WO2013050596A1 - Method of maintaining foxp3 expression in expanded t regulatory cell - Google Patents

Method of maintaining foxp3 expression in expanded t regulatory cell Download PDF

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WO2013050596A1
WO2013050596A1 PCT/EP2012/069857 EP2012069857W WO2013050596A1 WO 2013050596 A1 WO2013050596 A1 WO 2013050596A1 EP 2012069857 W EP2012069857 W EP 2012069857W WO 2013050596 A1 WO2013050596 A1 WO 2013050596A1
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inhibitor
cells
treg
cell
foxp3
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PCT/EP2012/069857
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French (fr)
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Guy Gorochov
Makoto MIYARA
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INSERM (Institut National de la Santé et de la Recherche Médicale)
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • C12N5/064—Immunosuppressive dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule

Definitions

  • the present invention concerns a method for expanding regulatory T cells using a combination of at least a m-TOR inhibitor, at least a histone deacetylase inhibitor and at least a DNA methylation inhibitor, for maintaining FoxP3 expression in regulatory T cells in order to maintain their suppressive function.
  • Treg cell based therapy seems in theory readily applicable in humans in order to treat autoimmune diseases [1].
  • the timing of Treg cell activation in relation to effector T cells activation is a key parameter to consider because effector T cells activated prior to Treg cells are resistant to in vivo suppression.
  • Treg cells are characterized in vivo, by their capacity to prevent autoimmunity, sometimes to delay the onset of disease but rarely to cure diseases completely[3,4].
  • Treg based therapy is more suitable to prevent disease onset or disease flares
  • autoimmune diseases for which Treg cell based therapy would be better appropriate can be determined based on their evolutionary patterns i.e. continuously progressive versus remitting/relapsing course of autoimmune diseases such as systemic lupus erythematosus, multiple sclerosis, vasculitites etc.
  • Treg cell based treatment may also be of interest for the treatment of allergic diseases and for induction of organ transplantation [1].
  • Treg cell based therapies may be beneficial when the disease is quiescent to prevent subsequent flares or organ rejection.
  • Treg-based manipulation strategies it is important to take into account the notion that Treg cells appear to exert a more preventive than curative effect on chronic inflammatory processes. Therefore, rather than considering Treg cell based treatment as a mere induction therapy that would be curative on its own, such treatment might better be considered as an adjuvant therapy in the management of autoimmune diseases.
  • Cyclophosphamide, mycophenolate and/or high dose steroids have proven efficient to treat disease flares.
  • Prevention of subsequent flares or of recurrence usually consists of immunosuppressors mostly given orally.
  • Such maintenance treatment might be replaced by treatments based on Treg biology.
  • Treg based therapeutic strategies seem readily feasible, one must keep in mind that Treg cells may convert in vitro into pathogenic cells such as TH- 17 cells [7]. Such converted Treg cells may theoretically make the diseases worse. Thus, it is of utmost importance to determine the conditions that preserve Treg phenotype and suppressive functions in vitro. The resolution of this key issue is a prerequisite for the development of clinical trials evaluating Treg cell based therapy in systemic autoimmune diseases, allergy or transplantation [8].
  • CD45RA-Treg cells are poorly proliferative even in the presence of high dose IL-2, human CD45RA+ Treg cells are the adequate population to isolate for expansion. However, even with high doses of IL-2, these cells lose FoxP3 expression after polyclonal stimulation. Treg cells require high levels of FoxP3 to be efficiently suppressive. Thus, there is a need for molecules that prevent the downregulation of FoxP3 in vitro and preserve Treg phenotype and suppressive functions for Treg based cell therapy.
  • TGF- ⁇ Treatment of T cells with the bioactive peptide TGF- ⁇ is known to induce de novo expression of Foxp3 [14].
  • WO2008071974 also describes use of phosphatidyl inositol-3-kinase (PI3K) inhibitors and/or mtTor inhibitors (such as rapamycin) and/or AKT inhibitors for inducing de novo expression of FoxP3 in T cells.
  • PI3K phosphatidyl inositol-3-kinase
  • mtTor inhibitors such as rapamycin
  • AKT inhibitors for inducing de novo expression of FoxP3 in T cells.
