7 th day after the start of the culture, a culture solution containing BALB/c-derived radiation-irradiated spleen cells and anti-CD 80 antibody/anti-CD 86 antibody was newly added under the same conditions as at the start of the culture. After 14 days, cells were recovered to obtain non-reactive cells.

In some experiments, after reacting PE fluorescently labeled anti-mouse CD8 antibody (53-6.7; eBioscience, #12-0081-85) with non-reactive cells, the cells were screened for CD8 positive and CD8 negative by auto-MACS (Miltenyi Biotec) using anti-PE magnetic beads (Miltenyi Biotec # 1300-10-639). In addition, the cells were screened for CD19 positive and CD19 negative by the same procedure using a PE fluorescently labeled CD19 antibody (1D 3; eBioscience, # 12-0193-85). These cells were used for an immune response suppressing ability test. Furthermore, in some experiments, in order to identify regulatory T cells (regT cells) expressing FOXP3 by expression of cell surface antigens, B6-derived mice (4) genetically engineered in such a manner that responder cells express both FoXP3 and human CD2 were used, and auto-MACS using PE fluorescent-labeled anti-human CD2 antibody (RPA-2.10; eBioscience, #12-0029-42) and anti-PE magnetic beads were screened for human CD2 positive cells (FoxP 3-expressing regT cells) and human CD2 negative cells for immune response suppression test.

(evaluation of immune response suppressing ability)

Experiment (3) was carried out according to the methods already described in the literature. Briefly, after the ratio of non-reactive cells to splenocytes responding to B6 was adjusted to 1/2 to 1/16, splenocytes obtained from freshly harvested B6 mice and BALB/c mice were added to a 96-well plate (manufactured by Corning Corp., Cat. No.3799) at 1X 10⁶1/mL of individual cells/mL: 1 Mixed culture (final 200. mu.L/well, 4 wells), 5% CO at 37 ℃₂The culture was carried out in an incubator (1X 10 spleen cells of B6 mouse and BALB/c mouse, respectively, in each well⁵The number of cells and non-reactive cells is 1X 10⁵The number of cells from 1/2 to 1/16). Adding on day 4 after the start of culture³H-thymidine (10. mu.L) on day 5 from the start of culture (addition)³After 16 to 20 hours of H-thymidine), the cultured cells were recovered by Cell Harvester (Molecular Devices), and the measurement was carried out by a scintillation counter³H-thymidine uptake. Non-stimulated original B6 lymphocytes³A graph was prepared with the H-thymidine uptake being 1, and comparative studies were performed.

(results)

The results are shown in FIG. 1. The value indicating that no stimulation is to be applied (initial)³The average value of the H-thymidine uptake was calculated assuming 1And (4) outputting the value. As shown in FIG. 1a, the fully cultured B6 spleen cells without any selection were confirmed to have the effect of inhibiting the reaction of fresh (primary) B6 spleen cells with BALB/c spleen cells after culturing with stimulation of BALB/c spleen cells in the presence of anti-CD 80/86 antibody (hereinafter referred to as "antibody/stimulator treatment"). The total cultured cells (cell mixture) which are predicted to induce anergy include cells (CD 19-positive B cells) other than anergy CD 8-positive cells, anergy CD 4-positive cells, regulatory T cells (regT cells).

When an experiment was performed using a sample obtained by purifying CD 8-positive cells (almost CD 44-positive anergic CD 8-positive cells) from the antibody/stimulus-treated cell mixture, it was found that: even the cell population with only anergic CD 8-positive cells had inhibitory effects (black), and the remaining cell population with anergic CD 8-positive cells removed had no inhibitory effect (gray) (fig. 1 b).

As shown in fig. 1c, even the cell population containing only regT cells had the inhibitory effect (black) and the remaining cell population from which regT cells were removed had the same inhibitory effect (gray) after purifying human CD 2-positive cells (regT cells) from the cell mixture after antibody/stimulus treatment.

On the other hand, as shown in fig. 1d, when CD19 positive cells (B cells) were purified from the cell mixture after antibody/stimulus treatment, only the cell population of the B cells had no inhibitory effect (black), but the remaining cell population from which the B cells were removed had an inhibitory effect (gray).

Thus, in the absence of CD 8-positive cells, suppression of immune response, i.e., induction of immune tolerance, was significantly reduced, and even when CD 8-positive cells alone were used, induction of immune tolerance was strongly shown, so it is understood that CD 8-positive cells are cells necessary for induction of effective immune tolerance. Thus, it can be understood that: in the production of an immune tolerance-inducing cell product, it is important to confirm the presence or absence of CD8 positive cells in quality control.

