METHODS FOR INCREASING EXPANSION AND IMMUNOSUPPRESSIVE CAPACITY OF A POPULATION OF CD8+CD45RCLOW/- TREGS

Abstract

A population of highly suppressive human CD8.sup.+CD45RC.sup.low/− Tregs is characterized by expressing Foxp3 and producing IFNγ, IL-10, IL-34 and TGFβ to mediate their suppressive activity. Accordingly, methods capable of increasing expansion and immunosuppressive capacity of such a population of CD8+CD45RC.sup.low/− Tregs are highly desirable for therapeutic purposes. Rapamycin has been shown to increase the expansion and immunosuppressive capacities of the population of CD8.sup.+CD45RC.sup.low/− Tregs. Accordingly, the method of increasing expansion and immunosuppressive capacity of a population of CD8.sup.+CD45RC.sup.low/− Tregs includes culturing the population of CD8.sup.+CD45RC.sup.low/− Tregs in presence of a rapamycin compound.

Claims

1-15. (canceled)

16. A method of increasing expansion and immunosuppressive capacity of a population of CD8+CD45RC.sup.low/− Treg cells, wherein said method comprises culturing the population of CD8+CD45RC.sup.low/− Treg cells in the presence of at least one immunosuppressive drug selected from the group consisting of a rapamycin compound and a combination of cyclosporine and methylprednisolone.

17. The method of claim 16, wherein the population of CD8+CD45RC.sup.low/− Treg cells is genetically modified to encode a T cell receptor or a subunit or functional equivalent thereof.

18. The method of claim 16, wherein the population of CD8+CD45RC.sup.low/− Treg cells is genetically modified to encode a chimeric antigen receptor (CAR) specific to an antigen of interest or a chimeric autoantibody receptor (CAAR) comprising an auto-antigen.

19. The method of claim 16, wherein the population of CD8+CD45RC.sup.low/− Treg cells is genetically modified and lacks expression of a functional T cell receptor (TCR) and/or human leukocyte antigen (HLA).

20. The method of claim 16, wherein the population of CD8+CD45RC.sup.low/− Treg cells is genetically modified and lacks expression of HLA class I and/or HLA class II.

21. The method of claim 16, wherein the population of CD8+CD45RC.sup.low/− Treg cells is allogeneic Tregs vis-à-vis the recipient patient to be treated.

22. The method of claim 16, wherein the population of CD8+CD45RC.sup.low/− Treg cells is cultured in the presence of antigen-presenting cells.

23. The method of claim 16, wherein the rapamycin compound is added in the culture medium at day 0 and 7 of culture or is added in the culture medium at day 14 of culture.

24. The method of claim 16, wherein the rapamycin compound is added in the culture medium at day 0 and 7 of culture and another immune suppressive drug is added to the culture medium.

25. The method of claim 24, wherein the other immune suppressive drug is methylprednisolone.

26. The method of claim 16, wherein the culture medium comprises an amount of rapamycin of about 45 ng/ml.

27. The method of claim 16, wherein cytokines are further added to the culture medium once, twice or three times or more.

28. The method of claim 27, wherein said cytokines are IL-2 and/or IL-15.

29. The method of claim 27, wherein said cytokines are added to the culture medium at day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and/or 20 of culture.

30. The method of claim 16, wherein an anti-CD3 antibody and/or an anti-CD28 antibody is added to the culture medium at day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and/or 20 of culture.

31. The method of claim 30, wherein said anti-CD3 antibody and/or said anti-CD28 antibody is added to the culture medium at day 0 and/or at day 11, 12, 13, 14 and/or 15 of culture.

32. The method of claim 16, wherein the culture is carried out for at least 12 days.

33. A method of preventing or reducing transplant rejection or GVHD in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a population of CD8+CD45RC.sup.low/− Treg cells in combination with at least one immunosuppressive drug selected from the group consisting of a rapamycin compound and a combination of cyclosporine and methylprednisolone.

34. A method of treating a genetic disease in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a population of CD8+CD45RC.sup.low/− Treg cells in combination with a rapamycin compound wherein the genetic disease is selected from the group consisting of a monogenic genetic disease affecting the immune system associated to autoimmunity, and a monogenic hereditary disease.