  • the present invention is based on the discovery that a combination of an inhibitor of mTOR (such as rapamycin), an inhibitor of histone deacetylases (such as vorinostat) and an inhibitor of DNA methylation (such as azacitidine) is efficient for the maintenance of high expression of FoxP3 and suppressive function in human CD4+ T regulatory cells expanded in vitro or ex vivo.
  • an inhibitor of mTOR such as rapamycin
  • an inhibitor of histone deacetylases such as vorinostat
  • an inhibitor of DNA methylation such as azacitidine
  • each molecule and either combinations of two molecules are not efficient for maintaining high expression of FoxP3 and suppressive function in human CD4+ T regulatory cells in vitro.
  • the present invention arises from the unexpected finding by the inventors that an inhibitor of mTOR, an inhibitor of histone deacetylases and an inhibitor of DNA methylation act synergistically for maintaining high expression of FoxP3 and suppressive function in T regulatory cells in vitro.
  • This combination has proved effective in human CD4+ T regulatory cells in vitro.
  • the novel combination is more efficient at maintaining highly FoxP3 expression on expanded cells than either drug alone or either combination of two molecules.
  • the combination of the invention enables the expansion of highly pure T regulatory cells displaying high levels of FoxP3.
  • the present invention concerns a novel method for the stable expression of FoxP3, the signature transcription factor of Tregs, which is maintained when Treg cells are activated in culture medium containing inhibitors of mTOR, inhibitors of histone deacetylases and inhibitors of DNA methylation.
  • the present invention provides an in vitro method for maintaining FoxP3 expression in expanded T regulatory cell comprising the steps of:
  • the Treg cell stimulated and treated with the combination of the invention is a naive Treg cell.
  • the in vitro method for maintaining Foxp3 expression in T regulatory cell comprises a preliminary step of isolating Treg cells from Peripheral Blood Mononuclear Cells (PBMCs)
  • PBMCs Peripheral Blood Mononuclear Cells
  • T regulatory cell also called “Tregs” or “Treg cells”
  • Regulatory T cells are involved in keeping effector T cells in check, and in the prevention of "self recognition" which can be a major factor in autoimmune disease.
  • Tregs have numerous acknowledged biomarkers known in the art. These include CD4+, CD25+, and Foxp3+.
  • a T regulatory cell shows Foxp3 expression (Foxp3+).
  • a Treg cell must preferably display regulatory function.
  • the demonstration of regulatory function may be determined by any suitable method known in the art.
  • examples of such tests are set out in the example section.
  • the tests embodied in example and figures 4 are regarded as standards in vitro tests for the assessment of regulatory T cell function.
  • Treg cells according to the present invention are mammalian Treg cells, most preferably human Treg cells.
  • na ' ive Treg cell T cells which express the following markers as CD4+CD45RA+CD25+.
  • FoxP3+ regulatory T cells comprise two subsets that distinguish each other by the expression of CD45RA defined as na ' ive Tregs that express FOXP3 and CD45RA and effector Tregs that also express FOXP3 and not CD45RA.
  • Cells that are suitable for expansion are naive Tregs since they are highly proliferative under stimulation conditions and in the presence of IL-2 while effector Tregs are poorly proliferative under such conditions.
  • the preferred target cells to expand are naive Tregs that can be separated as either CD4+CD25+CD45RA+ cells or CD4+CD25+CD45RO- cells.
  • Neive Treg cells may be isolated from PBMCs by any suitable method known in the art. Examples of such method are set out in the example section.
  • Treg cells By “stimulating Treg cells” is meant using biological means to obtain physiological activation and expansion of Treg cells using a) lymphokine and b) TCR stimulation.
  • Stimulation may be performed by any suitable means which renders the T cell sensistive to the action of m-TOR inhibitors histone, deacetylase inhibitors and DNA methylation inhibitors for the maintenance of the Treg phenotype as described herein.
  • the invention relates to the maintenance of Foxp3 expression in Treg cells together with contacting those Treg cells with said lyrnphokines.
  • the IL-2 lymphokine is used to enhance Treg cell expansion.
  • stimulation of Treg cell is also performed by stimulating the TCR receptor.