The same inhibitory effect was observed for both the purified FOXP3 positive cell population and the cell population without FOXP3 positive cells, which is not contradictory to the CD8 positive cells having the ability to induce immune tolerance. In addition, it also coincides with the case where FOXP3 positive cells, which are widely known as regulatory T cells, have immunosuppressive ability. It is therefore assumed that: of the total anergic cells after antibody/stimulant treatment, cells that were effective for inducing immune tolerance were FOXP3 positive cells, as did anergic CD8 positive cells, and this mixture was most effective in inducing immune tolerance.

From the experiments screening for CD19, CD19 positive cells (B cells) did not have inhibitory effect, and it is understood that CD19 positive cells were not required.

(example 2: test for obtaining inhibitory function based on CD80/86 blockade)

In this example, it was confirmed that the immune tolerance-inducing ability (immune rejection-suppressing ability) of CD 8-positive T cells was achieved by anergy induction by antibody/stimulant treatment, and the same experiment was performed on FOXP 3-positive T cells.

(materials and methods)

In the same manner as in example 1, non-responsive cells were obtained by subjecting splenocytes obtained from a wild-type B6 mouse and a B6-derived mouse genetically modified to express both FoxP3 and human CD2 to antibody/stimulus treatment. The anergic cells and non-stimulated primary splenocytes obtained from wild-type B6 mice and B6-derived mice genetically modified to express both FoxP3 and human CD2 were reacted with PE fluorescently labeled anti-mouse CD8 antibody or PE fluorescently labeled anti-human CD2 antibody, respectively. Then, the cells were screened for CD8 positive and CD8 negative, or human CD2 positive and human CD2 negative by auto-MACS using anti-PE magnetic beads. Each cell selected was added to a mixed culture system on a 96-well plate, and the immunosuppressive ability thereof was investigated.

(results)

The results are shown in FIG. 2.

As shown in fig. 2a (values indicating that no stimulation is to be applied (initial)³The average value of the H-thymidine uptake was calculated as 1.) Shown, it is shown that: no reactionSexual CD8 positive cells had inhibitory effect (black) and CD8 positive cells isolated from naive splenocytes without stimulation had no inhibitory effect (grey). Furthermore, as shown in fig. 2b, the antibody/stimulant treated regT cells (black) also exhibited immunosuppressive potency, which was stronger than the original regT cells (grey) without stimulation.

From these results, it can be seen that: when CD8 positive cells induce immune tolerance (suppression of immune response), it is necessary to induce anergy using stimulation of BALB/c spleen cells in the presence of anti-CD 80/86 antibody, and the same situation occurs when regT cells exert a more potent immunosuppressive function. Namely, it was revealed that the anergic CD 8-positive cells and regT cells induced by the antibody/stimulus treatment had a strong immune tolerance-inducing ability.

(example 3 purification of tolerance-inducing ability after transplantation of non-reactive cells)

In this example, it was confirmed whether or not the purified cells after induction of anergy had tolerance-inducing ability after transplantation.

(materials and methods)

In the same manner as in example 1, non-responsive cells were obtained from splenocytes of wild type B6 mice and B6-derived mice genetically modified to express both FoxP3 and human CD2 by co-culture with stimulated BALB/c splenocytes in the presence of anti-CD 80/86 antibody. In some experiments, among the obtained non-reactive cells (total non-reactive cells), CD 8-positive cells, CD 4-positive cells, or human CD 2-positive cells were selected in the same manner as in example 1. For wild-type B6 mice, hearts of BALB/c mice were transplanted 3 days after irradiation with 2Gy of radiation (γ rays), and rejection of hearts was observed by administering the primary B6 splenocytes or the resulting anergic cells from the tail vein immediately after transplantation (or on the same day as heart transplantation). Each group was tested with 5 or more.

(results)

Fig. 3 shows the results thereof. As shown in fig. 3a, in the case of non-transplanted anergic cells, the transplanted heart was rejected in all cases up to about 2 weeks. In contrast, in total no reactivity is fineIn the cell administration group, the survival rate of the heart was improved depending on the number of cells administered, and the survival rate was improved by injecting 6X 10⁶The transplanted hearts survived in all mice even after 100 days of total anergic cells judged to induce tolerance to the transplanted hearts. As shown in fig. 3b, the induction of tolerance was observed only in the case where the recipient mice were irradiated with radiation and injected with non-reactive cells, and was not observed at all in the case where non-reactive cells were administered without radiation irradiation and in the case where primary cells were injected into the radiation-irradiated mice. In the case of administering a cell population in which human CD 2-positive cells (FoxP 3-expressing regT cells) or anergic CD 8-positive cells were removed from anergic cells to radiation-irradiated mice, 100% survival of the transplanted heart after 100 days as in the case of injecting total anergic cells was not observed, tolerance induction ability was significantly reduced, and it was confirmed that administration of total anergic cells (anergic cell mixture) was superior (fig. 3 c). In addition, upon administration of purified naive CD8 positive T cells, transplanted hearts were rejected in all mice, whereas tolerance was induced in 40% of the mice receiving administration when injected with anergic CD8 positive T cells (fig. 3 d). Furthermore, while there were mice that induced tolerance when naive human CD2 positive cells (FoxP3 expressing regT cells) were injected, those given anergic human CD2 positive cells (FoxP3 expressing regT cells) induced tolerance more efficiently (fig. 3 d). Therefore, the administration of the anergic cell mixture more effectively suppresses rejection of the transplanted heart than the administration of each of the anergic CD8, CD4, and FoXP3 positive cells alone, and further shows that bone marrow suppression by irradiation of radiation to the receptor or the like can more effectively induce tolerance.

