阅读说明：本技术 用于免疫抑制的组合物和方法 (Compositions and methods for immunosuppression ) 是由 阿尼尔·钱德拉克 苏迪普塔·特里帕蒂 安娜·玛丽亚·瓦伽-伽瑟 于 2019-12-12 设计创作，主要内容包括：本文提供了能够特异性抑制针对供体同种异体抗原或自身抗原的免疫反应的调节性T细胞(Treg)、其组合物及其产生方法。任选地,这些Treg用于包括自然杀伤(NK)细胞的群体中。还描述了使用这些Treg或Treg和NK细胞的混合群的相关方法,包括用于促进移植受者的同种异体移植物接受性和治疗受试者的自身免疫性障碍的方法。这些Treg或Treg和NK细胞的混合群源自受试者的血细胞,并且可以减少或替代广效免疫抑制剂的使用。(Provided herein are regulatory T cells (tregs) capable of specifically suppressing an immune response against a donor alloantigen or autoantigen, compositions thereof, and methods of producing the same. Optionally, the tregs are used in a population comprising Natural Killer (NK) cells. Also described are related methods of using these tregs or mixed populations of tregs and NK cells, including methods for promoting allograft acceptance in transplant recipients and treating autoimmune disorders in subjects. These tregs or mixed populations of tregs and NK cells are derived from the subject's blood cells and may reduce or replace the use of broad-spectrum immunosuppressive agents.)

1. An isolated regulatory T cell Treg comprising a T cell receptor TCR that specifically binds:

(i) an alloantigen which is a human leukocyte antigen HLA molecule or fragment thereof and is not encoded by a nucleotide sequence present in the genome of the Treg, or

(ii) An autoantigen or fragment thereof that causes an autoimmune disorder.

2. The Treg of claim 1, wherein the TCR specifically binds the HLA molecule.

3. The Treg of claim 2, wherein the TCR specifically binds to a hypervariable region HVR of the HLA molecule.

4. The Treg of claim 3, wherein the TCR specifically binds to the β -chain HVR of the HLA molecule.

5. The Treg of claim 2, wherein the HLA molecule is an HLA-DR, HLA-DQ, HLA-DP, HLA-A, HLA-B or HLA-C molecule or fragment thereof.

6. The Treg of claim 5, wherein the HLA molecule is an HLA-DR, HLA-DQ or HLA-DP molecule or fragment thereof.

7. The Treg of claim 6, wherein the HLA-DR molecule is an HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR5, HLA-DR6, HLA-DR7, HLA-DR8, HLA-DR9, HLA-DR10, HLA-DR11, HLA-DR12, HLA-DR13, HLA-DR14, HLA-DR15, HLA-DR16, HLA-DR17, HLA-DR18, HLA-DR51, HLA-DR52 or HLA-DR53 molecule or fragment thereof.

8. The Treg of claim 1, wherein the Treg is capable of suppressing a T effector Teff response against the alloantigen or the autoantigen.

9. The Treg of claim 8, wherein the Treg is capable of suppressing Teff proliferation response to direct allorecognition, semi-direct allorecognition and/or indirect allorecognition.

10. The Treg of claim 8, wherein the Treg is capable of activating an adenosyllogic signaling pathway.

11. The Treg of claim 1, wherein the Treg expresses one or more markers selected from the group consisting of CD4, CD25, CD39, CD73, FOXP3, GITR, CLTA4, ICOS, GARP, LAP, PD-1, CCR6, and CXCR 3.

12. The Treg of claim 2, wherein the HLA molecule or fragment thereof to which the TCR specifically binds is encoded by a nucleotide sequence present in the genome of an organ or tissue donor.

13. An isolated Treg comprising a TCR that specifically binds to:

(i) an alloantigen which is an HLA molecule or fragment thereof and is not encoded by a nucleotide sequence present in the genome of the Treg, or

(ii) (ii) an autoantigen or fragment thereof that causes an autoimmune disorder;

wherein the tregs have been generated by a method comprising:

(a) contacting a population of immune cells comprising T cells obtained from a recipient subject with the HLA molecule or fragment of an autoantigen and autoantigen-presenting cells APCs; and

(b) expanding the population of immune cells of step (a) for a time and under conditions sufficient to form an expanded T cell line comprising a plurality of these tregs; and optionally

(c) Purifying the tregs from the immune cell population.

14. The Treg of claim 13, wherein the population of immune cells of (a) further comprises natural killer NK cells, and if step (c) is performed, step (c) comprises purifying the tregs and NK cells from the population of immune cells, thereby generating a mixed population of tregs and NK cells.

15. A mixed population of cells comprising the tregs and NK cells of claim 1.

16. A composition comprising the Treg of claim 1.

17. A composition comprising a mixed population of cells comprising the tregs and NK cells of claim 1.

18. A method of inhibiting an immune response in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 16 or 17.

19. The method of claim 18, wherein the immune response is a Teff response against the alloantigen or the autoantigen.

20. A method of treating or preventing transplant rejection or a method of treating an autoimmune disorder in a subject, the method comprising administering to the subject the composition of claim 16 or 17.

21. The method of claim 18, wherein the subject has an autoimmune disorder.

22. The method of claim 18, wherein the subject is an organ or tissue transplant recipient.

23. The method of claim 18, wherein the HLA molecule or fragment thereof to which the TCR specifically binds is encoded by a nucleotide sequence present in the genome of an organ or tissue donor.

24. The method of claim 18, wherein the method further comprises reducing the dose of immunosuppressive agent administered to the subject.

25. The method of claim 18, wherein the organ is a kidney, liver, heart, lung, pancreas, intestine, stomach, testis, penis, thymus, or facial, hand, or leg vascular composite allograft.

26. The method of claim 18, wherein the tissue comprises bone, tendon, cornea, skin, heart valve, neural tissue, bone marrow, langerhans islets, stem cells, blood, or blood vessels.

27. The method of claim 20, wherein the autoimmune disorder is autism, autism spectrum disorder, rheumatoid arthritis, lupus, focal segmental glomerulonephritis, or membranous nephropathy.

28. A method for generating the Treg of claim 1, comprising:

(a) contacting a population of immune cells comprising T cells obtained from a recipient subject with the HLA molecule or fragment of an autoantigen and autologous APCs; and

(b) expanding the population of immune cells of step (a) for a time and under conditions sufficient to form an expanded T cell line comprising a plurality of these tregs; and optionally

(c) Purifying the tregs from the immune cell population.

29. The method of claim 28, wherein the method comprises repeating steps (a) and (b) more than three times.

30. The method of claim 28, wherein the method comprises repeating steps (a) and (b) four or five times.

31. The method of claim 28, wherein step (a) is performed approximately every seven to ten days.

32. The method of claim 28, wherein the autologous APCs are peripheral blood mononuclear cells, PMBCs, dendritic cells, macrophages or B cells.

33. The method of claim 32, wherein the autologous APCs are PBMCs.

34. The method of claim 33, wherein the PBMCs are irradiated.

35. The method of claim 28, wherein the immune cell population comprising T cells is a PMBC population, a naive T cell population, or a purified Treg population.

36. The method of claim 35, wherein the population of immune cells is a population of PBMCs.

37. The method of claim 36, wherein step (a) further comprises contacting the population of PBMCs with IL-2.

38. The method of claim 37, wherein the concentration of IL-2 is about 50IU/ml to about 200 IU/ml.

39. The method of claim 38, wherein the concentration of IL-2 is about 100 IU/ml.

40. The method of claim 28, wherein the concentration of the HLA molecule or fragment of the autoantigen is about 25 μ g/ml to about 200 μ g/ml.

41. The method of claim 40, wherein the concentration of the HLA molecule or fragment of an autoantigen is about 50 μ g/ml.

42. The method of claim 28, wherein the fragment of an HLA molecule or autoantigen is a purified peptide or peptide mixture.

43. The method of claim 28, wherein the population of immune cells comprises NK cells.

44. The method of claim 28, wherein step (c) comprises purifying the tregs and NK cells from the population of immune cells, thereby generating a mixed population of tregs and NK cells.

45. A composition, comprising:

(a) the Treg of claim 1; and

(b) a fragment of the HLA molecule or autoantigen.

46. The composition of claim 45, wherein the composition further comprises IL-2.

47. The composition of claim 46, wherein the concentration of IL-2 is from about 50IU/ml to about 200 IU/ml.

48. The composition of claim 47, wherein the concentration of IL-2 is about 100 IU/ml.

49. The composition of claim 45, wherein the concentration of the HLA molecule or fragment of an autoantigen is about 25 μ g/ml to about 200 μ g/ml.

50. The composition of claim 49, wherein the concentration of the HLA molecule or fragment of an autoantigen is about 50 μ g/ml.

51. The composition of claim 45, wherein the HLA molecule or fragment of an autoantigen is a purified peptide or peptide mixture.

52. The composition of claim 45, further comprising NK cells.

Background

Kidney transplantation is currently the first treatment of patients with end stage renal disease (ESKD). According to U.S. renal data system annual reports, over 660,000 americans are receiving ESKD treatment. Of these patients, 468,000 were dialysis patients and more than 193,000 had functional kidney transplants. Over 89,000 ESKD patients die each year, with the U.S. medical insurance costs for treating renal failure amounting to approximately $ 310 million per year, accounting for approximately 7% of all medical insurance costs. By 2016, over 100,000 patients are currently awaiting kidney transplantation in the United states. The median latency for an individual's first transplant is 3.6 years, which may vary by health, compatibility, and availability of the organ. In 2017, approximately 20,000 kidney transplants were performed in the united states, two-thirds from deceased donors and one-third from live donors.

In recent years, despite improvements in short-term transplant survival rates, long-term transplant survival rates have not changed significantly. Currently, organ transplant patients receive a broad spectrum of immunosuppressive agents to prevent rejection of the donated organ. However, these immunosuppressive agents leave transplant recipients susceptible to serious infections, particularly because transplant recipients adhere to a long-term regimen of immunosuppressive agents. The development of infections and cancer due to immunosuppressant therapy is a significant problem for transplant recipients. Therefore, there is a need for alternative therapies to promote transplant tolerance and prevent rejection, thereby eliminating the toxicity associated with current immunosuppressive drugs, including those that affect allograft survival.

Disclosure of Invention

The invention described herein provides, inter alia, regulatory T cells (tregs) derived from patients specific for (i) a transplant donor alloantigen or (ii) an autoantigen. The invention also provides methods for inhibiting an immune response against an alloantigen or autoantigen, and methods for promoting allograft acceptance and for treating or preventing transplant rejection or an autoimmune disorder. Tregs can also be used in mixed populations of tregs and NK cells.

In one aspect, the invention provides an isolated regulatory T cell (Treg) comprising a T Cell Receptor (TCR) that specifically binds: (i) an alloantigen which is a Human Leukocyte Antigen (HLA) molecule or fragment thereof and is not encoded by a nucleotide sequence present in the Treg genome, or (ii) an autoantigen or fragment thereof which causes an autoimmune disorder.

In particular embodiments, the TCR specifically binds to an HLA molecule. In some embodiments, the TCR specifically binds a hypervariable region (HVR), e.g., a β -chain HVR of an HLA molecule. In some embodiments, the HLA molecule is an HLA-DR, HLA-DQ, HLA-DP, HLA-A, HLA-B, or HLA-C molecule or fragment thereof. In particular embodiments, the HLA molecule is an HLA-DR, HLA-DQ, or HLA-DP molecule or fragment thereof. In certain embodiments, the HLA-DR molecule is HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR5, HLA-DR6, HLA-DR7, HLA-DR8, HLA-DR9, HLA-DR10, HLA-DR11, HLA-DR12, HLA-DR13, HLA-DR14, HLA-DR15, HLA-DR16, HLA-DR17, HLA-DR18, HLA-DR51, HLA-DR52, or HLA-DR53 molecule, or any other HLA-DR serotype as described herein or known in the art. In some embodiments, the HLA molecule or fragment thereof to which the TCR specifically binds is encoded by a nucleotide sequence present in the genome of the organ or tissue donor.