  • stimulation is a persistent or sustained stimulation, which means that the stimulation is not necessarily withdrawn at the time of action of m-TOR, histone deacetylases and DNA methylation inhibitors.
  • a stimulated Treg cell is a Treg cell which has been stimulated via the T cell receptor (TCR).
  • the Treg cells are also stimulated via the TCR or the TCR-associated CD3 complex of signalling proteins.
  • TCR signal is in fact transmitted via the associated proteins.
  • to stimulate via the TCR it is possible to target the actual TCR itself (e.g. antigen specific TCR targeting) or to target CD3 (e.g the TCR associated protein(s)).
  • TCR stimulation is polyclonal and may be done via CD3.
  • CD28 is stimulated simultaneously with the stimulation of CD3 receptor(s).
  • a preferred embodiment stimulation is via simultaneous stimulation of TCR and/or CD3, and of CD28.
  • stimulation of CD2 can be added to CD3 and CD28 stimulation to strengthen the stimulation.
  • Preferably stimulating said Treg cell comprises contacting said Treg cell with anti- TC or anti-CD3 antibody.
  • stimulating said Treg cell further comprises contacting said Treg cell with anti-CD28 antibody, preferably contacting said T cell with anti-TCR or anti-CD3 antibody, and with anti-CD28 antibody simultaneously.
  • the mode of presentation of such antibodies may be chosen by the operator, for example this may be accomplished using plate-bound antibodies or by using beads coated with antibodies such as anti-CD3/anti- CD28 antibodies, or by any other means of presentation known to the operator.
  • TCR activation is performed by adding specifc antigen or by contacting Treg cell with activated Antigen Presenting cell (APC).
  • stimulating Treg cell comprises stimulating NTreg cell with Interleukine 2, anti CD3 antibody and anti CD28 antibody.
  • the Treg cell expanded by the method of the invention may be cultured between one and four weeks and most preferably during 2 weeks.
  • the major advantage of the present invention is the sustained expression of Foxp3. This is in contrast with the prior art where the major effect shown to date is the expansion of Foxp3 expressing T cells in a first time but followed by the loss of FoxP3 expression after polyclonal stimulation [see 17] .
  • FoxP3 also called “forkhead box P3"
  • FoxP3 a protein involved in responses of the immune system.
  • a member of the FOX protein family, FoxP3 appears to function as a master regulator in the development and function of regulatory T cells.
  • Foxp3 expression has been associated with regulatory T cell function, in certain settings, Foxp3 expression has been shown to be necessary and sufficient to induce regulatory T cell function. Indeed, Foxp3 expression has in some circumstances been regarded as an indicator or identifier of regulatory T cells as compared to other sub- populations of T cells, in the prior art, the only known way of inducing Foxp3 expression pharmacologically has been by treating a population of T cells with the bioactive peptide TGF- ⁇ associated with IL-2.
  • m-TOR inhibitor it meant a class of compounds that interfere with the function of the mammalian target of rapamycin (mTOR) also known as mechanistic target of rapamycin or FK506 binding protein 12-rapamycin associated protein 1 (FRAP1).
  • m-TOR is a protein which in humans is encoded by the FRAP 1 gene.
  • mTOR is a serine/threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis and transcription.
  • mTOR belongs to the phosphatidylinositol 3-kinase-related kinase protein family.
  • the m-TOR inhibitor in the frame of the present invention may be any known m-TOR inhibitor such as rapamycin (sirolimus), or an analogue thereof such as RADOOl (everolimus), CCI-779 (temsirolimus), AP23573,
  • the m-TOR inhibitor also may be wortmannin, and synthetic compounds such as PIK-90.
  • said m-TOR inhibitor is rapamycin.
  • the dose used for Rapamicin is between 0.5 and 5 Microg/mL, preferably 1 Microg/mL (i.e. 100 nM).
  • histone deacetylase inhibitor also called HDI
  • HDAC proteins are now also called lysine deacetylases (KDAC), to describe their function rather than their target, which also includes non-histone proteins.