35. The method of claim 34, wherein the monogenic genetic disease affecting the immune system associated to autoimmunity is selected from the group consisting of IPEX (immunodysregulation polyendocrinopathy enteropathy X-linked syndrome), APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy), B cell primary immunodeficiencies, Muckle-Wells syndrome, mixed autoinflammatory and autoimmune syndrome, NLRP12-associated hereditary periodic fever syndrome and tumor necrosis factor receptor 1 associated periodic syndrome, and wherein the monogenic hereditary disease is selected from the group consisting of Duchenne muscular dystrophy (DMD), cystic fibrosis, lysosomal diseases and alpha1-anti-trypsin deficiency.

Description

FIGURES

[0041] FIG. 1. CD8.sup.+CD45RC.sup.low/− Tregs expansion for cell therapy. CD8.sup.+CD45RC.sup.low/− Tregs were sorted from fresh blood of HVs, expanded for 14 d with anti-CD3 and anti-CD28 MAbs, then cultured for 7 d in presence of allogeneic APCs in medium supplemented or not with IL-2 and IL-15 and immunosuppressive drugs and assessed for survival. Values >1 means proliferation of cells. n=7.

[0042] FIG. 2. Effect of supplementation of culture medium with rapamycin on expansion yield, phenotype and suppressive activity of Tregs A. Results are expressed as number of cells harvested at days 7, 14 and 20 normalized to cell number plated at day 0, after culture in medium supplemented with rapamycin or not. B-D. Tregs cultured for 7 (B), 14 (C) or 20 (D) days in presence or absence of rapamycin were analyzed for Tregs associated markers expression. E. Left. Results are expressed as % of suppression mediated by Tregs after culture in presence of rapamycin normalized to % of suppression mediated by Tregs after culture in absence of rapamycin. Right. Representative histogram of responder T cells proliferation in response to allogeneic APCs in absence (w/o Tregs) or presence of Tregs cultured in absence (named w NT expanded Tregs) or in presence of rapamycin (named w rapa-expanded Tregs).

[0043] FIG. 3: Effect of supplementation of culture medium with CsA, MPA, MPr or tacrolimus on expansion yield and suppressive activity of TregsA. Results are expressed as number of cells harvested at days 7 normalized to cell number plated at day 0, after culture in medium supplemented with one IS drug or not. B-D. Tregs were cultured for 7 days in presence or absence of one IS drug, then cultured with the same drug or a different one, and analyzed for expansion yield (B) and suppressive activity on CD4+CD25− effector T cells stimulated with allogeneic APCs (C-D). B. Results are expressed as number of cells harvested at days 14 normalized to cell number plated at day 0. C. Results are expressed as % of suppression mediated by Tregs after culture with IS drugs normalized to % of suppression mediated by Tregs after culture without any IS drug. D. Results are expressed as mean of suppressive score in function of mean of expansion score for each IS drugs combination. 1:1 means suppression and expansion scores obtained with Tregs expanded without any IS drug.

[0044] FIG. 4. Effect of Rapamycin, CsA, MPA, MPr or tacrolimus on allogeneic activation and phenotype of Tregs. A. Results are expressed as number of cells harvested at days 21 normalized to cell number plated at day 0, after culture in medium supplemented with Is drug or not. B. Tregs were analyzed for Tregs associated markers expression at day 21 (14 days drug free expansion and 7 days IS-supplemented medium culture).

EXAMPLE 1

[0045] Material & Methods

[0046] CD8.sup.+CD45RC.sup.low/− Tregs from fresh or thawed PBMCs were seeded at 3×10.sup.5/ml in RPMI1640 medium supplemented with 10% AB serum, Penicillin (100 U/ml), Streptomycin (0.1 mg/ml), Sodium pyruvate (1 mM), Glutamine (2 mM), Hepes Buffer (1 mM), non-essential amino acids (1×), IL-2 (1000 U/ml) and IL-15 (10 ng/ml), and were stimulated with coated anti-CD3 mAb (1 μg/ml), soluble anti-CD28 mAb (1 μg/ml) and/or allogeneic APCs at 1:4 Tregs: APCs ratio. At day 7, expanded cells were diluted at 1.5×10.sup.5/ml and stimulated again with coated anti-CD3 and soluble anti-CD28 mAbs (1 μg/ml each). IL-2 and IL-15 cytokines were freshly added at days 0, 7, 10 and 12 Immunosuppressive drugs, such as cyclosporine A (45 ng/ml), rapamycin (45 ng/ml), methylprednisolone (500 pg/ml), tacrolimus (2 ng/ml) or mycophenolate mofetil (1 μg/ml) were added in culture medium at day 0 and 7 of the expansion or after 14 days drug-free expansion to assess their toxicity and effect on suppression on CD8.sup.+CD45RC.sup.low/− Tregs. At day 14, expanded Tregs were washed with PBS before use. Suppressive activity was tested on CD8.sup.+CD45RC.sup.low/− Tregs expanded more than 10 fold in 7 days. For long-term expansion, CD8.sup.+CD45RC.sup.low/− Tregs and CD4.sup.+CD25.sup.highCD127.sup.low/− Tregs were stimulated again with coated anti-CD3 (1 μg/ml) and soluble anti-CD28 MAbs (1 μg/ml) at days 14 and 21 and IL-2 and IL-15 cytokines were freshly added every 2 days from day 7 to 28. Culture medium 1× cytokines (IL-2 (1000 U/ml) and IL-15 (10 ng/ml)) was added when required.