In some embodiments, the tregs are capable of suppressing T effector cell (Teff) responses against alloantigens or autoantigens. In further embodiments, the tregs are capable of suppressing Teff proliferation responses to direct allorecognition, semi-direct allorecognition, and/or indirect allorecognition.

In some embodiments, the Treg comprises activating an adenoergic signaling pathway.

In some embodiments, the tregs express one or more markers selected from the group consisting of CD4, CD25, CD39, CD73, FOXP3, GITR, CLTA4, ICOS, GARP, LAP, PD-1, CCR6, and CXCR 3.

In another aspect, the invention features an isolated Treg that includes a TCR that specifically binds to: (i) an alloantigen which is an HLA molecule or fragment thereof and is not encoded by a nucleotide sequence present in the Treg genome, or (ii) an autoantigen or fragment thereof that causes an autoimmune disorder; wherein the tregs have been generated by a method comprising (a) contacting a population of immune cells comprising T cells obtained from a recipient subject with an HLA molecule or fragment of an autoantigen and an Autoantigen Presenting Cell (APC); and (b) expanding the immune cell population of step (a) for a time and under conditions sufficient to form an expanded T cell line comprising a plurality of tregs; and optionally (c) purifying the tregs from the immune cell population. In some embodiments, the population of immune cells of (a) further comprises Natural Killer (NK) cells, and if step (c) is performed, step (c) comprises purifying the tregs and NK cells from the population of immune cells, thereby generating a mixed population of tregs and NK cells.

In another aspect, the invention features a mixed population of cells including tregs and NK cells as described in any one of the preceding aspects.

In another aspect, the invention features a composition including a Treg as described in any of the preceding embodiments.

In another aspect, the invention features a composition that includes a mixed population of tregs and NK cells as described in the previous aspect.

In another aspect, the invention features a method of suppressing an immune response in a subject, the method including administering to the subject a Treg, a mixed population of tregs and NK cells, or a pharmaceutical composition as described in any one of the preceding aspects. In some embodiments, the immune response is a Teff response against an alloantigen or autoantigen.

In another aspect, the invention features a method of treating or preventing transplant rejection or treating an autoimmune disorder in a subject, the method including administering to the subject a Treg, a mixed population of tregs and NK cells, or a composition as described in any one of the preceding aspects.

In some embodiments, the subject has an autoimmune disorder (e.g., autism spectrum disorder, rheumatoid arthritis, lupus, focal segmental glomerulonephritis, or membranous nephropathy).

In some embodiments, the subject is an organ or tissue transplant recipient. In some embodiments as described in any of the preceding aspects, the HLA molecule or fragment thereof to which the TCR specifically binds is encoded by a nucleotide sequence present in the genome of the organ or tissue donor. In additional embodiments, the method further comprises reducing the dose of the immunosuppressant administered to the subject (e.g., by 10%, by 20%, by 30%, by 40%, by 50%, by 60%, by 70%, by 80%, by 90%, or by 100%). Preferably, the dose of immunosuppressive agent is reduced by up to 50% (e.g., by 10%, by 20%, by 30%, by 40%, or by 50%).

In some embodiments, the organ is a kidney, liver, heart, lung, pancreas, intestine, stomach, testis, penis, thymus, or facial, hand, or leg vascular composite allograft. In some embodiments, the tissue comprises bone, tendons, cornea, skin, heart valves, neural tissue, bone marrow, islets of langerhans, stem cells, blood, or blood vessels.

In another aspect, the invention features a method for producing a Treg as in any of the preceding aspects, the method comprising (a) contacting a population of immune cells comprising T cells obtained from a recipient subject with an HLA molecule or a fragment of an autoantigen and an autoantigen-presenting cell (APC); and (b) expanding the immune cell population of step (a) for a time and under conditions sufficient to form an expanded T cell line comprising a plurality of tregs; and optionally (c) purifying the tregs from the immune cell population.

In some embodiments, the method comprises repeating steps (a) and (b) more than once. In some embodiments, the method comprises repeating steps (a) and (b) more than three times, for example four or five times. In further embodiments, step (a) is performed approximately every seven to ten days.

In some embodiments, the autologous APC is a Peripheral Blood Mononuclear Cell (PBMC), a dendritic cell, a macrophage, or a B cell. In certain embodiments, the autologous APCs are PBMCs. In some embodiments, the PBMCs are irradiated.

In some embodiments, the immune cell population comprising T cells is a PBMC population, naiveA population of T cells or a purified population of tregs. In particular embodiments, the population of immune cells is a population of PBMCs. In further embodiments, step (a) further comprises contacting PBMCs of the recipient subject with IL-2. In some embodiments, the concentration of IL-2 is from about 50IU/ml to about 200IU/ml, such as about 100 IU/ml. In other embodiments, the concentration of the HLA molecule or fragment of the autoantigen is from about 25 μ g/ml to about 200 μ g/ml, e.g., about 50 μ g/ml. In some embodiments, the fragment of an HLA molecule is a purified peptide or a mixture of peptides. In some embodiments, the population of immune cells comprises NK cells. In some embodiments, step (c) comprises purifying the tregs and NK cells from the population of immune cells, thereby generating a mixed population of tregs and NK cells.

In another aspect, the invention features a composition that includes: (a) the Treg of any one of the preceding aspects; and (b) a fragment of an HLA molecule or autoantigen.

In some embodiments, the composition further comprises IL-2. In some embodiments, the concentration of IL-2 is from about 50IU/ml to about 200IU/ml, such as about 100 IU/ml. In some embodiments, the concentration of the HLA molecule or fragment of the autoantigen is about 25 μ g/ml to about 200 μ g/ml, e.g., about 50 μ g/ml. In some embodiments, the fragment of an HLA molecule or autoantigen is a purified peptide or peptide mixture. In some embodiments, the composition further comprises NK cells.

Definition of

For convenience, the meanings of some of the terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise indicated or implied from the context, the following terms and phrases include the meanings provided below. These definitions are provided to help describe particular embodiments and are not intended to limit the claimed technology, as the scope of the technology is limited only by the claims. 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 technology belongs. To the extent that there is a clear difference between the usage of a term in the art and its definition provided herein, the definition provided in the specification controls.

Definitions of terms commonly used in immunology and molecular biology can be found in the following documents: the Merck Manual of Diagnosis and Therapy [ Merck handbook ], 19 th edition, published by Merck Sharp & Dohme Corp., 2011(ISBN 978-0-911910-19-3); robert S.Porter et al (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd, 1999 2012(ISBN 9783527600908); and Robert a. meyers (ed.), Molecular Biology and Biotechnology a Comprehensive Desk Reference [ Molecular Biology and Biotechnology: integrated desk reference ], published by VCH publishing company (VCH Publishers, Inc.), 1995(ISBN 1-56081-; immunology by Werner Luttmann [ Verner Luttmann Immunology ], published by Esevirel corporation (Elsevier), 2006; janeway's Immunobiology [ simple Immunobiology ], Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor-Francis publishing group (Taylor & Francis Limited),2014(ISBN 0815345305,9780815345305); lewis's Genes XI [ Gene XI for Lewis ], published by Jones and Bartlett Publishers, 2014 (ISBN-1449659055); michael Richard Green and Joseph Sambrook, Molecular Cloning: Laboratory Manual [ Molecular Cloning: A Laboratory Manual ], 4 th edition, Cold Spring Harbor Laboratory Press (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, New York, USA (2012) (ISBN 1936113414); davis et al, Basic Methods in Molecular Biology [ Basic Methods in Molecular Biology ], Elsevier Science Publishing Co., Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); laboratory Methods in Enzymology DNA [ Methods in Enzymology: DNA ], Jon Lorsch (editors) eisweil (Elsevier),2013(ISBN 0124199542); current Protocols in Molecular Biology (CPMB) [ modern methods of Molecular Biology (CPMB) ], Frederick M.Ausubel (eds.), John Wiley and Sons, 2014(ISBN 047150338X, 9780471503385); current Protocols in Protein Science (CPPS) [ Protein Science modern methods (CPPS) ], John e.coligan (editors), John willi father corporation (John Wiley and Sons, Inc.), 2005; and Current Protocols in Immunology (CPI) [ immunologically Current methods (CPI) ] (John e. coligan, Ada m. kruisbeam, David h. margulies, Ethan m. shevach, Warren Strobe (editors) John Wiley and Sons, Inc., 2003(ISBN 0471142735,9780471142737), the contents of which are all incorporated herein by reference in their entirety.

The term "about" as used herein in reference to a measurable value such as an amount or concentration is intended to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5% or ± 0.1% of the specified value as well as the specified value. For example, "about X," where X is a measurable value, is intended to include X as well as variations of X by ± 10%, ± 5%, ± 1%, ± 0.5%, or ± 0.1%. Ranges of measurable values provided herein may include any other range and/or individual value therein.

As used herein, the term "administering" refers to administering a composition (e.g., an isolated Treg, a pharmaceutical composition thereof, any additional therapeutic agent, and/or any pharmaceutical composition including an additional therapeutic agent) to a subject. Administration to an animal subject (e.g., to a human) can be by any suitable route. For example, administration may be bronchial (including by bronchial instillation), buccal, enteral, parenteral, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intratumoral, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal or vitreal. Administration may be systemic or local.

As used herein, "allogeneic" refers to cells, tissues, organs, nucleic acids (e.g., DNA), or polypeptides (e.g., proteins), or other molecules derived or obtained from a different subject of the same species (e.g., a subject from the same species as the transplant recipient). "alloantigen" refers to an antigen that occurs in some but not all members of the same species.

The term "antigen presenting cell" or "APC" refers to a cell (e.g., an immune system cell, such as a helper cell (e.g., B cell, dendritic cell, or macrophage)) that displays an antigen (e.g., a foreign antigen) complexed at its surface to a Major Histocompatibility Complex (MHC). In some embodiments, the APC can be a professional APC (e.g., a cell expressing MHC class II molecules, including B cells, dendritic cells, or macrophages). In other embodiments, the APC may be a non-professional APC (e.g., a cell that expresses MHC class I molecules, such as a fibroblast, glial, or endothelial cell). The APC processes antigens and presents them to T cells. T cells can recognize these complexes using their T Cell Receptor (TCR).

As used herein, an "autoantigen" or "self-antigen" is any substance normally found in a subject that under abnormal circumstances is no longer recognized by lymphocytes or antibodies of the subject as part of the subject's own body and is therefore attacked by the immune system as if it were a foreign substance. The autoantigen may be a naturally occurring molecule, such as a protein that is typically produced and used by the subject itself, to elicit an immune response that may result in an autoimmune disease or disorder in the subject.

As used herein, an "autoimmune disease" or "autoimmune disorder" is characterized by an inability of a person's immune system to distinguish between foreign cells and healthy cells. This results in programmed cell death of a person's immune system against a person's healthy cells.

The term "autologous" as used herein refers to cells, tissues, organs, nucleic acids (e.g., DNA) or polypeptides (e.g., proteins) derived or obtained from the same subject or patient.

As used herein, the term "fragment" refers to less than 100% of the amino acid sequence of a reference protein (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of a reference length sequence), but includes, for example, 5, 6, 7, 8, 9, 10, 15, or more amino acids. Fragments may be of sufficient length to retain the desired function of the reference protein.

As used herein, "immune response" refers to the response of the immune system of an organism to a substance, including but not limited to a foreign protein or a self-protein. Three general types of "immune responses" include mucosal, humoral, and cellular immune responses. The immune response may include at least one of: antibody production, inflammation, immunity, development of hypersensitivity to antigen, antigen-specific lymphocyte response to antigen, and transplant or graft rejection.