This result is not in conflict with example 1, and does not negate the possibility of: even when purified non-reactive CD8, CD4, FoxP 3-positive cells, and initial FoxP 3-positive cells were administered alone, the number of cells was large, and the rejection of the transplanted heart was suppressed (tolerance induction was 100 days or more after survival of the transplanted heart) as compared with the injection of the non-reactive cell mixture. In addition, in theGenetically engineered mice were used in the experiments, and therefore Foxp 3-expressing cells, i.e., suppressor T cells, could be purified under cell survival conditions by expression of human CD2 on the cell surface, but intracellular Foxp3 expression was generally not detected by viable cells. It is known that: in the case of using CD 25-positive CD 4-positive T cells as substitutes, the immunosuppressive function of the cells is weak, containing a large amount of activated CD 4-positive T cells as compared with suppressive T cells. In addition, purification of anergic Foxp 3-expressing suppressor cells was very difficult because of the low number of Foxp 3-positive suppressor T cells. In practice, to obtain a1 × 10 display as shown in FIG. 3d⁶Individual human CD 2-expressing cells, FoxP 3-expressing cells, required 1 × 10⁷More than one total anergic cell. Thus, the results shown here indicate that the mixture is more practical and effective.

Example 4 ability of anergic cells derived from human PBMC)

Next, in this example, the ability of non-reactive cells derived from human PBMC was evaluated.

(materials and methods)

(Generation of non-reactive cells)

Experiments were carried out according to methods already described in the literature (5-8). Mononuclear Cells (PBMC) were isolated from human peripheral blood of 4 volunteers (2 humans as stimulators and 2 humans as effectors) using a Lymphocyte separation Media (Cat. No. C-44010, manufactured by Promo cell Co., Ltd.) or a Ficoll-Paque PREMIUM (GE Healthcare #17-5442-02), a Lymphocyte separation Solution (Nacalai Tesque #20828) and adjusted to 4X 10 by using a culture medium (ALyS 505N-0, manufactured by Biowest Corporation (CSTI)1020P10) to which 2% human AB-type serum (Bank) was added⁶Individual cells/ml. Stimulation PBMCs were irradiated with 30Gy of radiation (γ rays) at a rate of 1: 1 were mixed with responding PBMCs. To the above-mentioned mixed PBMC, mouse anti-human CD80 antibody (2D10.4) (Cat.No.16-0809-85) manufactured by eBioscience and mouse anti-human CD86 antibody (IT2.2) (Cat.No.16-0869-85) were added so that the final concentrations were 10. mu.g/mL, and 12-well plates (Corning, #3513), 6-well plates (Corning, Cat.No.3516) (, 6cm petri dishes (Greiner CELLSTAR (registered trademark) dish, Cat.No.628160), or 10cm dishes (Corning, Cat.No. 4) were used30167) At 37 ℃ and 5% CO₂The incubation was started in the incubator. After removing the culture solution by centrifugation on the 7 th day after the start of the culture, a culture solution containing radiation irradiation stimuli PBMC and anti-CD 80 antibody/anti-CD 86 antibody was added under the same conditions as at the start of the culture. Cells were recovered on day 14 of culture and the culture broth was washed away by centrifugation to obtain non-reactive cells. In some experiments, after staining non-reactive cells with PE fluorescently labeled mouse anti-human CD25 antibody (BC 96; eBioscience #12-0259-42), FITC fluorescently labeled mouse anti-human CD4 antibody (RPA-T4; eBioscience #11-0049-42), APC fluorescently labeled mouse anti-human CD8 antibody (RPA-T8; eBioscience #17-0088-42), each cell was purified using a JSAN cell sorter (Bay bioscience Co., Ltd.) and added to the mixed culture system.