[0047] Results

[0048] We tested the effect of immunosuppressive drugs (at concentrations used in the clinic) on CD8.sup.+CD45RC.sup.low/− Tregs survival 7 days following the 14 days expansion (FIG. 1) or on the expansion yield and suppressive function score of CD8.sup.+CD45RC.sup.low/− Tregs during the 14 days expansion (FIG. 3D). To test the effect of the immunosuppressors on the in vitro expansion, they were added either alone during 14 days or subsequently (i.e. day 0 to 7 cyclosporine A (CsA), then day 7 to 14 methylprednisolone (MPr) is labeled CsA-MPr in the upper left corner of FIG. 3D) or without any immunosuppressor (NT). We observed that 14 days expansion in presence of only rapamycin (Rapa) was the most efficient to increase both expansion fold and suppression capacity and first rapamycin and followed by methylprednisolone was also an efficient combination whereas the presence of mycophenolate mofetil (MPA) inhibited the expansion by rapamycin and decreased suppression (FIG. 3D), therefore, rapamycin might be beneficial to improve expansion and function of CD8.sup.+CD45RC.sup.low/− Tregs in vitro but also in vivo.

EXAMPLE 2

[0049] Methods: Fresh blood was taken from 1 healthy volunteer, PBMCs were isolated by Ficoll centrifugation, washed in PBS, adjusted at 2×10.sup.8 PBMC/ml in PBS-FCS-EDTA and were incubated with anti-CD3-PeCy7, anti CD4-PerCPCy5.5 and anti-CD45RC FITC 30′ 4° C. Cells were washed with PBS-FCS-EDTA, filtered on 60 μm tissue, labeled with Dapi and FACS Aria sorted on lymphocyte morphology, exclusion of doublet cells, and DAPI.sup.−CD3.sup.+CD4.sup.−CD45RC.sup.low/− expression. After sorting, cells were washed in medium, plated at 10.sup.6Tregs/well/3 ml in p6 plate previously coated with anti-CD3 (OKT3 clone, 1 μg/ml in PBS, 1 ml/well, 1 h at 37° C. then washed with PBS 3 times), in RPMI1640 medium supplemented with 10% AB serum, Penicillin (100 U/ml), Streptomycin (0.1 mg/ml), Sodium pyruvate (1 mM), Glutamine (2 mM), Hepes Buffer (1 mM), non-essential amino acids (1×), IL-2 (1000 U/ml) and IL-15 (10 ng/ml) anti-CD28 mAbs (clone CD28.2, soluble, 1 μg/ml), and supplemented or not with 50 nM rapamycin. At days 7 and 14, Tregs were harvested, counted, washed, and plated at 5×10.sup.5Tregs/well/3 ml in p6 plate previously coated with anti-CD3 (OKT3 clone, 1 μg/ml in PBS, 1 ml/well, 1 h at 37° C. then washed with PBS 3 times), in RPMI1640 medium supplemented with 10% AB serum, Penicillin (100 U/ml), Streptomycin (0.1 mg/ml), Sodium pyruvate (1 mM), Glutamine (2 mM), Hepes Buffer (1 mM), non-essential amino acids (1×), IL-2 (1000 U/ml) and IL-15 (10 ng/ml), anti-CD28 mAbs (clone CD28.2, soluble, 1 μg/ml), and supplemented or not with 50 nM rapamycin. From day 7 to 20, cytokines were freshly added every 2 days and fresh medium (RPMI1640 medium supplemented with 10% AB serum, Penicillin (100 U/ml), Streptomycin (0.1 mg/ml), Sodium pyruvate (1 mM), Glutamine (2 mM), Hepes Buffer (1 mM), non-essential amino acids (1×), IL-2 (1000 U/ml) and IL-15 (10 ng/ml)) was added when required, depending on proliferation rate. Expanded Tregs were harvested at day 14 for suppressive activity assessment. PBMCs from the same HV donor were thawed, washed in PBS, adjusted at 2×10.sup.8 PBMC/ml in PBS-FCS-EDTA and were incubated with anti-CD3-PeCy7, anti CD4-PerCPCy5.5, and anti-CD25-APC-Cy7 30′ 4° C. Cells were washed with PBS-FCS-EDTA, filtered on 60 μm tissue, labeled with Dapi and FACS Aria sorted on lymphocyte morphology, exclusion of doublet cells, and DAPI-CD3.sup.+CD4±CD25.sup.− expression, and CFSE labeled. APCs were obtained by CD3.sup.+ cells depletion and 35Gy irradiation. Expanded Tregs were plated at 1:1:1 Tregs:Teff:APCs ratio in RPMI 1640 medium supplemented with 10% AB serum, Penicillin (100 U/ml), Streptomycin (0.1 mg/ml), Sodium pyruvate (1 mM), Glutamine (2 mM), Hepes Buffer (1 mM), and non-essential amino acids (1×). After 5 days culture, Teff proliferation was analyzed by CFSE analysis in DAPI.sup.−CD4±CD3.sup.+ T cells.