An "immunosuppressive agent" or "immunosuppressive agent" is any agent that prevents, delays the development of, or reduces the intensity of an immune response against foreign cells (particularly transplanted cells) in a host. Examples of immunosuppressive agents include, but are not limited to, cyclosporine, cyclophosphamide, prednisone, dexamethasone, methotrexate, azathioprine, mycophenolate mofetil, thalidomide, FK-506, systemic steroids, and a wide range of antibodies, receptor agonists, receptor antagonists, and other such agents known to those skilled in the art.

As used herein, the term "isolated" refers to a product, compound, or composition that is separate from at least one other product, compound, or composition with which it is associated in its naturally occurring state (whether made in nature or synthetically).

As used herein, the terms "major histocompatibility complex" and "MHC" refer to a specific cluster of genes, many of which encode evolutionarily related cell surface proteins involved in antigen presentation, which are among the most important determinants of histocompatibility. MHC molecules are also known in the art as major histocompatibility antigens. Class I MHC or MHC-I function is mainly directed to CD8⁺T lymphocytes present antigens. MHC class II or MHC-II function is predominantly directed to CD4⁺T lymphocytes present antigens. MHC class I molecules are heterodimers of heavy chains (also called α chains) and β 2-microglobulin (β 12M) encoded in the MHC. The extracellular region of the heavy chain folds into three domains (β 01, β 22 and β 33), while β 52M constitutes the fourth domain. The peptide binding site of class I MHC molecules is composed primarily of the β 41 and β 82 domains, which form a notch for binding antigenic peptides. MHC class II molecules are also heterodimers, but do not include β 62M, but rather include the α and β 7 chains, both encoded in the MHC. Class II MHC alpha chains are transmembrane proteins including extracellular alpha 1 and alpha 2 domains, while beta 9 chains are transmembrane proteins including extracellular beta 1 and beta 2 domains. The α 1 and β 1 domains form peptide binding sites for MHC class II molecules. For a review of MHC and their function, see, e.g., Janeway's Immunobiology]The same as above.

In humans, MHC genes are referred to as "human leukocyte antigens" or "HLA" genes. For example, humans have three MHC class I α -chain genes, designated HLA-A, HLA-B and HLA-C, and three pairs of MHC class II α -and β -chain genes, designated HLA-DR, HLA-DP and HLA-DQ. The HLA-DR cluster may contain an additional beta-chain gene, the product of which can be paired with the DR alpha chain.

As used herein, the term "organ" refers to a body tissue structure that is dedicated as a whole to performing a specific bodily function. Within the meaning of the invention described herein, transplanted organs include, for example, but are not limited to, the heart, kidney, liver, lung, bladder, ureter, stomach, intestine (e.g., small and large intestine), skin, tongue, esophagus, endocrine glands (e.g., pancreas, adrenal gland, salivary gland, thyroid, pituitary, etc.), bone marrow, spleen, thymus, lymph nodes, tendons, ligaments, muscle, uterus, vagina, ovary, fallopian tube, penis, testis, cornea, crystalline lens, retina, middle ear, external ear, cochlea, iris, and vein. Organs for transplantation may also include vascular composite allografts, such as the face, hands or legs.

The term "peripheral blood mononuclear cells" or "PBMCs" refers to any blood cell with a circular nucleus, such as lymphocytes, monocytes or dendritic cells.

As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent, optionally in combination with one or more pharmaceutically acceptable excipients, diluents and/or carriers, for administration to a subject (such as a mammal, e.g., a human), to prevent, treat or control a particular disease or condition that affects or may affect the subject. The pharmaceutical composition may comprise an isolated Treg as described herein.

As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are suitable for contact with the tissue of a subject, such as a mammal (e.g., a human), without excessive toxicity, irritation, allergic response, and/or other problem complications commensurate with a reasonable benefit/risk ratio.

As used herein, the term "mixed population of tregs and NK cells" refers to a mixture of tregs and NK cells that have been stimulated with alloantigens or autoantigens and expanded according to the procedures described herein. The cells may be present in a ratio of Treg cells to NK cells of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:10, 1:20, 1:50, 1:100, 100:1, 50:1, 20:1, 10:1, 6:1, 5:1, 4:1, 3:1, or 2: 1. In a preferred embodiment, the cells are present in a ratio of Treg cells to NK cells of 6:1 to 2: 1. As described herein, the mixed population of tregs and NK cells includes a minimal amount of other cell types, e.g., less than 2% (or 1% or less, or 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%) of the population.

The term "preventing" (prevention and prevention) refers to the administration of an agent or composition to a clinically asymptomatic individual who is predisposed to a particular adverse condition, disorder or disease, and thus relates to preventing the occurrence of at least one sign or symptom of disease. As used herein, the term "symptom" includes signs and symptoms unless otherwise indicated.

As used herein, the term "rejection" or "transplant rejection" refers to one or more processes in which an immune response of an organ transplant recipient elicits a response against the transplanted organ, cell, or tissue (whether natural or bioartificial, such as recellularized tissue), sufficient to impair or destroy the normal function of the organ. The immune system response may involve specific (antibody and T cell dependent) or non-specific (phagocytosis, complement dependent, etc.) mechanisms, or both. In one example, rejection or acceptance of a kidney transplant can be measured by creatinine levels in the blood, where creatinine levels ≧ 1.6mg/dl indicate chronic rejection, and creatinine levels ≦ 1.6mg/dl indicate stable renal function.

As used herein, the term "specific binding and specific binding" refers to a physical interaction between two molecules, compounds, cells and/or particles, wherein a first entity binds to a second target entity with a higher specificity and affinity than a third entity that binds to a non-target. In some embodiments, specific binding may refer to an affinity of a first entity for a second, target entity that is at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or higher than the affinity for a third entity that is not a target under the same conditions. An agent specific for a given target is one that exhibits specific binding to that target under the assay conditions used. Non-limiting examples include antibodies or ligands that recognize and bind to a cognate binding partner (e.g., a stimulatory and/or co-stimulatory molecule present on a T cell) protein.

As used herein, the term "subject" refers to any organism to which a composition according to the invention can be administered, e.g., for experimental, diagnostic, prophylactic and/or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, dogs, cats, non-human primates, and humans). Preferably, the subject is a human. The subject may seek or require treatment, be receiving treatment, will receive treatment in the future, or be a human or animal under the care of a professional trained for a particular disease or condition. The subject may be a patient (e.g., a transplant recipient).

As used herein, "inhibiting" a function or activity is decreasing the function or activity when compared to the same other condition other than the condition or parameter of interest, or alternatively, when compared to another condition (e.g., an immune response of a subject). An "immunosuppressive" effect or response generally refers to the production or expression of cytokines or other molecules by an APC to reduce, suppress, or prevent an immune response. When an APC produces an immunosuppressive effect on immune cells that recognize an antigen presented by the APC, the immunosuppressive effect is said to be specific for the antigen presented.

As used herein, the term "T cell" refers to a type of lymphocyte that plays a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of a T Cell Receptor (TCR) on the cell surface. T cells do not present antigen and rely on other lymphocytes (e.g., natural killer cells, B cells, macrophages, and dendritic cells) to assist in antigen presentation. There are several T cell subsets (e.g., helper T cells, memory T cells, regulatory T cells, cytotoxic T cells, natural killer T cells, γ δ T cells, and mucosa-associated constant T cells), each with different functions.

As used herein, the terms "regulatory T cells" and "tregs" refer to a subset of immunosuppressive T cells, which are generally characterized by expression of the markers CD4, FOXP3, and CD 25. Tregs regulate the immune system, maintain tolerance to self-antigens, prevent autoimmune diseases, and also suppress anti-tumor immune responses.

"tissue" refers to a group of cells having similar morphology and function. Within the meaning of the invention described herein, tissues capable of transplantation include, but are not limited to, bone, tendon, cornea, skin, heart valves, neural tissue, bone marrow, langerhans islets, stem cells, blood vessels, cartilage, ligaments, nerves, and middle ear.

As used herein, the term "transplant" refers to an organ, portion of an organ, tissue, engineered tissue, or cell that has been transferred from a site of origin in one subject to a recipient site in the same or a different subject. For example, in an allograft transplantation procedure, the site of origin of the transplant is in the donor individual and the recipient site is in another recipient individual.

As used herein, a "transplant donor" is a mammal from which an organ, a portion of an organ, a tissue, an engineered tissue, or cells are removed for transplantation into a recipient. By "transplant recipient" is meant a mammal that receives an organ, portion of an organ, tissue, engineered tissue, or cells from a donor.

As used herein, the terms "treat," "treatment," "treating," or "ameliorating" refer to a therapeutic treatment wherein the objective is to reverse, alleviate, ameliorate, contain, slow, or stop the progression or severity of a condition associated with a disease or disorder (e.g., transplant rejection or GHVD). The term "treating" includes reducing or alleviating at least one side effect or symptom of a condition, disease, or disorder. A treatment is typically "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is effective if progression of the disease is slowed or stopped. That is, treatment includes not only improvement of symptoms or markers, but also stopping or at least slowing the progression or worsening of symptoms as compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or complete), and/or reduction in mortality, whether detectable or undetectable. The term "treating" a disease also includes causing the symptoms or side effects of the disease to be alleviated (including palliative treatment).

The present invention provides a number of advantages. For example, the present invention provides an immunotherapy that promotes allograft acceptance with the potential to reduce or eliminate the need for immunosuppressive drug therapy. The therapy can be individualized for each patient and does not require donor tissue, and is therefore suitable for subjects who have received a deceased or live donor transplant. The therapy can be initiated at any time after transplantation and is a method suitable for any type of solid organ transplantation. Furthermore, the invention is applicable to the suppression of immune responses to indirect, semi-direct and/or direct alloantigen recognition. The present invention can also be used to provide protection against both acute and chronic transplant rejection. Furthermore, the invention may be used to treat autoimmune disorders, for example by stimulating tregs with autoantigens.

Another feature of the present invention is that it is not broadly immunosuppressive in nature, but rather promotes allograft acceptance by modulating the body's own immune response. Alloantigen-specific or autoantigen-specific methods are strategically safer due to less interference with the overall response to the pathogen, and are therefore associated with low infection tolerance to third party antigens.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Drawings

FIGS. 1A-1D are a series of diagrams showing CD4 from transplant recipients⁺And CD4⁺CD25^-Graph of proliferation of T cells in response to donor-specific alloantigens. Cell proliferation is measured by the replication index, which is the average number of divisions all cells undergo after being stained with a cell proliferation dye.

FIG. 2 is a graph showing CD4 before and after Treg stimulation with donor-specific antigen⁺Graphs of T cell proliferation. Proliferation was measured after tregs received one, two or three stimulations.

Figure 3 is a series of graphs showing the suppressive ability of tregs regardless of different combinations of immunosuppressive drug regimens that a subject receives.

Fig. 4 is a graph showing the difference in Treg inhibitory potency between specific HLA-DR allopeptides. No significant difference was observed.

Fig. 5A-5F are a series of graphs showing the suppressive ability of tregs in the presence (contact-dependent) and absence (non-contact-dependent) of a rotating pore membrane (transwell membrane). Fig. 5A is a series of graphs showing contact-dependent immunosuppression in subject 035. Fig. 5B is a series of graphs showing contact-dependent immunosuppression in subject 008. Figure 5C is a series of graphs showing contact-dependent immunosuppression with tregs and T cell clones 10E9, 1G11, 10H9 and 10B 5. Figure 5D is a series of graphs showing contact-dependent immunosuppression in subject 022. Fig. 5E is a series of graphs showing contact-dependent immunosuppression in subject 002. FIG. 5F is a series of graphs showing contact-dependent immunosuppression in subject 036, who had two HLA mismatches (HLA-DR1 and HLA-DR 15). T line after Treg3-3 stimulations; t lines after Treg4-4 stimulations; treg5-5 post-stimulation T lines.