(evaluation of immune response suppressing ability)

The obtained non-responsive cells were adjusted to 1/16 from 1/2, and added to 96-well plates (Cat. No.3799, manufactured by Corning) using newly collected PBMC of the same volunteers at 2X 10 each⁵4 wells per 200. mu.L/well in a mixed culture system at 37 ℃ with 5% CO₂Culturing in a thermostat. Adding on day 4 after the start of culture³H-thymidine (10. mu.L) on day 5 from the start of culture (addition)³After 16 to 20 hours of H-thymidine), the cultured cells were recovered by Cell Harvester (Molecular Devices), and the measurement was carried out by a scintillation counter³H-thymidine uptake. Non-irritating initial response to PBMC³A graph was prepared with the H-thymidine uptake being 1, and comparative studies were performed.

(results)

The results are shown in FIG. 4.

FIG. 4a shows the dose-dependent immune response suppression capacity of "total anergic cells after antibody/stimulus treatment". It was confirmed that the higher the ratio of total anergic cells after antibody/stimulus treatment, the higher the inhibitory effect.

FIG. 4b is: immunosuppressive ability was compared using a cell population (gray) obtained by purifying CD 25-positive cells including CD 8-positive CD 44-positive non-reactive cells and regT cells, which was obtained by sorting total non-reactive cells after antibody/stimulus treatment, a cell population (vertical streaks) obtained by purifying CD 4-positive CD 25-positive regT cells, and a cell population (black) obtained by adding purified CD 8-positive cells to purified CD 4-positive CD 25-positive regT cells, in this order from the left 3 rd histogram to the right. The results shown here confirm that: the regT cells do not have immunosuppressive ability when used alone, and exhibit immunosuppressive ability by coexisting with CD8 positive T cells.

FIG. 4c is: total non-reactive cells after antibody/stimulus treatment were screened for CD4 positive cells (grey) and CD4 negative cells (diagonal) and compared for immunosuppressive ability to the total non-reactive cells (black). The value indicating that no stimulation is to be applied (initial)³The average value of the H-thymidine uptake was calculated as 1. The CD 4-positive cells include regT cells, and the CD 4-negative cells include CD 8-positive cells. Both CD4 positive cells and CD4 negative cells showed immunosuppression.

From the results in fig. 4b that the regT cells alone failed to exhibit immunosuppressive ability and the regT cells exhibited immunosuppressive ability in the presence of CD8 positive cells, it is suggested that: in order to exert sufficient immunosuppressive ability of regT cells, it is necessary to have CD 8-positive cells or CD 4-positive cells other than regT cells. It is believed that the immunosuppressive ability of CD4 negative cells is derived from the anergic CD8 positive T cells contained therein.

(example 5: experiment showing antigen-specific inhibition)

In this experiment, donor-specific induction of tolerance was confirmed in total anergic cells after antibody/stimulus treatment.

(materials and methods)

BALB/c mouse-derived (H-2) was used in the presence of anti-CD 80/86 antibody in the same manner as in example 1^b) Stimulation of splenocytes by wild type B6 mice (H-2)^b) The obtained splenocytes were cultured to obtain non-reactive cells. BALB/c or CBA-transplanted mice (H-2) were treated 3 days after 2Gy irradiation^k) Wild type B6 mice of cardiac calm from the tailPulse administration of 5X 10⁶Rejection of the heart was observed for these anergic cells.

(results)

As shown in fig. 5, when B6 mice were injected with B6 mouse-derived anergic cells stimulated with splenocytes of Balb/C mice in the presence of anti-CD 80/86 antibody, rejection of the heart of transplanted Balb/C mice was 100% inhibited, and the heart was also survived, i.e., tolerance was induced, after 100 days. The heart of this third-party CBA mouse elicited rejection rapidly after transplantation, with 100% rejection occurring in about 50 days.

The above results show that: host-derived lymphocytes that have been reacted with an antigen in the presence of an anti-CD 80/86 antibody have the ability to suppress an immune response specific to the antigen.

(example 6 suppression of immune response based on initial cells with Selective early adhesion of anergic cells)

In this experiment, it was confirmed that: since the anergic cells bind to the donor (stimulus) cells more rapidly than the primary cells, the response and proliferation of the primary cells are inhibited.

(materials and methods)

In the same manner as in example 1, splenocytes obtained from B6 mice were stimulated with splenocytes obtained from BALB/c mice in the presence of anti-CD 80/86 antibody to obtain anergic cells. In this experiment, splenocytes newly obtained from B6 mice genetically modified to stably express fluorescent pigment GFP were used as effectors. 1X 10 wells were each contained in a 12-well plate (Cat. No.3799, manufactured by Corning Co., Ltd.)⁶The above-mentioned non-reactive cells were added to a 4ml mixed culture system of the above-mentioned response B6 spleen cells and stimulation (donor) BALB/c spleen cells at a ratio of 1/2 to response B6 spleen cells at 37 ℃ with 5% CO₂Culturing in a thermostat. The plate was observed from 1 day to 3 days later and photographs were taken.