  • the histone deacetylase inhibitor is selected from the group consisting of
  • HDIs hydro xamic acids (or hydro amates), such as trichostatin A; cyclic tetrapeptides (such as trapoxin B), and depsipeptides; benzamides; electrophilic ketones; and aliphatic acid compounds such as phenylbutyrate and valproic acid.
  • HDIs include the hydroxamic acids vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat (LBH 89); and the benzamides : entinostat (MS-275), CI994, and mocetinostat (MGCD0103).
  • Vorinostat is preferred histone deacetylase inhibitor in the frame of the present invention.
  • the dose used for Vorinostat are between 0,1 and 5 microM, preferably 0.5 microM or 1 microM.
  • DNA methylation inhibitor also called “Demethylating agents”
  • Demethylating agents is meant a class of compounds that interfere with DNA methylation which is the addition of a methyl group to the 5- position of the cytosine pyrimidine ring or the nitrogen in position 6 of the adenine purine ring. DNA methylation stably alters the gene expression pattern in cells i.e. decrease gene expression
  • Demethylating agents are compounds that can inhibit methylation, resulting in the expression of the previously hypermethylated silenced genes.
  • Cytidine analogs such as 5- azacytidine (azacitidine) and 5-azadeoxycytidine (decitabine) are the most commonly used demethylating agents . These compounds work by binding to the enzymes that catalyse the methylation reaction, i.e. DNA methyltransferases.
  • Azacitidine is preferred DNA methylation inhibitor that can be used in the frame of the present invention.
  • the dose used for Azacitidine is between 1 and 10 microM, preferably 2.5 microM or 5 micro M.
  • Another aspect of the invention is an in vitro method of maintaining Foxp3 expression in a previously stimulated Treg cell comprising treating said Treg cell with a combination of at least a m-TOR inhibitor, at least a histone deacetylase inhibitor and at least a DNA methylation inhibitor.
  • Another aspect of the invention is the in vitro and/or ex-vivo use of mTOR inhibitor, histone deacetylase inhibitor and DNA methylation inhibitor in order to maintain the differentiation of expanded regulatory T cell.
  • the present invention also relates to a culture medium comprising Treg cell stimulant- TOR inhibitor and histone deacetylase inhibitor and DNA methylation inhibitor.
  • the culture medium of the present invention is suitable for use to expand regulatory T cells for maintaining high expression of FoxP3 and suppressive function in Treg cells in vitro
  • the term "culture medium” as used herein refers to a liquid medium suitable for the in vitro culture of Treg cells, preferably manufactured at clinical grade.
  • the culture medium of the invention contains:
  • a source of carbon as energy substrate such as glucose, galactose or sodium pyruvate
  • vitamins such as biotin, folic acid, B 12... ;
  • the culture medium may also contain pH buffers in order to maintain the pH of the medium at a value suitable for cell growth
  • the culture medium of the invention may be based on a commercially available medium such as X-VIVO 15 or MACOPHARMA HP01.
  • Treg cell stimulant means any mean previously describe in the "stimulation of Treg cell". Most preferably the Treg cell stimulants are Interleukine 2 and anti-CD28 and anti-CD3 antibodies.
  • Another aspect of the invention relates to an in vitro method for expanding Treg cell wherein said method comprises the step of culturing Treg cells with the culture medium as described above.
  • the step of culturing Treg cells (preferably na ' ive Treg cell) with the culture medium of the invention shall be carried out for the necessary time required for the production of functional Treg cell.
  • the culture of Treg cells with the medium of the invention shall be carried out for at least 7 days, preferably at least 10 days, even more preferably at least 14 days.
  • the culture medium of the invention can be renewed, partly or totally, at regular intervals.
  • the culture medium of the invention can be replaced with fresh culture medium of the invention every other day, for 14 days.
  • kits comprising: (i) a Treg cell stimulant; and (ii) combination of at least a mTOR inhibitor, at least a histone deacetylase inhibitor and at least a DNA methylation inhibitor.
  • the m-TOR inhibitor is Rapamycin
  • the histone deacetylase inhibitor is Voronistat
  • the DNA methylation inhibitor is Azacitidin.
  • a further aspect of the invention relates to a method of treating a subject in need of regulatory T cell(s) comprising
  • the Treg cell removed and stimulated are naive Treg cell.