[0050] Results: Supplementation of culture medium for Tregs culture with rapamycin improved expansion yield and suppressive function without affecting cell phenotype (FIG. 2).

EXAMPLE 3

[0051] Methods: Fresh blood was taken from 1 healthy volunteer, PBMCs were isolated by Ficoll centrifugation, washed in PBS, adjusted at 2×10.sup.8 PBMC/ml in PBS-FCS-EDTA and were incubated with anti-CD3-PeCy7, anti CD4-PerCPCy5.5 and anti-CD45RC FITC 30′ 4° C. Cells were washed with PBS-FCS-EDTA, filtered on 60 μm tissue, labeled with Dapi and FACS Aria sorted on lymphocyte morphology, exclusion of doublet cells, and DAPI-CD3.sup.+CD4.sup.−CD45RC.sup.low/− expression. After sorting, cells were washed in medium, plated at 10.sup.6Tregs/well/3 ml in p6 plate previously coated with anti-CD3 (OKT3 clone, 1 μg/ml in PBS, 1 ml/well, 1 h at 37° C. then washed with PBS 3 times), in RPMI1640 medium supplemented with 10% AB serum, Penicillin (100 U/ml), Streptomycin (0.1 mg/ml), Sodium pyruvate (1 mM), Glutamine (2 mM), Hepes Buffer (1 mM), non-essential amino acids (1×), IL-2 (1000 U/ml) and IL-15 (10 ng/ml), anti-CD28 mAbs (clone CD28.2, soluble, 1 μg/ml), and supplemented or not with an immunosuppressive drug (cyclosporine A (45 ng/ml), rapamycin (45 ng/ml), methylprednisolone (500 pg/ml), tacrolimus (2 ng/ml) or mycophenolate mofetil (1 μg/ml)). At days 7, Tregs were harvested, counted, washed, and plated at 5×10.sup.5Tregs/well/3 ml in p6 plate previously coated with anti-CD3 (OKT3 clone, 1 μg/ml in PBS, 1 ml/well, 1 h at 37° C. then washed with PBS 3 times), in RPMI1640 medium supplemented with 10% AB serum, Penicillin (100 U/ml), Streptomycin (0.1 mg/ml), Sodium pyruvate (1 mM), Glutamine (2 mM), Hepes Buffer (1 mM), non-essential amino acids (1×), IL-2 (1000 U/ml) and IL-15 (10 ng/ml), anti-CD28 mAbs (clone CD28.2, soluble, 1 μg/ml), and supplemented or not with an immunosuppressive drug (cyclosporine A (45 ng/ml), rapamycin (45 ng/ml), methylprednisolone (500 pg/ml), tacrolimus (2 ng/ml) or mycophenolate mofetil (1 μg/ml)), the same drug as from day 0 to 7 or a different one. At days 10 and 12, cytokines were freshly added. Expanded Tregs were harvested at day 14 for suppressive activity assessment. PBMCs from the same HV donor were thawed, washed in PBS, adjusted at 2×10.sup.8 PBMC/ml in PBS-FCS-EDTA and were incubated with anti-CD3-PeCy7, anti CD4-PerCPCy5.5, and anti-CD25-APC-Cy7 30′ 4° C. Cells were washed with PBS-FCS-EDTA, filtered on 60 μm tissue, labeled with Dapi and FACS Aria sorted on lymphocyte morphology, exclusion of doublet cells, and DAPI-CD3.sup.+CD4±CD25.sup.− expression, and CFSE labeled. APCs were obtained by CD3.sup.+ cells depletion and 35Gy irradiation. Expanded Tregs were plated at 1:1:1 Tregs:Teff:APCs ratio in RPMI 1640 medium supplemented with 10% AB serum, Penicillin (100 U/ml), Streptomycin (0.1 mg/ml), Sodium pyruvate (1 mM), Glutamine (2 mM), Hepes Buffer (1 mM), and non-essential amino acids (1×). After 5 days culture, Teff proliferation was analyzed by CFSE analysis in DAPI.sup.−CD4±CD3.sup.+ T cells.