Figure 6 is a series of graphs showing the suppressive effect of Treg in response to direct allorecognition and indirect allorecognition.

Figures 7A-7C are a series of graphs showing the inhibitory effect of tregs in response to donor-specific allogeneic stimulation (allostimulation) in autologous and third-party responders. Figure 7A is a series of graphs showing immune responses in subjects 038(HLA-DR4 mismatch), 011(HLA-DR4 mismatch) and 023(HLA-DR15 mismatch) with tregs from subject 038. FIG. 7B is a series of graphs showing immune responses in subjects 002(HLA-DR1 mismatch), 035(HLA-DR1 mismatch) and 037(HLA-DR4 mismatch) with Tregs from subject 002. Figure 7C is a series of graphs showing immune responses in subjects 004(HLA-DR1 mismatch), 023(HLA-DR15 mismatch) and 011(HLA-DR4 mismatch) with tregs from subject 004. It was observed that tregs suppressed the immune response specifically against the donor alloantigen.

Figures 8A-8C are a series of graphs showing the bystander suppressive effect of tregs. In FIG. 8A, subject 036 has HLA-DR1 and HLA-DR15 mismatches. In FIG. 8B, subject 004 had HLA-DR1 and HLA-DR15 mismatches. In FIG. 8C, subject 022 had HLA-15 and HLA-17 mismatches. Tregs were observed to exhibit bystander immunosuppressive effects in subjects with more than one HLA mismatch.

Figure 9 is a series of graphs showing the effect of anti-IL-10 antibodies on Treg immunosuppressive activity. No effect of anti-IL-10 antibodies on Treg inhibitory activity was observed.

Figure 10 is a series of graphs showing the effect of the A2A receptor antagonist, istradefylline, on Treg immunosuppressive activity. Istradefylline has been shown to abolish the suppressive activity of tregs.

Figure 11 is a series of graphs showing the effect of istradefylline on the immunosuppressive activity of tregs in subjects 016, 018, 037 and 037.

Figures 12A-12H are a series of graphs showing phenotypic markers associated with characteristic Treg phenotypes as analyzed by flow cytometry for tregs generated from subjects 023, 035, 046, and 052. CD4⁺T cells up-regulate CD25 and Foxp3, while down-regulating CD 127. CD4⁺T cells also upregulate GITR, CTLA4, ICOS, GARP, LAP, PD-1, CD39, CD73, CD45RA, CXCR3, and CCR 6.

Detailed Description

Disclosed herein are personalized immunotherapy for transplant patients using patient regulatory T cells (tregs) specific for one or more donor alloantigens. Tregs are capable of suppressing T effector cell (Teff) immune responses to donor alloantigens, thereby promoting allograft acceptance without the need for immunosuppressive agents. This approach using patient-generated tregs allows for individualized therapy without the need for donor tissue. In addition, the methods provide for the generation and use of tregs specific for self-antigens in the treatment of autoimmune disorders. The use of tregs avoids non-specific immunosuppression, thereby protecting patients from the risk of infection resulting from immunosuppressive therapy. Furthermore, the tregs described herein may also be used in populations comprising tregs and natural killer cells (NK cells).

Tregs are an important component of the immune system and act as "professional" inhibitors of the immune response. Their importance in maintaining allograft function has been demonstrated in several in vitro and in vivo modelsTo prove (see, e.g., Duran-struck et al, Transplantation]101(2) 274-283,2017; lam et al, Transplantation]101(10):2277-2287,2017). According to the classical phenotypic description, Treg is CD4⁺Cells that constitutively express high levels of Interleukin (IL) -2 receptor alpha-chain CD25 and the transcription factor Foxp3, Foxp3, are considered to be an important component for the development and maintenance of regulatory function (see, e.g., vaikunthan et al, clin]189(2):197-210,2017). Another surface marker, CD127, is negatively correlated with Foxp3 expression and can be used to identify tregs (see, e.g., Liu et al, j.exp.med. [ journal of experimental medicine ]]203(7):1701-11,2006). The potential use of tregs in therapy for inducing tolerance to allografts or as immunomodulation has led to interest in increasing the number of tregs, including the development of different expansion protocols by antigen-specific or non-specific means. In antigen-specific expansion, tregs are typically exposed to alloantigens by either direct methods of presentation of the alloantigen by donor B cells or dendritic cells, or by indirect presentation using autologous dendritic cells (see, e.g., veerapathtran et al, Blood [ Blood ] for example]118(20):5671-80,2011). Donor alloantigen-specific tregs have been shown to be five to ten times more potent than non-specific polyclonal tregs (see, e.g., vaikunthan et al, clin. exp. immunol. [ clinical and experimental immunology ]]189(2):197-210,2017)。

Isolated Treg and NK cells

The isolated tregs and NK cells of the invention are derived from T cells and NK cells of a subject (e.g., a transplant recipient or a subject with an autoimmune disorder). T cells and NK cells useful in the present invention include autologous T cells and NK cells (e.g., human T cells and NK cells) obtained from a subject, which cells will subsequently be administered to the subject after ex vivo modification and expansion. T cells and NK cells are typically obtained from peripheral blood collected from a subject by, for example, venipuncture or aspiration through an implanted port or catheter. Optionally, the blood may be obtained by a process including leukopheresis, wherein leukocytes are obtained from the subject's blood, while other blood components are returned to the subject. Blood or leukapheresis products (fresh or cryopreserved) can be treated to enrich for T cells using methods known in the art. Thus, for example, density gradient centrifugation (using, e.g., Ficoll) and/or countercurrent centrifugal elutriation may be performed to enrich for monocytes (including T cells). A T cell stimulation step using, for example, IL-2 can be further performed to stimulate T cells and deplete other cells. The T cells of the enriched T cell preparation may then be modified ex vivo.

In some embodiments, the tregs and NK cells described herein are specific for a donor alloantigen and are capable of suppressing an immune response against a particular alloantigen. For example, the donor alloantigen may be an MHC molecule that is present in the transplant donor but not in the transplant recipient. In particular, the donor alloantigen for which Treg and NK cells are specific may be Human Leukocyte Antigen (HLA) present in the transplant donor but absent from the transplant recipient. When the transplant donor and recipient have different HLA, this is called HLA mismatch. The transplant recipient may have more than one HLA mismatch with the donor. The tregs and NK cells described herein are specific for HLA mismatches in transplant recipients.

For example, Treg and NK cells may be specific for HLA-DR proteins such as HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR5, HLA-DR6, HLA-DR7, HLA-DR8, HLA-DR9, HLA-DR10, HLA-DR11, HLA-DR12, HLA-DR13, HLA-DR14, HLA-DR15, HLA-DR16, HLA-DR17, HLA-DR18, HLA-DR51, HLA-DR52, or HLA-DR53 proteins, or any other HLA-DR serotype known in the art. In another example, tregs and NK cells may be specific for HLA-DQ proteins, such as HLA-DQ2, HLA-DQ3, HLA-DQ4, HLA-DQ5, HLA-DQ6, HLA-DQ7, HLA-DQ8, or HLA-DQ9 peptides, or any other HLA-DQ serotype known in the art. In another example, the tregs and NK cells may be specific for HLA-DP proteins, such as HLA-DPw1, HLA-DPw2, HLA-DPw3, HLA-DPw4, HLA-DPw5, or HLA-DPw6 proteins, or any other HLA-DP serotype known in the art. In another example, the Treg and NK cells may be specific for HLA-a peptides, such as HLA-a1, HLA-a2, HLA-A3, HLA-a9, HLA-a10, HLA-a11, HLA-a19, HLA-a23, HLA-a24, HLA-a25, HLA-a26, HLA-a28, HLA-a29, HLA-a30, HLA-a31, HLA-a32, HLA-a33, HLA-a34, HLA-a36, HLA-a43, HLA-a66, HLA-a68, HLA-a69, HLA-a74, or HLA-a80 protein, or any other HLA-a serotype known in the art. In another example, the Treg and NK cells may be specific for HLA-B proteins, such as HLA-B5, HLA-B7, HLA-B8, HLA-B12, HLA-B13, HLA-B14, HLA-B15, HLA-B16, HLA-B17, HLA-B18, HLA-B21, HLA-B22, HLA-B27, HLA-B35, HLA-B37, HLA-B38, HLA-B39, HLA-B40, HLA-B41, HLA-B42, HLA-B44, HLA-B45, HLA-B46, HLA-B47, HLA-B4672, HLA-B465, HLA-B, HLA-B, HLA-B-4672, HLA-B-465, HLA-B, HLA-B, HLA-B, HLA-B, HLA-B, HLA-B, HLA-B-583, HLA-B, HLA-B, HLA-B, HLA, HLA-B62, HLA-B63, HLA-B64, HLA-B65, HLA-B67, HLA-B70, HLA-B71, HLA-B72, HLA-B73, HLA-B75, HLA-B76, HLA-B77, HLA-B78, HLA-B81, HLA-B82, or HLA-B83 protein, or any other HLA-B serotype known in the art. In another example, the Treg and NK cells may be specific for HLA-C proteins, such as HLA-Cw1, HLA-Cw2, HLA-Cw3, HLA-Cw4, HLA-Cw5, HLA-Cw6, HLA-Cw7, HLA-Cw8, HLA-Cw9, HLA-Cw10, or HLA-Cw11 proteins, or any other HLA-C serotypes known in the art.

In another example, the tregs and NK cells described herein are specific for an autoantigen that causes an autoimmune disorder. The autoantigen may be, for example, an autoantigen that causes rheumatoid arthritis, lupus, or membranous nephropathy. The autoantigen may also be, for example, an autoantigen that causes autism or an autism spectrum disorder.

In any of the foregoing examples, the tregs and NK cells may be specific for full-length HLA peptides or autoantigens. Alternatively, tregs and NK cells may be specific for fragments of HLA peptides (e.g., β -chain fragments of HLA peptides) or fragments of autoantigens. In particular, tregs and NK cells may suppress Teff immune responses against any of the aforementioned HLA peptides or autoantigens or fragments thereof.

The tregs produced by the methods described herein may be characterizedIn the presence or absence of one or more additional molecular markers, which can be readily assessed by standard methods known in the art (e.g., flow cytometry). Tregs produced by the methods described herein may express one or more markers selected from the group consisting of CD4, CD25, Foxp3, GITR, CTLA4, ICOS, GARP, LAP, PD-1, CD39, CD73, CD45RA, CXCR3, and CCR 6. Furthermore, tregs may down-regulate or lack the expression of CD 127. For example, tregs may have CD4⁺CD25⁺CD127^-Phenotype. Tregs may also have CD4⁺CD25⁺CD39⁺Phenotype. In another example, the Treg may have CD4⁺CD25⁺CD73⁺Phenotype. In any of the foregoing examples, the Treg may further express one or more markers selected from GITR, CTLA4, ICOS, GARP, LAP, PD-1, CD39, CD73, CD45RA, CXCR3, and CCR 6. NK cells are also characterized by the presence or absence of one or more additional molecular markers, such as CD56 or CD 16.