  • a subject denotes a mammal, such as a rodent, a feline, a canine, and a primate.
  • a subject according to the invention is a human.
  • the method and combination for uses according to the invention find use in a patient in need of regulatory T cell(s) which means for the treatment of autoimmune diseases (such as systemic lupus erythematosus, multiple sclerosis, vasculitites etc. ), allergic diseases and in transplantation (Graft-versus-host (GVH) disease, .).
  • autoimmune diseases such as systemic lupus erythematosus, multiple sclerosis, vasculitites etc.
  • allergic diseases and in transplantation Graft-versus-host (GVH) disease, .
  • the combination of the invention enables the expansion of highly pure human T regulatory cells with sustained high expression of FoxP3 said Treg cells having a high suppression activities in vitro (in cell culture) and in vivo (in a mice model of Graft-versus-host (GVH) disease)
  • FIGURE 1 Rapamycin increases FoxP3 levels in expanded nTregs but does not completely prevent the downregulation of FoxP3 expression
  • nTreg sorted as shown in ref 13 from a healthy donor PBMCs were cultured for 7 days in the presence of IL-2 and anti-CD3/CD28 beads 300 IL-2 with rapamycin (right) or without rapamycin (left). FoxP3 and Ki-67 levels were analyzed by FACS. Data are representative of 10 independent experiments
  • FIGURE 2 Azacitidine and Vorinostat increase FoxP3 levels upon activation
  • FIGURE 3 Combination of rapamycin with Azacitidine and Vorinostat enables the expansion of highly pure Treg cells displaying high levels of FoxP3
  • nTregs isolated from a healthy donor PBMCs following the gating strategy shown in ref [13] were cultured for 14 days in the presence of anti-CD3/CD28 beads, 3001 U/mL IL-2 and of rapamycin (R), Azacytidine (A), Vorinostat (B) alone or in the presence of a combination of R with A, R with B or A with B, or in the presence of the 3 molecules.
  • CD4+CD25-CDR45RA+ T cells were also cultured in order to set the threshold for FoxP3 expression (bottom FACS plot). Ki-67 and FoxP3 expression were analyzed by FACS at day 14-16 of culture. Data representative of 8 independent experiments.
  • FIGURE 4 Combination of Rapamycin (R) with Azacitidine (A) and Vorinostat
  • nTreg cultured for 14 days in conditions described in Figure 3 were cocultured with autologous CD4+CD25-CD45RA+ responder cells labeled with CFSE in the presence of plate bound anti-CD3 (0.5 microg/mL) and autologous irradiated B cells and monocytes.
  • CFSE dilution in responder cells was analyzed after 96 hours of culture on gated CD4+CFSE+ T cells. % suppression are indicated. Data representative of 4 independent experiments.
  • FIGURE 5 Azacytidin/vorinostat/rapamycin combination preserves suppressive nTreg function in a xenoGVH model in NSG mice
  • PBMCs were isolated through Ficoll gradient separation from freshly drawn blood.
  • CD4+T cells were first magnetically isolated using a CD4 T cell separation kit (Miltenyi) and subsequently surface stained using a combination of flurochrome conjugated mAbs: anti-CD4-PErCP 5.5, anti-CD25-PE, anti-CD 127- AF647 and anti-CD45RA-FITC obtained from BD bioscience.
  • NTreg cells Na ' ive CD25++CD45RA+ Treg cells (NTreg cells) were isolated from PBMCs following flow isolation according to the gating strategy we validated (see ref.[13]).
  • Isolated na ' ive Treg cells were immediately distributed into U bottom well for culture and expansion. To analyze the effect of tested molecules or combination of molecules, 10 to 30 * 10 3 nTreg cells were collected per U bottom
  • NTreg cells were first polyclonally stimulated using anti-CD3/anti-CD28 beads (concentration: 40 000 beads/microL; 2 microL/well) or anti-CD2/anti-CD3/anti-CD28 beads in the presence of 300 IU/mL IL-2 in culture media in the presence of a combination of Rapamycin diluted in culture medium (1 microg/mL), Azacytidine first diluted in PBS and then in culture medium (5 microM) and Vorinostat first diluted in DMSO and then in culture medium (1 microM). 300 IU/mL IL-2 was added every 3-4 days and cells were split every week and restimulated by additional anti-CD3/anti-CD28 beads every week.