(results)

Fig. 6 shows a representative image of a photograph. In the culture system to which only the lowermost row of non-reactive cells was added, clumps (clusters) of cells formed from 1 day after the culture, and in the system having only the primary cells and donor cells (row 2 from the top), clumps of cells were not observed. Therefore, it is presumed that the non-reactive cells may form a cell mass centering on the BALB/c-derived cells. After 2 days in culture, the primary B6 splenocytes also began to form a cell mass with increased fluorescence at this site. It is considered that this is caused by an increase in initial cells which fluoresce and adhere to BALB/c spleen cells and react, and such an increase in fluorescence was not observed in the upper row of initial cell culture alone and in the culture system to which non-reactive cells were added. Therefore, it is suggested that the proliferation reaction of the initial cells is suppressed by adding the non-reactive cells. The results show that: the anergic cells adhere to the donor cells more rapidly than the naive cells, inhibiting recognition (adhesion) of the naive cells to the donor cells, thereby inhibiting the naive cells from responding to proliferation.

Example 7 Properties of cells exerting immunosuppressive ability (non-response-inducing ability)

In this experiment, a confirmation experiment was performed that, among the anergic cells, the cells exhibiting immunosuppressive ability (anergic induction ability) were positive for CD 44.

(materials and methods)

In the same manner as in example 1, splenocytes obtained from wild-type B6 mice were stimulated with BALB/c cells in the presence of anti-CD 80/86 antibody to obtain anergic cells. After staining the non-reactive cells with APC fluorescently labeled anti-mouse CD8 antibody (53-6.7; eBioscience #17-0081-82 or BioLegend; #100730), PerCP/Cy5.5 fluorescently labeled anti-mouse CD4 antibody (RM 4-5; eBioscience #45-0042-82 or GK 1.5; BioLegend; #100434), and APC/Cy7 fluorescently labeled anti-mouse CD44 antibody (BioLegend; #1003028), CD8 positive CD44 negative cells or CD4 positive CD44 negative cells were removed using a JSAN cell sorter (Bay bioscience Co., Ltd.) and added to the mixed culture system. Furthermore, an experiment in which CD 8-positive CD 44-positive cells or CD 4-positive CD 44-positive cells were purified using a JSAN cell sorter and added to the mixed culture system was also performed.

(results)

As shown in fig. 7a, even when CD 8-positive CD 44-negative cells or CD 4-positive CD 44-negative cells were removed from the non-reactive cells, the proliferation of the primary B6 spleen cells as response cells in the new mixed culture system was observed to be inhibited, as in the case of adding total non-reactive cells. The results show that: CD44 negative cells do not have immunosuppressive effects, i.e., no reactivity-inducing ability. Further, as shown in fig. 7b, it was confirmed that the purified CD 8-positive CD 44-positive cells or CD 4-positive CD 44-positive cells had immunosuppressive ability even when used alone: immunosuppression of the anergic CD 8-positive cells or anergic CD 4-positive cells shown in the examples so far was exerted by CD 44-positive cells.

Meanwhile, as a result of examining the phenotype of the non-reactive cells by FACS, as shown in fig. 7c, 16.61% of non-reactive CD44 positive cells having a strong immunosuppressive function among non-reactive CD8 positive T cells and 31.01% of non-reactive CD44 positive cells having a strong immunosuppressive function among non-reactive CD4 positive T cells were obtained.

Example 8 Induction of immune tolerance Using various suppressors

In the present embodiment, there are shown: immune tolerance can be induced even with various suppressive factors.

(Generation of non-reactive cells)

Basically, experiments (1 to 3) were carried out according to the method described in example 1 and according to the method already described in the literature. For recipient PBMC and donor PBMC, freshly isolated from human peripheral blood or rapidly thawed from cryopreserved at-80 ℃ were used, respectively, and these cells were adjusted to 4X 10 by RPMI1640 medium (Sigma; R8758-500MK) containing autologous plasma or 10% inactivated Fetal Calf Serum (FCS) (SIGMA #172012-⁶Individual cells/mL. Donor PBMC were irradiated with 20Gy of radiation in advance. Mixing the raw materials in a ratio of 1: 1 mixing the recipient PBMC and the donor PBMC, adding inhibitors (e.g., anti-CD 80 antibody/anti-CD 86 antibody) to the mixture at a final concentration of 10. mu.g/ml, respectively, and adding bleiracept (belatacept) or aberracept (aberracept), respectivelyAdded at a final concentration of 10. mu.g/ml to 40. mu.g/ml). 5% CO at 37 ℃ in a 6cm dish (Greiner CELLSTAR (registered trademark) dish, Cat. No.628160) (culture volume 3-6 mL) or a 10cm dish (Corning, Cat. No.430167) (culture volume 10-15 mL)₂The culture was carried out in an incubator for 7 days (cell density at the beginning of the culture was 4X 10)⁶Individual cells/mL).