[0052] Results: Supplementation of culture medium for Tregs culture with rapamycin, CsA, or Tacrolimus improved expansion yield and suppressive activity of Tregs, supplementation with MPA is deleterious for activity and expansion, and with methylprednisolone improved function (FIG. 3).

EXAMPLE 4

[0053] Methods: Fresh blood was taken from 1 healthy volunteer, PBMCs were isolated by Ficoll centrifugation, washed in PBS, adjusted at 2×10.sup.8 PBMC/ml in PBS-FCS-EDTA and were incubated with anti-CD3-PeCy7, anti CD4-PerCPCy5.5 and anti-CD45RC FITC 30′ 4° C. Cells were washed with PBS-FCS-EDTA, filtered on 60 μm tissue, labeled with Dapi and FACS Aria sorted on lymphocyte morphology, exclusion of doublet cells, and DAPI-CD3.sup.+CD4.sup.−CD45RC.sup.low/− expression. After sorting, cells were washed in medium, plated at 10.sup.6Tregs/well/3 ml in p6 plate previously coated with anti-CD3 (OKT3 clone, 1 μg/ml in PBS, 1 ml/well, 1 h at 37° C. then washed with PBS 3 times), in RPMI1640 medium supplemented with 10% AB serum, Penicillin (100 U/ml), Streptomycin (0.1 mg/ml), Sodium pyruvate (1 mM), Glutamine (2 mM), Hepes Buffer (1 mM), non-essential amino acids (1×), IL-2 (1000 U/ml) and IL-15 (10 ng/ml), anti-CD28 mAbs (clone CD28.2, soluble, 1 μg/ml). At days 7, Tregs were harvested, counted, washed, and plated at 5×10.sup.5Tregs/well/3 ml in p6 plate previously coated with anti-CD3 (OKT3 clone, 1 μg/ml in PBS, 1 ml/well, 1 h at 37° C. then washed with PBS 3 times), in RPMI1640 medium supplemented with 10% AB serum, Penicillin (100 U/ml), Streptomycin (0.1 mg/ml), Sodium pyruvate (1 mM), Glutamine (2 mM), Hepes Buffer (1 mM), non-essential amino acids (1×), IL-2 (1000 U/ml) and IL-15 (10 ng/ml), anti-CD28 mAbs (clone CD28.2, soluble, 1 μg/ml). At days 10 and 12, cytokines were freshly added. At day 14, expanded Tregs were harvested and plated at 5×10.sup.5Tregs/well/200 μl with allogeneic APCs (Tregs:APC 1:4) in p96 plate in RPMI1640 medium supplemented with 10% AB serum, Penicillin (100 U/ml), Streptomycin (0.1 mg/ml), Sodium pyruvate (1 mM), Glutamine (2 mM), Hepes Buffer (1 mM), non-essential amino acids (1×), and supplemented or not with an immunosuppressive drug (cyclosporine A (45 ng/ml), rapamycin (45 ng/ml), methylprednisolone (500 pg/ml), tacrolimus (2 ng/ml) or mycophenolate mofetil (1 μg/ml)) and with or without cytokines (IL-2 (1000 U/ml) and IL-15 (10 ng/ml) added at days 14, 16, 18 and 20). Tregs were analyzed at day 21 for survival/expansion yield and phenotype.

[0054] Results: Supplementation of culture medium for Tregs culture did not affect Tregs survival, neither Tregs proliferation in response to allogeneic APCs (FIG. 5). Tacrolimus and methylprednisolone significantly decreased IFNg and CD154 expression by Tregs respectively compared to drug free medium (FIG. 4).

REFERENCES

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