  • Expanded cells were counted and flow analyzed every week for the expression of FoxP3 and Ki-67 after fixation and permeabilization. Because FoxP3 expression cutoff that differentiates positive cells from negative cells is different in cultured cells compared to ex vivo isolated cells, we also cultured as control cells CD4+CD25-CD45RA+ cells. The expression of FoxP3 in these cells enabled us to set the right threshold for FoxP3 expression in expanded nTreg cells.
  • Expanded cells were assessed for suppressive function between 10 and 14 days of culture.
  • 1 x 10 4 CFSE-labeled responder CD25-CD45RA+CD4+ T cells were cocultured with 1 x 10 4 unlabeled cells assessed for their suppressive capacity and 1 x 10 5 irradiated autologous accessory cells and were stimulated with 0.5 ⁇ g mL plate-bound anti-CD3 (OKT3 mAb) in 96-well round-bottom plate in supplemented RPMI medium.
  • Proliferation of CFSE- labeled cells was assessed by flow cytometry after 84-90 hr of culture.
  • Percent suppression was calculated as follows: 100 - (number of proliferating CFSE-diluting responder cells in the presence of suppressor cells at a 1 to 1 ratio / number of proliferating responder cells when cultured alone) and multiplied by 100.
  • mice NOD.Cg-Prkd cscld H2rg tmlwjl/SzJ (NSG) (stock ⁇ 005557, The Jackson Laboratory) mice were bred in our own animal facilities in Specific Pathogen-Free conditions (accreditation number from the Veterinary services: A75-13-10). with an enriched fat regime and addition of Bactrim in drinking water every other week. The colony was regularly checked for ⁇ -c deficiency by PCR according to the Jackson Laboratory protocol. All procedures were approved by the Regional Ethical Commitee on Animal Experimentation.
  • mice Youg adult NSG female mice (11 weeks old) were irradiated (2 Gy) and received 2x10 6 PBMCs from a healthy donor with or without 10 6 autologous Treg cells cultured in IL- 2 + RAV conditions for 14 days. Animals were euthanasied if their weight dropped below 80% of their initial weight.
  • CD45RA+ Treg cells are the adequate population to isolate for expansion [9].
  • Treg cells lose FoxP3 expression after polyclonal stimulation [10].
  • Treg cells require high levels of FoxP3 to be efficiently suppressive [11].
  • inhibitors of mTOR such as rapamycin enables the preferential expansion of FoxP3 expressing cells by eliminating other effector cells that do not express FoxP3 primarily [12]
  • rapamycin at commonly reported concentration that is 1 microg/mL
  • FoxP3negative cells were indeed less prevalent, that FoxP3 losing CD4+ T cells were still detectable upon culture (FIGURE 1).
  • nTreg cells expanded with Rapamycin, Azacitidine and Vorinostat together were highly suppressive (figure 4).
  • nTregs expanded in the presence of each molecule alone or in the presence of RA, VR or VA combinations showed less potent suppression than nTreg cells expanded under the three inhibitor combination regimen
  • nTreg cells require the addition of these 3 molecules in commonly accepted Treg expanding conditions.
  • nTreg cells expanded in the presence of RAV combination in vivo as their capacity to delay xeno GVHD (Graft vs. Host Disease) in NOD common gamma chain (-/-) SCID mice (NSG) mice. Irradiated NSG mice develop severe GVH disease within 3 weeks after the transfer of human PBMCs. In comparison, when the same amount of human PBMCs was co-transferred with 1 million nTreg cells expanded for 14 days under RAV condition in vitro, we observed that GVHD was significantly delayed (FIGURE 5).
  • FOXP3 iso forms in the generation of human CD4+ Tregs. J Clin Invest 115: 3276-3284.

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Abstract

The present invention concerns a method for expanding regulatory T cells using a combination of am-TOR inhibitor, a histone deacetylase inhibitor and a DNA methylation inhibitor, for maintaining Foxp3 expression in order to maintain their suppressive function.