Cultured recipient PBMC were recovered by centrifugation on the 7 th day after the start of culture and adjusted to 4X 10 with the above-mentioned medium⁶Individual cells/mL. In the cultured recipient PBMC, the ratio of the number of cells was 2: the freshly prepared irradiated donor PBMC is added as described in the above 1, and further inhibitors (for example, 5. mu.g/ml to 10. mu.g/ml at the final concentration in the case of anti-CD 80 antibody/anti-CD 86 antibody; and 10. mu.g/ml to 40. mu.g/ml at the final concentration in the case of belicep (belacapt) or abatacept) are added). The culture was carried out for 7 days under the same conditions as described above (cell density: 4X 10)⁶Individual cells/mL).

(evaluation of immune response suppressing ability)

Induced cells were recovered by centrifugation on the 14 th day from the start of culture, and lymphocyte mixture test (3) was performed basically according to the method already described in the literature. At 37 deg.C, 5% CO₂Co-culturing the cell suspension in an incubator. Addition on day 4 from the start of co-cultivation³H-Thymidine (10. mu.l) on day 5 from the start of co-cultivation (addition)³After 16 to 20 hours of H-thymidine) is removed from the culture medium³H-thymidine assay³The immune response inhibitory activity was confirmed by the amount of H-thymidine taken.

Example 9 quality control of cell preparations

The method for producing a cell preparation is described in examples 1 to 7 above. For the cell preparations produced according to the examples, the following quality control was performed.

Representative of the quality criteria to be met are as follows.

[ Table 4]

Quality standard of final product

(quality control test of cell preparation)

According to the method described in the present specification, for example, in order to examine whether or not the non-reactive cells produced according to the descriptions of examples 1 to 7 meet the quality standard of the final product, the following test was performed.

Appearance of

The appearance of non-reactive cells suspended in physiological saline was examined visually. Suspensions meeting the quality criteria should be composed of cells that are yellowish-white to yellowish-yellow.

Purity of the cell phenotype and of the non-reactive cells

To examine each phenotype of an anergic cell by a flow cytometer, for example, the following antibodies were used and analyzed by multiplex staining:

CD 3: FITC fluorescently labeled anti-human CD3 antibody (UCHT 1; eBioscience #11-0038-42) or Pacific Blue fluorescently labeled anti-human CD3 antibody (UCHT 1; Invitrogen # CD0328)

CD 4: PE fluorescent-labeled anti-human CD4 antibody (RPA-T4; eBioscience #25-0049-42)

CD 8: APC fluorescence labeled anti-human CD8 antibody (RPA-T8; eBioscience #17-0088-42)

CD 25: PerCP fluorescent-labeled anti-human CD25 antibody (MEM-181; eBioscience # A15802)

CD 44: PE-Cy7 fluorescent-labeled anti-human CD44 antibody (IM 7; eBioscience #25-0441-82)

CD 45: bright purple fluorescence labeled anti-human CD45 antibody (HI 30; BioLegend #304032)

CD45 RA: FITC Fluorescently labeled anti-CD 45RA antibody (ALB 11; Beckman Coulter A07786) or PE Fluorescently labeled anti-CD 45RA antibody (ALB 11; Beckman Coulter IM1834U)

CD45 RO: ECD fluorescently labeled anti-CD 45RO antibody (UCHL 1; Beckman Coulter IM2712U) or PE fluorescently labeled anti-CD 45RO antibody (UCHL 1; Beckman Coulter A07787) or APC fluorescently labeled anti-CD 45RO antibody (UCHL 1; Bay biosciences 20-0457)

Step (ii) of

CD 3-positive cell ratio, CD 45-positive cell ratio in living cells, CD 8-positive CD 44-positive cell ratio in CD 3-positive cells, CD 4-positive CD 44-positive cell ratio in CD 3-positive cells, CD 8-positive CD45 RA-negative cell ratio in CD 3-positive cells, CD 8-positive CD45 RA-negative CD45 RO-positive cell ratio in CD 3-positive cells, CD 4-positive CD45 RA-negative CD45 RO-positive cell ratio in CD 3-positive cells, and CD 4-positive CD 25-positive cell ratio in CD 3-positive cells

After reacting the above antibody with non-reactive cells suspended in physiological saline, dead cells were stained using the Zombie NIR Fixable visualization Kit (BioLproduced # 423106). For the cells subjected to multiplex fluorescent staining, the ratio of CD3 positive cells among all living cells was determined in FACS Verse (BD Bioscience).