Methods of generating Treg and NK cells

Treg and NK cell generation

The tregs of the invention are typically produced from a population of immune cells (e.g., PBMCs obtained from a subject) comprising T cells derived from the subject (e.g., a transplant recipient or a subject having an autoimmune disease or disorder). Optionally, the population of immune cells further comprises NK cells. Generally, methods for ex vivo stimulation of T cells are known in the art. For the methods described herein, T cells are stimulated by contacting the cells with HLA molecules (e.g., HLA peptides) or fragments of self antigens (e.g., β -chain fragments) and autologous APCs (e.g., PBMCs, dendritic cells, macrophages, or B cells). The immune cell population can be a PBMC population, a naive T cell population, or an isolated population of tregs derived from a subject (e.g., a transplant recipient or a subject having an autoimmune disease or disorder), and optionally including NK cells. For example, the immune cell population can be contacted with HLA peptides or autoantigens at the following concentrations: about 25 μ g/ml to about 200 μ g/ml, such as about 25 μ g/ml to about 150 μ g/ml, about 25 μ g/ml to about 100 μ g/ml, about 25 μ g/ml to about 75 μ g/ml, about 25 μ g/ml to about 50 μ g/ml, about 30 μ g/ml to about 200 μ g/ml, about 30 μ g/ml to about 150 μ g/ml, about 30 μ g/ml to about 100 μ g/ml, about 30 μ g/ml to about 75 μ g/ml, about 40 μ g/ml to about 200 μ g/ml, about 40 μ g/ml to about 150 μ g/ml, about 40 μ g/ml to about 100 μ g/ml, about 40 μ g/ml to about 75 μ g/ml, about 50 μ g/ml to about 200 μ g/ml, about 50 μ g/ml to about 150 μ g/ml, From about 50 μ g/ml to about 100 μ g/ml, or from about 50 μ g/ml to about 75 μ g/ml. In a particular example, the concentration of the HLA peptide or autoantigen is 50 μ g/ml.

To expand T cell lines, immune cell populations can be stimulated in the presence of IL-2. The concentration of IL-2 used in the process can be, for example, from about 50IU/ml to about 200IU/ml, e.g., from about 50IU/ml to about 150IU/ml, from about 50IU/ml to about 100IU/ml, from about 70IU/ml to about 200IU/ml, from about 70IU/ml to about 150IU/ml, from about 100IU/ml to about 200IU/ml, from about 100IU/ml to about 150IU/ml, or from about 150IU/ml to about 200 IU/ml. In a specific example, the concentration of IL-2 is 100 IU/ml.

The immune cell population may be stimulated once with HLA peptides or autoantigens and autologous APCs in the presence of IL-2. In other cases, the cells are stimulated more than once, e.g., two, three, four, or five times. The time interval between each stimulation is, for example, between seven and ten days, such as seven, eight, nine or ten days.

The methods described herein for providing tregs can be performed on a population of T cells or a population of immune cells that includes both tregs and NK cells. In some embodiments, the tregs are subsequently purified from a population of T cells or from a population of immune cells. In further embodiments, a mixed population of tregs and NK cells is purified from the immune cell population. Methods for isolating Treg and NK cells are known in the art. For example, tregs can be purified from mixed populations using a number of commercially available separation kits (laboratory scale separations) and FACS cell sorters (GMP separations). In the standard formulations described herein, tregs are purified and such purification typically involves NK cells.

HLA molecules

As noted above, tregs that are useful for treating or preventing transplant rejection or promoting allograft acceptance are specific for alloantigens (e.g., HLA proteins) that are present in an organ or tissue transplant donor but not in the recipient. HLA proteins found in the donor but not in the recipient are referred to as HLA protein mismatches. These tregs recognizing mismatched HLA proteins can be generated by contacting the tregs with one or more HLA peptide fragments, which may or may not overlap. Such HLA peptide fragments are generated from mismatched HLA protein sequence portions that are present in the donor HLA protein but not in the recipient HLA protein. For example, HLA peptides can be synthesized based on the sequences of, for example, the hypervariable regions of the β -chain sequences of any known HLA serotype (e.g., any of the HLA serotypes described above) or fragments thereof. In some embodiments, the HLA peptide fragments are generated from the HLA-DRB sequence of the UniProt accession nos: p04229, P01912, P13760, P13761, Q30134, Q9TQE0, Q30167, P20039, Q95IE3, Q5Y7a7, Q9GIY3, P01911, or Q29974. HLA fragments can be peptides of about 10-100, 15-50, or 18-22 amino acids in length. Table 1 provides a table of known HLA genotypes and their corresponding serotypes.

TABLE 1 HLA class I and II genotypes and serotypes

In addition to those listed above, any other HLA protein and peptide fragments thereof may also be used to prepare the inventive tregs described herein. Peptides can be readily synthesized by methods known to those skilled in the art (e.g., solid phase synthesis), or they can be synthesized or obtained from a variety of commercial sources. One or more HLA peptide fragments corresponding to HLA proteins can be used to stimulate tregs as described herein. Exemplary HLA-DR peptide fragment sequences can be found in Vella et al, Transplantation 27; 64(6) 795-800,1997, which are incorporated herein by reference in their entirety.

In one working example, at least one HLA-DR protein mismatch is identified, wherein the HLA-DR protein is found in the transplant donor but not in the recipient. A set of HLA-DR peptide fragments based on one or more mismatched HLA-DR proteins is synthesized, wherein the peptides are unique to the mismatched HLA-DR protein of the donor and do not overlap with the HLA-DR protein of the recipient. These peptide fragments are synthesized based on the β -chain hypervariable region of mismatched HLA-DR proteins and may, for example, correspond to the following sequences:

TABLE 2 exemplary beta-chain HLA-DR peptide fragment sequences

Although the sequences in table 2 are provided as examples of peptide fragments that can be used in the present invention, other peptide fragments can be synthesized according to the procedures described herein. The peptide fragments provided in table 2 should not be construed as limiting the range of HLA peptide fragment sequences that can be used in the procedure for generating tregs as described herein.

Exemplary suppression mechanisms

Several contact-independent and contact-dependent mechanisms have been described that contribute to Treg function, which usually work simultaneously. Without being bound by theory, the tregs described herein may suppress an immune response by one or more of the mechanisms described below.

Non-contact dependent mechanisms that have been described include the production of anti-inflammatory cytokines (e.g., IL-10, IL-35, and TGF-. beta.) (see, e.g., Maloy et al, J.Exp.Med. [ J. Experimental medicine ]197(1):111-9,2003) and the transfer of miRNAs, which can silence specific genes in T cells via exosomes, thereby preventing proliferation and cytokine production (see, e.g., Okoye et al, Immunity [ immune ]41(1):89-103,2014).

Contact-dependent mechanisms include, but are not limited to, the interaction of CTLA-4 with its ligands B7.1 and B7.2 on APC, leading to negative signals that prevent T cell activation (see, e.g., Vasu et al, j.immunol. [ journal of immunology ]173(4): 2866-; cell surface LAG-3 expression that binds to MHC class II molecules and prevents maturation of APCs and the ability to activate effector T cells; expression of membrane-binding active TGF-beta 1 on the Treg population (see, e.g., Savage et al, J.Immunol. [ J. Immunol ]181(3): 2220-; induction of apoptosis via conjugation of CTLA-4 and programmed cell death 1(PD-1) (see, e.g., Francisco et al, J.Exp.Med. [ J.EXPERIMENT MEDICAL ]206(13): 3015-; granzyme A/B expression (see, e.g., Grossman et al, Blood [ Blood ]104(9):2840-8, 2004); IL-2 deprivation (see, e.g., Pandiyan et al, nat. Immunol. [ Natural immunology ]8(12): 1353-; and by disrupting adenosine-capable metabolic pathways.

Inhibition of human tregs by the adenosine-capable pathway involves conversion of ATP to AMP and adenosine in turn, which binds to the A2a receptor on effector T cells. This activates the immunosuppressive loop through elevation of cytoplasmic cAMP, resulting in decreased production and proliferation of pro-inflammatory cytokines (see, e.g., Mandapathil et al, J.biol.chem. [ J.Biochem ]285(10): 7176-.

Pharmaceutical composition

The tregs described herein can be incorporated into a vehicle for administration to a subject (e.g., a human patient receiving an organ, tissue, or cell transplant, or a patient having an autoimmune disorder). Pharmaceutical compositions containing Treg cells can be prepared using methods known in the art. In addition, the pharmaceutical composition may include a mixed population of tregs and NK cells. Such compositions can be prepared using a variety of pharmaceutically acceptable carriers as determined to be suitable by those skilled in The art (see, e.g., Gennaro, Remington: The Science and Practice of pharmacy [ Remington: Science and Practice of Pharmacology ] 22 nd edition, Allen, L. editor (2013); Ansel et al, Pharmaceutical Dosage Forms and Drug Delivery Systems [ Pharmaceutical Dosage Forms and Drug Delivery Systems ], 7 th edition, Lipocot-Williams-Wilkins publishing company (Lippincott, Williams and kins) (2004); Wilbbe et al, Handbook of Pharmaceutical Excipients [ Pharmaceutical Excipients ], 3 rd edition, Pharmaceutical Press (Pharmaceutical Press) (2000)). A variety of pharmaceutically acceptable carriers can be used, including vehicles, adjuvants, and diluents. In addition, a variety of pharmaceutically acceptable auxiliary substances may be used, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizing agents, wetting agents, and the like. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

Methods of treatment and uses

The tregs described herein (optionally in populations that include NK cells) can be used to suppress an immune response to promote allograft acceptance in patients undergoing organ or tissue transplantation, or to treat or prevent transplant rejection. The organ can be any implantable organ including, but not limited to, heart, kidney, liver, lung, bladder, ureter, stomach, intestine (e.g., small or large intestine)), skin, tongue, esophagus, endocrine glands (e.g., pancreas, adrenal gland, salivary gland, thyroid, pituitary, etc.), bone marrow, spleen, thymus, lymph nodes, tendons, ligaments, muscles, uterus, vagina, ovary, fallopian tube, testis, penis, cornea, lens, retina, middle ear, outer ear, cochlea, iris, and veins. Organs that can be transplanted also include vascular composite allografts, such as the face, hands or legs. The tissue may be any tissue that is implantable, including, but not limited to, bone marrow, islets of langerhans, stem cells, blood vessels, neural tissue, cartilage, tendons, ligaments, cornea, heart valves, nerves and/or veins, middle ear, cultured tissue (e.g., differentiated cells that may function as an organ or tissue), and/or 3D engineered tissue. The cell may be any cell that is transplantable (e.g., a stem cell (e.g., a hematopoietic stem cell)).

There are currently a number of accepted protocols for promoting tolerance of transplant recipients to donor organs or tissues. Typically, these regimens include administration of an immunosuppressive agent to prevent rejection of the transplanted organ or tissue by the recipient. In one example, for donor-matched transplant recipient HLA, the protocol includes administration of cyclophosphamide, thymus irradiation, and antithymocyte globulin. In another example, for donor mismatched transplant recipient HLA, the protocol includes administration of cyclophosphamide, anti-CD 2 antibody, thymus and bone marrow irradiation, and may or may not use rituximab. In another example, for donor-matched transplantation recipient HLA, the protocol involves administration of cyclophosphamide, fludarabine and CD34+ cells. Other methods for promoting allograft acceptance are known to those skilled in the art.

The tregs or mixed population of tregs and NK cells may be administered in addition to or in lieu of any accepted regimen for promoting allograft acceptance. In certain instances, administration of immunosuppressive agents is reduced following administration of tregs. The dose of immunosuppressive agent can be reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% following administration of tregs or a mixed population of tregs and NK cells. In some examples, the dose of immunosuppressive agent is reduced by about 50% following treatment with tregs or a mixed population of tregs and NK cells. In another example, administration of the immunosuppressive agent is discontinued after administration of the Treg or mixed population of tregs and NK cells.