Non-responsive cells meeting the quality criteria should be more than 50% positive for CD 3. Meanwhile, based on fluorescence, the ratio of CD 45-positive cells in live cells, the ratio of CD 8-positive CD 44-positive cells, the ratio of CD 4-positive CD 44-positive cells, the ratio of CD 8-positive CD45 RA-negative cells, the ratio of CD 8-positive CD45 RA-negative CD45 RO-positive cells, the ratio of CD 4-positive CD45 RA-negative CD45 RO-positive cells, and the ratio of CD 4-positive CD 25-positive cells in all the CD 3-positive cells that survived were determined.

The non-reactive cells meeting the quality standard should be more than 95% of the living cells positive for CD45, and contain no large amount of impurities such as red blood cells and platelets. Furthermore, non-reactive cells meeting the quality criteria should be cells in which 5% or more of the population of CD 3-positive cells are CD 8-positive CD 44-positive, 5% or more of the population of CD 4-positive CD 44-positive, 5% or more of the population of CD 8-positive CD45 RA-negative, 5% or more of the population of CD 8-positive CD45 RA-negative CD45 RO-positive, 5% or more of the population of CD 4-positive CD45 RA-negative CD45 RO-positive, and 5% or more of the population of CD 4-positive CD 25-positive.

Safety

Basically, the test is carried out according to the record of the japanese pharmacopoeia or pharmacopoeias of the corresponding countries. Exemplary embodiments are described below.

Sterility test method

The suspension of non-reactive cells was gently centrifuged and the supernatant was subjected to sterility test. In the direct method, which is one of typical sterility tests in the Japanese pharmacopoeia, a supernatant is inoculated into a soybean/casein/digestion medium or a liquid thioglycolic acid medium and cultured at 30 to 35 ℃ or 20 to 25 ℃ for 14 days or more, respectively. Then, the culture was observed several times during the culture. In another membrane filter method, which is a typical aseptic test, a supernatant is diluted with a sterile diluent (e.g., a 1g/L meat or casein peptone solution (pH 7.1. + -. 0.2)), and the diluted supernatant is transferred to a membrane filter and filtered. Then, the membrane filter was placed in each of the 2 kinds of culture media and cultured for 14 days or longer. In the preparation meeting the quality criteria, there was no proliferation of microorganisms visible to the naked eye in the culture medium during the culture period and on the last day.

Endotoxin test method

The non-reactive cell suspension is diluted with physiological saline to a pH of 6.0 to 8.0. Then, the mixture was mixed with a lysis reagent, and the endotoxin concentration in the sample was quantitatively determined using the gel formation of the lysis reagent as an index (gelation method), the turbidity change during gelation of the lysis reagent as an index (turbidimetry), or the color development of the synthetic matrix due to hydrolysis as an index (colorimetry). If necessary, a preliminary test for confirming the display sensitivity of the cleavage reagent is performed. In a preparation meeting the quality standards, the endotoxin concentration must be less than 0.25 EU/mL.

Mycoplasma negative test

The culture broth, or suspension of non-reactive cells, was gently centrifuged and the supernatant was subjected to a mycoplasma negative test. In a culture method which is one of representative mycoplasma negative tests in the japanese pharmacopoeia, a sample is inoculated into an agar plate medium, cultured at 35 to 37 ℃ for 14 days or more at an appropriate humidity in nitrogen gas containing 5 to 10% of carbon dioxide, or the sample is inoculated into a container containing a liquid medium, cultured at 35 to 37 ℃, and when discoloration of the liquid medium is observed or an aliquot is taken out from the liquid medium at regular intervals from the start of culture, inoculated into a new agar plate medium, and culture is continued. Then, on the 7 th day and the 14 th day, the presence or absence of mycoplasma colonies was examined with respect to all agar plate media using a microscope with a magnification of 100 times or more. In another DNA staining method using indicator cells, which is a representative mycoplasma negative test, typically Vero cells and a specified mycoplasma strain are used as indicator cells. In this method, indicator cells are seeded in a culture dish or the like with a cover glass pressed thereon, and proliferated in air containing 5% carbon dioxide at 35 to 38 ℃ for one day. Then, the sample (culture solution or supernatant) is added thereto, and the culture is continued for 3 to 6 days under the same conditions. After fixing the cultured cells on the cover glass, DNA fluorescent staining is performed by a stain such as bisbenzimide, microscopic examination is performed by a fluorescence microscope (magnification of 400 to 600 times or more), and when 0.5% or more of cells having a microscopic extra-nuclear fluorescent spot surrounding the cell nucleus are present, the cells are judged to be mycoplasma positive, compared with a negative (non-inoculated) control and a mycoplasma positive control. Products meeting the quality criteria should be mycoplasma negative.

Cell number

For non-reactive cells suspended in physiological saline, the number of cells was determined under a microscope using a hemocytometer or using an automatic cell counter. The number of non-reactive cells suitable for administration meeting the quality criterion was 1X 10⁸～30×10⁸When the amount of the cells is less than the above range (for example, 100mL of physiological saline), the cells should be added appropriately.