The tregs or mixed populations of tregs and NK cells of the invention may also be used to treat autoimmune disorders such as autism, autism spectrum disorders, rheumatoid arthritis, lupus, focal segmental glomerulonephritis and membranous nephropathy. Additional non-limiting examples of autoimmune diseases or disorders include, but are not limited to, inflammatory arthritis, type 1 diabetes, multiple sclerosis, psoriasis, inflammatory bowel disease and vasculitis, allergic inflammation (e.g., allergic asthma, atopic dermatitis, contact hypersensitivity), Graves 'disease (hyperthyroidism), Hashimoto's thyroiditis (hypothyroidism), celiac disease, Crohn's disease and ulcerative colitis, Guillain-Barre syndrome (Guillain-Barre syndrome), primary biliary/liver cirrhosis, sclerosing cholangitis, autoimmune hepatitis, Raynaud's phenomenon, scleroderma, Sjogren's syndrome, Goodpasture's syndrome (Goodpasture's syndrome), Wegener's granuloma (Wegener's granulomatosis), Wegener's granuloma, Polymyalgia rheumatica, temporal arteritis/giant cell arteritis, Chronic Fatigue Syndrome (CFS), autoimmune Addison's Disease, ankylosing spondylitis, acute disseminated encephalomyelitis, antiphospholipid antibody syndrome, aplastic anemia, idiopathic thrombocytopenic purpura, myasthenia gravis, ocular clonic myoclonic syndrome, optic neuritis, Ord's thyroiditis, pemphigus, pernicious anemia, canine polyarthritis, Reiter's syndrome, Takayasu's arteritis, warm autoimmune hemolytic anemia, and Fibromyalgia (FM).

Combination therapy

The tregs or mixed populations of tregs and NK cells described herein may be used in combination with other known agents and therapies. As used herein, "administration in combination" refers to delivery of two (or more) different treatments to a subject during the time the subject has a disorder (e.g., a disease or condition), e.g., after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or otherwise discontinued treatment. In some embodiments, when delivery of the second therapy begins, delivery of the first therapy is still ongoing, so there is overlap with respect to administration. This is sometimes referred to herein as "simultaneous delivery" or "concurrent delivery". In other embodiments, delivery of one therapy ends before delivery of another therapy begins. In some embodiments of either case, the treatment is more effective as a result of the combined administration. For example, the second treatment is more effective than the results observed when the second treatment is administered in the absence of the first treatment, e.g., an equivalent effect is observed with less of the second treatment, or the second treatment reduces symptoms to a greater extent, or a similar condition is observed for the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than the observed result for one treatment delivered in the absence of the other treatment. The effects of the two treatments may be partially additive, fully additive, or more than additive. The delivery may be such that the effect of the delivered first therapy is still detectable when the second therapy is delivered. The tregs or mixed population of tregs and NK cells and at least one additional therapeutic agent described herein may be administered simultaneously, in the same or separate compositions, or sequentially. For sequential administration, the tregs or mixed population of tregs and NK cells may be administered first, and then the additional agent may be administered. Alternatively, the order of administration may be reversed, and the additional agent may be administered first, and then the tregs or mixed population of tregs and NK cells may be administered. Tregs or mixed population cell therapies of tregs and NK cells and/or other therapeutic agents, procedures or modalities may be administered during periods of mobility impairment or during periods of remission or less active disease. Tregs or a mixed population cell therapy of tregs and NK cells may be administered before another treatment, concurrently with a treatment, after a treatment, or during the remission of the disorder.

When administered in combination, the tregs or mixed population of tregs and NK cells and the additional agent (e.g., second or third agent) or all described herein may be administered in a higher, lower, or the same amount or dose as the amount or dose of each agent used alone (e.g., as a monotherapy). In certain embodiments, the administered amount or dose of tregs or a mixed population of tregs and NK cells, additional agent (e.g., second or third agent), or all is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dose of each agent used alone. In other embodiments, the amount or dose of tregs or a mixed population of tregs and NK cells, additional agent (e.g., a second or third agent), or all that produces a desired effect (e.g., treating cancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dose of each agent alone needed to achieve the same therapeutic effect.

For example, the one or more additional therapeutic agents may include one or more immunosuppressive agents commonly used in organ or tissue transplants. The one or more immunosuppressive agents may be an agent administered immediately after transplantation to prevent acute rejection (e.g., methylprednisolone, antithymocyte gamma globulin (atgam), thymoglobulin, basiliximab, or alemtuzumab) or one or more immunosuppressive agents for maintenance (e.g., prednisone, a calmodulin inhibitor (e.g., cyclosporine or tacrolimus), mycophenolate mofetil, azathioprine, sirolimus, or everolimus). Other immunosuppressive agents administered after organ transplantation include CTLA-4 fusion proteins (e.g., belicept or abamectin), corticosteroids (e.g., methylprednisolone, dexamethasone, or prednisolone), cytotoxic immunosuppressive agents (e.g., azathioprine, chlorambucil, cyclophosphamide, mercaptopurine, or methotrexate), immunosuppressive antibodies (e.g., anti-thymocyte globulin, basiliximab, or infliximab), sirolimus derivatives (e.g., everolimus or sirolimus), and antiproliferative agents (e.g., mycophenolate mofetil, mycophenolate sodium, or azathioprine). Additional immunosuppressive agents suitable for use with the invention described herein are known to those skilled in the art, and the invention is not limited in this regard.

Furthermore, in view of the results described herein, the inhibitory activity of donor peptide-driven T cell lines generated from kidney transplant patients involves an adenoergic pathway. In view of these results, additional combination therapies involve combining Treg infusion with additional therapies such as adenosine receptor agonists (e.g., regadenoson) or increasing CD39 expression in the transplant (e.g., by administering immunomodulatory therapies such as interferon beta, fingolimod, alemtuzumab, and corticosteroids) to achieve transplant tolerance.

Route of administration

An effective amount of a therapeutic agent described herein for treating or preventing a disease or disorder (e.g., transplant rejection or an autoimmune disorder) or for promoting allograft acceptance (e.g., tregs or a mixed population of tregs and NK cells specific for a donor alloantigen or autoantigen) can be administered to a subject by standard methods. For example, the agent can be administered by any of a number of different routes, including, e.g., intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, transdermal injection, oral, transdermal (topical), intraarterial, intratumoral, intranodal, intramedullary, or transmucosal administration. In some embodiments, an agent (e.g., a Treg or a mixed population of tregs and NK cells specific for a donor alloantigen or autoantigen) can be administered directly (e.g., by injection or infusion) into a transplanted organ or tissue. In one embodiment, the compositions described herein are administered into a body cavity or body fluid (e.g., ascites, pleural fluid, peritoneal fluid, or cerebrospinal fluid). For example, a therapeutic agent (e.g., a Treg or a mixed population of tregs and NK cells specific for a donor alloantigen or autoantigen) can be administered by injection or infusion, e.g., intramuscular, subcutaneous, intraperitoneal, or intravenous. The most suitable route of administration in any given case will depend on the particular agent being administered, the patient, the particular disease or condition being treated, the pharmaceutical formulation method, the method of administration (e.g., time of administration and route of administration), the age, weight, sex of the patient, the severity of the disease being treated, the diet of the patient and the rate of excretion from the patient. The agent (e.g., tregs or a mixed population of tregs and NK cells specific for a donor alloantigen or autoantigen) can be encapsulated or injected, e.g., in a viscous form, for delivery to the site of selection. The agent may be provided in the form of a matrix capable of delivering the agent to the selected site. The matrix may provide a slow release of the agent and provide proper presentation and a suitable environment for cellular infiltration. The matrix may be formed from materials currently used for other implanted medical applications. The selection of the matrix material is based on any one or more of the following: biocompatibility, biodegradability, mechanical properties, and appearance and interface properties. One example is a collagen matrix.

Therapeutic agents (e.g., tregs or a mixed population of tregs and NK cells specific for a donor alloantigen or autoantigen) can be incorporated into a pharmaceutical composition suitable for administration to a subject (e.g., a human). Such compositions generally comprise a pharmaceutical agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used herein. Supplementary active compounds may also be incorporated into the compositions.

Administration of drugs

The term "unit dosage form" as used herein refers to a dosage suitable for one administration. For example, the unit dosage form may be a quantity of therapeutic agent disposed in a delivery device (e.g., a syringe or an intravenous drip bag). For example, the unit dosage form is administered in a single administration. In another example, more than one unit dosage form may be administered simultaneously.

In some embodiments, the tregs or mixed population of tregs and NK cells are administered as a monotherapy, i.e., another treatment for the disorder is not administered to the subject concurrently. The tregs or mixed population composition of tregs and NK cells may be administered to the patient at one time. The Treg cell composition may also be administered multiple times if desired. Tregs or a mixed population of Tregs and NK cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al, New England Journal of Medicine 319:1676 (1988)).

The dosage of such treatment administered to a patient will vary with the exact nature of the condition being treated and the recipient treated. Scaling (scaling) of the dose administered to a human can be performed according to art-accepted practice.

In some embodiments, a single treatment regimen is required. In other embodiments, administration of one or more subsequent doses or treatment regimens may be performed. For example, after every two weeks of treatment for three months, the treatment may be repeated once a month for six months or a year or more. In some embodiments, no additional treatment is administered after the initial treatment.

The dosage of the compositions as described herein can be determined by a physician and adjusted as necessary to accommodate the observed therapeutic effect. With respect to the duration and frequency of treatment, a skilled clinician will typically monitor the subject to determine when treatment provides a therapeutic benefit, and to determine whether to administer additional cells, stop treatment, resume treatment, or make other changes to the treatment regimen. The dose should not be so large as to cause adverse side effects, such as cytokine release syndrome. In general, the dosage will vary with the age, condition and sex of the patient and can be determined by one skilled in the art. In the case of any complication, the dosage may also be adjusted by the individual physician.

Therapeutic effect

The efficacy of treatment with tregs or a mixed population of tregs and NK cells (e.g., in treating transplant rejection or autoimmune disorders, or promoting allograft acceptance) can be determined by the skilled clinician. However, treatment is considered to be "effective treatment" (as the term is used herein) under the following conditions: one or more signs or symptoms of the conditions described herein are altered in a beneficial manner, other clinically acceptable symptoms are ameliorated or even alleviated, or a desired response is induced, e.g., by at least 10% after treatment according to the methods described herein. For example, efficacy may be assessed by measuring the incidence of markers, indicators, symptoms, and/or conditions treated according to the methods described herein, or any other suitable measurable parameter. Treatment according to the methods described herein can reduce the level of a marker or symptom of a disorder by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

Efficacy may also be measured by the individual being assessed in a hospital without worsening or requiring medical intervention (i.e., cessation of progression of the disease). Methods of measuring these indices are known to those skilled in the art and/or described herein.

Treatment includes any treatment of a disease in an individual, and includes: (1) suppression of disease, e.g., prevention of worsening of symptoms (e.g., pain or inflammation); or (2) reducing the severity of the disease, e.g., causing regression of symptoms. An effective amount for treating a disease refers to an amount sufficient to result in effective treatment of the disease (as that term is defined herein) when administered to a subject in need thereof. The therapeutic effect of an agent can be determined by assessing a physical indicator of the condition or desired response. It is well within the ability of the person skilled in the art to monitor the efficacy of an administration and/or treatment by measuring any one of such parameters or any combination of parameters. The efficacy of a given method can be assessed in an animal model of the conditions described herein. When using experimental animal models, the efficacy of the treatment can be demonstrated when statistically significant changes in the markers are observed.

Exemplary non-limiting symptoms of transplant rejection include elevated serum creatinine, reduced eGFR (estimated glomerular filtration rate), flu-like symptoms, fever, reduced urine output, weight gain, pain, and fatigue.

Exemplary, non-limiting symptoms of an autoimmune disease or disorder include fatigue, joint pain and swelling, skin problems, abdominal pain or digestive problems, repeated fever, proteinuria, and swollen glands.

All such modifications are intended to be included within the scope of the appended claims.

The invention is further described in the following numbered paragraphs:

1. an isolated regulatory T cell (Treg) comprising a T Cell Receptor (TCR) that specifically binds:

(i) an alloantigen which is a Human Leukocyte Antigen (HLA) molecule or fragment thereof and is not encoded by a nucleotide sequence present in the genome of the Treg, or

(ii) An autoantigen or fragment thereof that causes an autoimmune disorder.