Viable cell rate

The non-reactive cells suspended in physiological saline are mixed with 0.3 to 0.5% trypan blue staining solution (for example, catalog #35525-02, Nacalai Tesque), and the number of viable cells is measured under a microscope or using an automatic cell counter using a hemocytometer. Products meeting the quality criteria should have more than 70% of the cells that are viable.

(examination)

From the above results, it is evident that the compositions of the invention:

1) the immunosuppressive cell population and mixture having CD 44-positive CD 8-positive T cells and CD 44-positive CD 4-positive T cells as the core can exhibit their performances to a high degree.

2) This cell population was shown to contain both FoxP 3-positive regT cells, but the inhibitory ability of the regT cells was also enhanced in a culture system that induces anergy such as antigen stimulation in the presence of anti-CD 80/86 antibody, and could exert its inhibitory ability more strongly in the presence of CD 44-positive CD 8-positive cells and CD 44-positive CD 4-positive cells.

3) The selection of non-reactive CD8 positive cells and non-reactive CD4 positive cells that have achieved antigen specificity by reacting with antigen in the presence of anti-CD 80/86 cells recognize and cover antigen earlier than the initial cells is a mechanism of immunosuppression. This result is a very important finding that has not been confirmed in the conventional immune rejection suppression experiment.

These findings may be important confirmation points in quality control of cell preparations for suppressing immune rejection.

The final product criteria shown in table 4 are typical examples, and the reference values such as cell phenotype may be changed as appropriate, and examples of modifications thereof include those shown in table 3.

Reference to the literature

In the examples and the like, the following references are referred to as basic techniques, and these documents are not considered to constitute prior art of the present invention. These are incorporated by reference.

1.Bashuda H,Kimikawa M,Seino K,Kato Y,Ono F,Shimizu A,et al.Renal allograft rejection is prevented by adoptive transfer of anergic T cells in nonhuman primates.The Journal of clinical investigation.2005；115(7):1896-902.

2.Bashuda H,Shimizu A,Uchiyama M,Okumura K.Prolongation of renal allograft survival by anergic cells:advantages and limitations.Clin Transplant.2010；24Suppl 22:6-10.

3.Luo Z,Gotoh M,Grochowiecki T,Tanaka T,Kimura F,Kawashima H,et al.Anergic T cells generated in vitro suppress rejection response to islet allografts.Transplantation.2000；69(10):2144-8.

4.Miyao T,Floess S,Setoguchi R,Luche H,Fehling HJ,Waldmann H,et al.Plasticity of Foxp3(+)T cells reflects promiscuous Foxp3 expression in conventional T cells but not reprogramming of regulatory T cells.Immunity.2012；36(2):262-75.

5.Davies JK,Barbon CM,Voskertchian A,Nadler LM,Guinan EC.Ex vivo alloanergization with belatacept:a strategy to selectively modulate alloresponses after transplantation.Cell transplantation.2012；21(9):2047-61.

6.Davies JK,Gribben JG,Brennan LL,Yuk D,Nadler LM,Guinan EC.Outcome of alloanergized haploidentical bone marrow transplantation after ex vivo costimulatory blockade:results of 2phase 1studies.Blood.2008；112(6):2232-41.

7.Davies JK,Nadler LM,Guinan EC.Expansion of allospecific regulatory T cells after anergized,mismatched bone marrow transplantation.Science translational medicine.2009；1(1):1ra3.

8.Davies JK,Yuk D,Nadler LM,Guinan EC.Induction of alloanergy in human donor T cells without loss of pathogen or tumor immunity.Transplantation.2008；86(6):854-64.

(remarks)

As above, the present invention has been exemplified using the preferred embodiments thereof, but it is understood that the present invention is to be interpreted only by the claims. It is to be understood that the contents of patents, patent applications, and other documents cited in this specification are incorporated by reference into this specification as if fully set forth herein. The present application claims the benefit of priority from japanese patent application kokai 2018-119001 (application No. 6/22/2018), it being understood that the contents of this application, which may be all, are incorporated by reference into the present application. In addition, Japanese patent applications laid-open No. 2018-118996 and laid-open No. 2018-119003 (both laid-open No. 6/22 in 2018), and a part or the whole of the contents of these international applications claiming priority are incorporated by reference in the present specification

Industrial applicability

The present invention provides pharmaceutical compositions comprising cells that induce specific immune tolerance to a particular antigen. Provided is a technology that can be used in industries (pharmaceutical industries) related to preparations and the like based on such technologies.

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Antibody for inducing immune tolerance using cell mixture having complex state, and induced lymphocyte, and cell therapeutic agent and therapeutic method using induced lymphocyte

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