2. The Treg of paragraph 1, wherein the TCR specifically binds the HLA molecule.

3. The Treg of paragraph 2, wherein the TCR specifically binds to a hypervariable region (HVR) of the HLA molecule.

4. The Treg of paragraph 3, wherein the TCR specifically binds to the β -chain HVR of the HLA molecule.

5. The Treg of any of paragraphs 2-4, wherein the HLA molecule is an HLA-DR, HLA-DQ, HLA-DP, HLA-A, HLA-B or HLA-C molecule or fragment thereof.

6. The Treg of paragraph 5, wherein the HLA molecule is an HLA-DR, HLA-DQ or HLA-DP molecule or fragment thereof.

7. The Treg of paragraph 6, wherein the HLA-DR molecule is an HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR5, HLA-DR6, HLA-DR7, HLA-DR8, HLA-DR9, HLA-DR10, HLA-DR11, HLA-DR12, HLA-DR13, HLA-DR14, HLA-DR15, HLA-DR16, HLA-DR17, HLA-DR18, HLA-DR51, HLA-DR52 or HLA-DR53 molecule or a fragment thereof.

8. The Treg of any of paragraphs 1-7, wherein the Treg is capable of inhibiting a T effector cell (Teff) response against the alloantigen or the autoantigen.

9. The Treg of paragraph 8, wherein the Treg is capable of suppressing Teff proliferation response to direct allorecognition, semi-direct allorecognition and/or indirect allorecognition.

10. The Treg of paragraph 8 or 9, wherein the Treg is capable of activating an adenosylergic signaling pathway.

11. The Treg of any of paragraphs 1-10, wherein the Treg expresses one or more markers selected from the group consisting of CD4, CD25, CD39, CD73, FOXP3, GITR, CLTA4, ICOS, GARP, LAP, PD-1, CCR6, and CXCR 3.

12. The Treg of any of paragraphs 2-11, wherein the HLA molecule or fragment thereof to which the TCR specifically binds is encoded by a nucleotide sequence present in the genome of the organ or tissue donor.

13. An isolated Treg comprising a TCR that specifically binds to:

(i) an alloantigen which is an HLA molecule or fragment thereof and is not encoded by a nucleotide sequence present in the genome of the Treg, or

(ii) (ii) an autoantigen or fragment thereof that causes an autoimmune disorder;

wherein the tregs have been generated by a method comprising:

(a) contacting a population of immune cells comprising T cells obtained from a recipient subject with the HLA molecule or fragment of a self antigen and autologous Antigen Presenting Cells (APCs); and

(b) expanding the population of immune cells of step (a) for a time and under conditions sufficient to form an expanded T cell line comprising a plurality of these tregs; and optionally

(c) Purifying the tregs from the immune cell population.

14. The tregs of paragraph 13, wherein the population of immune cells of (a) further comprises Natural Killer (NK) cells, and if step (c) is performed, step (c) comprises purifying the tregs and NK cells from the population of immune cells, thereby generating a mixed population of tregs and NK cells.

15. A mixed population of cells comprising the tregs and NK cells of any one of paragraphs 1-12.

16. A composition comprising a Treg as described in any of paragraphs 1-14.

17. A composition comprising the mixed population of cells of paragraph 15.

18. A method of suppressing an immune response in a subject, the method comprising administering to the subject a Treg of any of paragraphs 1-14, a mixed population of cells of paragraph 15, or a pharmaceutical composition of paragraphs 16 or 17.

19. The method of paragraph 18, wherein the immune response is a Teff response against the alloantigen or the autoantigen.

20. A method of treating or preventing transplant rejection or a method of treating an autoimmune disorder in a subject, the method comprising administering to the subject a Treg of any of paragraphs 1-14, a mixed population of cells of paragraph 15, or a composition of paragraphs 16 or 17.

21. The method of paragraphs 18 or 19, wherein the subject has an autoimmune disorder.

22. The method of any of paragraphs 18-20, wherein the subject is an organ or tissue transplant recipient.

23. The method of any of paragraphs 18-20 or 22, wherein the HLA molecule or fragment thereof to which the TCR specifically binds is encoded by a nucleotide sequence present in the genome of the organ or tissue donor.

24. The method of any of paragraphs 18-20, 22, or 23, wherein the method further comprises reducing the dose of immunosuppressive agent administered to the subject.

25. The method of any of paragraphs 18-20 or 22-24, wherein the organ is a kidney, liver, heart, lung, pancreas, intestine, stomach, testis, penis, thymus, or vascular composite allograft for the face, hand or leg.

26. The method of any of paragraphs 18-20 or 22-24, wherein the tissue comprises bone, tendon, cornea, skin, heart valve, neural tissue, bone marrow, islets of Langerhans, stem cells, blood, or blood vessels.

27. The method of paragraph 20 or 21, wherein the autoimmune disorder is autism, autism spectrum disorder, rheumatoid arthritis, lupus, focal segmental glomerulonephritis, or membranous nephropathy.

28. A method for generating a Treg as described in any of paragraphs 1-12, the method comprising:

(a) contacting a population of immune cells comprising T cells obtained from a recipient subject with the HLA molecule or fragment of an autoantigen and autologous APCs; and

(b) expanding the population of immune cells of step (a) for a time and under conditions sufficient to form an expanded T cell line comprising a plurality of these tregs; and optionally

(c) Purifying the tregs from the immune cell population.

29. The method of paragraph 28, wherein the method comprises repeating steps (a) and (b) more than three times.

30. The method of paragraph 28 or 29, wherein the method comprises repeating steps (a) and (b) four or five times.

31. The method of any of paragraphs 28-30, wherein step (a) is performed approximately every seven to ten days.

32. The method of any of paragraphs 28-31, wherein the autologous APCs are peripheral blood mononuclear cells (PMBC), dendritic cells, macrophages or B cells.

33. The method of paragraph 32, wherein the autologous APCs are PBMCs.

34. The method of paragraph 33, wherein the PBMCs are irradiated.

35. The method of any of paragraphs 28-34, wherein the population of immune cells comprising T cells is a PMBC population, a naive T cell population, or a purified Treg population.

36. The method of paragraph 35, wherein the population of immune cells is a population of PBMCs.

37. The method of paragraph 36, wherein step (a) further comprises contacting the population of PBMCs with IL-2.

38. The method of paragraph 37, wherein the concentration of IL-2 is from about 50IU/ml to about 200 IU/ml.

39. The method of paragraph 38, wherein the concentration of IL-2 is about 100 IU/ml.

40. The method of any of paragraphs 28-39, wherein the concentration of the HLA molecule or fragment of self antigen is from about 25 μ g/ml to about 200 μ g/ml.

41. The method of paragraph 40, wherein the concentration of the HLA molecule or fragment of the autoantigen is about 50 μ g/ml.

42. The method of any of paragraphs 28-41, wherein the fragment of an HLA molecule or autoantigen is a purified peptide or peptide mixture.

43. The method of any of paragraphs 28-42, wherein the population of immune cells comprises NK cells.

44. The method of any one of paragraphs 28-43, wherein step (c) comprises purifying the Tregs and NK cells from the population of immune cells, thereby generating a mixed population of Tregs and NK cells.

45. A composition, comprising:

(a) the Treg of any of paragraphs 1-14; and

(b) a fragment of the HLA molecule or autoantigen.

46. The composition of paragraph 45, wherein the composition further comprises IL-2.

47. The composition of paragraph 46, wherein the concentration of IL-2 is from about 50IU/ml to about 200 IU/ml.

48. The composition of paragraph 47, wherein the concentration of IL-2 is about 100 IU/ml.

49. The composition of any of paragraphs 45-48, wherein the concentration of the HLA molecule or fragment of the autoantigen is from about 25 μ g/ml to about 200 μ g/ml.

50. The composition of paragraph 49, wherein the concentration of the HLA molecule or fragment of the autoantigen is about 50 μ g/ml.

51. The composition of any of paragraphs 45-50, wherein the HLA molecule or fragment of an autoantigen is a purified peptide or peptide mixture.

52. The composition of any one of paragraphs 45-51, further comprising NK cells.

The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise forms disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order or may perform the functions substantially in parallel. The teachings of the disclosure provided herein may be applied to other programs or methods as appropriate. The various embodiments described herein may be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ compositions, functions and concepts of the above-described references and applications to provide yet further embodiments of the disclosure. Furthermore, for reasons of biological functional equivalence, some changes can be made to the protein structure without affecting the kind or amount of biological or chemical action. These and other changes can be made to the disclosure in light of the detailed description.

The techniques described herein are further illustrated by the following examples, which should not be construed as further limiting in any way.

Examples of the invention

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be practiced in view of the description provided herein.

Example 1 study patients

A total of 45 kidney transplant recipients (with one or more HLA-DR mismatches with the donor) were included in the study. In addition to the three patients who received everolimus or placido instead of tacrolimus, other patients received dual or triple immunosuppressive therapy including tacrolimus. After informed consent, blood samples were obtained in multiple visits following transplantation and nineteen T cell lines were generated from seventeen patients. The local institutional ethics committee approved the study protocol.

Example 2 Generation of HLA-DR specific T cell lines

Synthesis of peptides

A set of 18-22 amino acid long non-overlapping peptides corresponding to the full-length beta-chain hypervariable regions of HLA-DRB 1x 0101, HLA-DRB 1x 1501, HLA-DRB 1x 0301 and HLA-DRB 1x 0401 were synthesized (seeRittemol, uk) as previously reported (Tsaur et al, Kidney Int. [ international Kidney ]]79(9):1005-12,2011)。

Generation of T cell lines

Peripheral blood samples from kidney transplant recipients were collected at multiple visits following transplantation and used with LYMPHOPREP^TM(Stemcell Technologies) Peripheral Blood Mononuclear Cells (PBMC) were isolated by density gradient centrifugation. The cells were then expanded ex vivo or frozen in LN2 for future use.

PBMC (10X 10)⁶) In IMMUNOCULT^TMSerum-free medium (Stem cell technology), containing 100U/mL penicillin, 100. mu.g/mL streptomycin, 100. mu.g/mL L-glutamine, 5mmol/L HEPES, 1% nonessential amino acids, and 1mmol/L sodium pyruvate (Gibco) and 2-mercaptoethanol. PBMCs were repeatedly stimulated at 7-10 day intervals with mismatched donor-derived HLA-DR allopeptides (50. mu.g/mL) and autologous irradiated (10-15Gy) PBMCs as Antigen Presenting Cells (APC) in the presence of IL-2 (10. mu.g/mL), as in Tsuur et al, Kidney Int [ International Kidney Int]79(9) 1005-12 (2011). All T cell lines were incubated at 37 ℃ in humidified 5% CO₂Cultured in an incubator and harvested after four to five stimulation cycles. The immunomodulatory function of the expanded cells was assessed by inhibition assays and was shown to be able to inhibit CD4 in response to the same donor antigen⁺T cells proliferated (fig. 2).

EXAMPLE 3 proliferation and inhibition assays

Will be 1x10⁶Carboxyfluorescein succinimidyl ester (CFSE) stained PBMC were used as responders and donor mismatched HLA-DR allopeptides and autologous irradiated PBMC were used as APC (2X 10)⁶) In 96 hole U bottom plate in wet 5% CO₂Stimulating in an incubator for 72 h. Proliferation was assessed by dilution of CFSE. In the inhibition assay, stimulated PBMC are cultured at a PBMC to T cell line ratio ranging from 1:2 to 1:16 in the presence or absence of a T cell line.

For the non-contact dependent inhibition assay, a rotary well plate was used instead of a 96-well U-bottom plate. Experiments involving suppression of inhibition included the addition of istradefylline (20. mu.g/mL) or anti-IL-10 (10. mu.g/mL) and anti-TGF-beta (10. mu.g/mL) neutralizing antibodies. All assays were performed in triplicate.

Example 4 flow cytometry analysis

Immunophenotypic analysis of T cell lines was performed against various T cell markers having fluorophore conjugated human anti-CD 3, anti-CD 4, anti-CD 25, anti-CD 127, anti-CD 39, and anti-CD 73. Using Canto II cytometer (BD)) Acquiring data and usingAnd (6) carrying out analysis. The gating strategy for phenotypic analysis (gating strategy) involves initial gating of a population of live PBMCs, followed by CD3⁺CD4⁺And (4) clustering. Expression levels of CD25, CD127, CD39, and CD73 are expressed as CD3⁺CD4⁺Percentage of clusters.

T cell proliferation and inhibition were determined by CFSE dye dilution of responder cells. Analysis of CFSE distribution inOn the proliferation platform and data is replicatedIndex (RI). RI only determines fold expansion of responder cells (Roederer, Cytometry a. [ Cytometry a.)]79(2) 95-101(2011) and is defined as the average number of divisions all cells undergo after staining with a cell proliferation dye. Percent inhibition was calculated from proliferation and inhibition values.

Example 5 statistical analysis

Results are expressed as mean ± s.d. The student's t-test is used to compare patient characteristic, phenotypic and functional data as appropriate. Each experimental condition was repeated three times. p <0.05 was considered significant.

Example 6 proliferation of CD4+ CD25-T cells from transplant recipients in response to donor-specific alloantigens

CD4⁺CD25⁺Cells were depleted from PBMCs of kidney transplant recipients. Will CD4⁺CD25^-Cells (i.e., Treg depleted T cell pool) and CD4⁺Cells were stimulated with donor alloantigens. Proliferation was measured in a flow cytometer using the dye dilution method.

Depletion of tregs in the T cell pool leads to an enhanced Teff response to alloantigens. This reaction varied from recipient to recipient (FIGS. 1A-1D).

EXAMPLE 7 immunosuppressive Activity of the generated T cell lines

Demographic and clinical characteristics

The study included a total of 45 kidney transplant recipients (with one or more donor DR mismatches). A total of 19 single T cell lines were created and expanded ex vivo from Peripheral Blood Mononuclear Cells (PBMCs) of 17 subjects. The demographics of these 17 subjects are presented in table 3 below.

TABLE 3 demographic data of patients who generated T cell lines

HLA-human lymphocyte antigen; c1-first blood collection; values for age, HLA mismatch, serum creatinine, and time between the date of transplantation and C1 were expressed as mean ± (s.d.).

A total of 82.35% of patients received thymoglobulin as an induction therapy. In terms of maintenance immunosuppressive therapy, a total of fourteen patients (82.3%) received tacrolimus therapy, of which ten patients received combined therapy with Mycophenolate Mofetil (MMF) or mycophenolic acid (MPA) and steroids. One patient received everolimus and MMF, while two other patients received placido and a steroid, one of the two patients also received MMF, and the other patient received azathioprine. All patients had stable renal function, although four of them had previously been treated for acute rejection.

Generation and morphological characterization of T cell lines

T cell lines were generated by repeated stimulation (4-5 times) of PBMCs from kidney transplant recipients with donor-specific HLA-DR allopeptides (HLA-DR1, HLA-DR4, HLA-DR15 or HLA-DR17) as described in example 2. Cell surface markers for each T cell line generated were analyzed to define ex vivo expanded cells. It was observed that 20% -50% of the cells in the ex vivo expanded lines were CD3⁺CD4⁺T cells. Resulting CD3⁺CD4⁺Some of the T cells also up-regulate the expression of CD25, while down-regulating the expression of CD 127. In addition, CD3 was observed⁺CD4⁺T cells consistently expressed CD39 and CD 73. Table 4 shows CD4 from each ex vivo expanded T cell line⁺T cell, CD4⁺CD25⁺CD125^-、CD4⁺CD39⁺And CD4⁺CD73⁺Percentage of cells. Flow cytometry data for four representative T cell lines are presented in fig. 12A-12H.

CD4 in all ex vivo expanded T cell lines⁺T cell subsets express a regulatory phenotype (CD 25)⁺CD127^-CD39⁺And CD25⁺CD127^-CD73⁺). Expression of CD4⁺CD39⁺And CD4⁺CD73⁺Cell ofThe ratio varies from 20% to 60%. It was further observed that CD39 and CD73 are not on the same CD4⁺Co-expression on a population of T cells.

TABLE 4 phenotypic analysis of the resulting T cell lines

Functional characterization of T cell lines

Their functional characterization was next determined by assessing the immunosuppressive function of ex vivo expanded T cell lines in suppressing antigen-specific and non-specific T cell proliferation. It was observed that all 19T cell lines were able to suppress donor antigen specific T cell proliferation. Table 5 presents the proliferative responses of recipient PBMCs to donor-specific HLA-DR allopeptides and the immunosuppressive capacity of T cell lines. All ex vivo expanded T cell lines generated from 17 transplant recipients showed inhibitory capacity regardless of the different combinations of immunosuppressive drug regimens that the subjects received (table 5, figure 3). The T cell line did not inhibit non-specific T cell proliferation.

The data shown in table 5 below are presented using replication index, defined as the average number of divisions all cells undergo after staining with cell proliferation dye. Percent inhibition was calculated from proliferation and inhibition values (Roederer, Cytometry a. [ Cytometry a ]79(2):95-101 (2011)).

TABLE 5 proliferation response of PBMCs and suppressive capacity of T cell lines

Rep index-replication index; fk-tacrolimus; MMF-mycophenolate mofetil; MPA-mycophenolic acid;

azathioprine; HLA-human lymphocyte antigens

No significant difference was observed between the specific HLA-DR allopeptides and the percent inhibition (figure 4). Likewise, CD4 was not present in the resulting T cell line⁺CD25⁺CD127^-A correlation was observed between the percentage of cells and the percentage of inhibition, nor was a correlation observed between the percentage of inhibition and the renal function of these patients. CD4 was also analyzed in T cell lines between transplanted patients with or without acute rejection therapy⁺CD25⁺CD127^-The difference between the percentage of cells and the percentage of inhibition. Also, no statistically significant difference was observed.

Example 8 contact-dependent immunosuppression

To further understand the mechanism of inhibition, a classical inhibition assay, a rotating well system, was used as described in example 3. It was observed that the T cell line lost the ability to inhibit donor antigen-specific T cell proliferation when separated by a semi-permeable membrane, indicating that T cell line-mediated inhibition was dependent on cell-cell contact (fig. 5A-5F).

Example 9 inhibitory Capacity in response to direct and Indirect allorecognition

CD4 from a renal transplant recipient⁺T cells were stimulated by either donor cells (direct allorecognition) or autologous APC loaded with donor antigen (indirect allorecognition) and proliferation/inhibition was measured by dye dilution. Ex vivo expanded immunomodulatory T cell lines effectively inhibit CD4⁺Proliferative responses of T cells to both direct and indirect allorecognition (fig. 6). This is desirable in allograft recipients to provide protection against both acute and chronic rejection.

Example 10 antigen specificity of Tregs

Antigen-specific inhibition of ex vivo expanded T cell lines was determined using standard inhibition assays. Measurement of CD4 from Kidney transplant recipients and third-party responders by dye dilution⁺Proliferative response of T cells to donor antigens.

It was observed that T cell lines selectively suppress T cell proliferative immune responses against specific donor alloantigens, and that tregs have no effect on the proliferative responses of third party responders to different donor antigens. As shown in fig. 7A, subjects 038 and 023 had different HLA-DR mismatches from their respective donors (HLA-DR4 mismatch and HLA-DR15 mismatch, respectively). The T line expanded from subject 038 suppressed the subject's immune response against the donor alloantigen, but did not suppress the third party immune response against a different alloantigen in subject 023. On the other hand, subjects 038 and 011 had the same HLA-DR mismatch. T lines expanded from subject 038 showed the ability to partially inhibit T responder activation against the same antigen in different subjects. In FIG. 7B, subjects 002 and 035 had the same HLA-DR mismatch (HLA-DR1), while subject 037 had a different HLA mismatch (HLA-DR 4). In FIG. 7C, subjects 004, 023, and 011 all had different HLA mismatches (HLA-DR1, -DR15, and-DR 4, respectively).

Example 11 bystander inhibiting action

Bystander inhibition was determined in subjects with more than one HLA mismatch to their donor. Antigen-specific T cell lines were expanded separately for each mismatch. The bystander inhibitory effect of each T cell line was determined using a standard inhibition assay in which antigenic stimulation was provided by the same or different donor peptides.

Although T cells are usually specific for a particular antigen, in this case tregs have been shown to be able to suppress the immune response to antigens co-expressed with their specific antigens, demonstrating an example of linkage (bystander) suppression.

For example, in figure 8A, subject 036 has two HLA mismatches, HLA-DR1 and HLA-DR 15. As shown in the previous two figures, tregs against HLA-DR1 and tregs against HLA-DR15 show immunosuppressive effects against APCs presenting their respective antigens. In the bottom two panels, both Treg lines are also able to suppress the immune response against APCs presenting the co-expressed alloantigens. HLA-DR1 Treg inhibits immune responses against both HLA-DR1 and HLA-DR 15; similarly, HLA-DR15 Treg suppresses immune responses against both HLA-DR1 and HLA-DR 15. This effect was also demonstrated in fig. 8B and 8C, respectively, for subject 004 with HLA-DR1 and HLA-DR15 mismatches and for subject 022 with HLA-DR15 and HLA-DR17 mismatches.

Example 12 immunosuppressive mechanisms

Upregulation of expression of both CD39 and CD73 in the generated T cell lines was detected as described above. Both CD39 and CD73 have previously been shown to be involved in the immunosuppressive mechanism of mouse tregs, where ATP-derived adenosine production from CD39/CD 73-mediated extracellular ATP degradation to AMP increases intracellular cyclic AMP (camp) levels in tregs. This is transferred to T effector cells via gap junctions (gap junctions), resulting in upregulation of Inducible CAMP Early Repressor (ICER), and in turn, suppression of the transcription of activated T Nuclear Factor (NFAT) and IL-2 (Deaglio et al, J.Exp.Med. [ J.Experimental Med ]204(6): 1257-.

To determine whether the immunosuppressive mechanisms of the generated T cell lines involved activation of an adenosylergic pathway similar to mouse tregs, standard inhibition assays were performed in the presence or absence of an A2A receptor (A2Ar) antagonist. Suppression of the adenosylergic pathway using the A2Ar antagonist, istradefylline, resulted in elimination of the inhibition and an increase in antigen-specific T cell proliferation (figure 10). This suggests that regulatory function of the T cell line is mediated by upregulation of CD39 and CD73, which results in the production of adenosine and activation of the adenoergic pathway. In addition, upregulation of surface PD-1 expression on T cell lines was detected, another cell surface marker associated with activation of the adenosine-capable signaling pathway (fig. 11).

To determine whether IL-10 contributes to the inhibitory mechanism of T cell lines, neutralizing IL-10 monoclonal antibodies were used in standard inhibition assays. Ex vivo expanded T cell lines showed no change in inhibition of antigen specific T cell proliferation in the presence or absence of neutralizing IL-10 monoclonal antibodies (figure 9). Furthermore, TGF-. beta.neutralizing antibodies also had no effect on the immunosuppressive capacity of the T cell line. It has previously been reported that high concentrations of both IL-10 and TGF- β neutralizing antibodies can abrogate Treg-mediated inhibition. However, no change in T cell line mediated inhibition was observed in the presence of both IL-10 and TGF- β neutralizing antibodies in the standard inhibition assay.

Other embodiments

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Sequence listing

<110> Brigham and Women Hospital, Inc. (The Brigham and Women's Hospital, Inc.)

<120> compositions and methods for immunosuppression

<130> 51329-002WO2

<150> US 62/778,538

<151> 2018-12-12

<160> 15

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