Description

METHOD OF MAINTAINING FOXP3 EXPRESSION IN EXPANDED
T REGULATORY CELL
RELATED APPLICATION
The present application claims priority to European Patent Application
No. EP 11 306 296, which was filed on October, 6 2011. The European patent application is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION:
The present invention concerns a method for expanding regulatory T cells using a combination of at least a m-TOR inhibitor, at least a histone deacetylase inhibitor and at least a DNA methylation inhibitor, for maintaining FoxP3 expression in regulatory T cells in order to maintain their suppressive function.
BACKGROUND OF THE INVENTION:
Based on recent progress in the understanding of FoxP3-expressing Treg cell immunobiology, Treg cell based therapy seems in theory readily applicable in humans in order to treat autoimmune diseases [1]. Thus, it is now well established that the timing of Treg cell activation in relation to effector T cells activation is a key parameter to consider because effector T cells activated prior to Treg cells are resistant to in vivo suppression. [2]. Moreover, as shown in most if not all models Treg cells are characterized in vivo, by their capacity to prevent autoimmunity, sometimes to delay the onset of disease but rarely to cure diseases completely[3,4].
Thus, based on the postulate that Treg based therapy is more suitable to prevent disease onset or disease flares, autoimmune diseases for which Treg cell based therapy would be better appropriate can be determined based on their evolutionary patterns i.e. continuously progressive versus remitting/relapsing course of autoimmune diseases such as systemic lupus erythematosus, multiple sclerosis, vasculitites etc. Treg cell based treatment may also be of interest for the treatment of allergic diseases and for induction of organ transplantation [1]. Treg cell based therapies may be beneficial when the disease is quiescent to prevent subsequent flares or organ rejection. In the process of designing Treg-based manipulation strategies it is important to take into account the notion that Treg cells appear to exert a more preventive than curative effect on chronic inflammatory processes. Therefore, rather than considering Treg cell based treatment as a mere induction therapy that would be curative on its own, such treatment might better be considered as an adjuvant therapy in the management of autoimmune diseases. Cyclophosphamide, mycophenolate and/or high dose steroids have proven efficient to treat disease flares. Prevention of subsequent flares or of recurrence usually consists of immunosuppressors mostly given orally. One can imagine that such maintenance treatment might be replaced by treatments based on Treg biology.
In line with this strategy, it seems plausible that all autoimmune diseases would benefit from Treg biology based therapy, considering that an ideal strategy would be to reestablish dominant self-tolerance [5] based on the elimination of pathogenic effector or memory cells as much as possible and the reinstallation of functional Treg cells via ex vivo expansion [1,6].
However, although Treg based therapeutic strategies seem readily feasible, one must keep in mind that Treg cells may convert in vitro into pathogenic cells such as TH- 17 cells [7]. Such converted Treg cells may theoretically make the diseases worse. Thus, it is of utmost importance to determine the conditions that preserve Treg phenotype and suppressive functions in vitro. The resolution of this key issue is a prerequisite for the development of clinical trials evaluating Treg cell based therapy in systemic autoimmune diseases, allergy or transplantation [8].
Given that CD45RA-Treg cells are poorly proliferative even in the presence of high dose IL-2, human CD45RA+ Treg cells are the adequate population to isolate for expansion. However, even with high doses of IL-2, these cells lose FoxP3 expression after polyclonal stimulation. Treg cells require high levels of FoxP3 to be efficiently suppressive. Thus, there is a need for molecules that prevent the downregulation of FoxP3 in vitro and preserve Treg phenotype and suppressive functions for Treg based cell therapy.
Treatment of T cells with the bioactive peptide TGF-β is known to induce de novo expression of Foxp3 [14].
WO2008071974 also describes use of phosphatidyl inositol-3-kinase (PI3K) inhibitors and/or mtTor inhibitors (such as rapamycin) and/or AKT inhibitors for inducing de novo expression of FoxP3 in T cells. However, it has been shown that most induced FOXP3 expressing CD4+ T cells are not suppressive in vitro. Moreover, such combinations have not been proposed for the maintenance of FOXP3 expression in naturally occurring FOXP3 expressing Tregs. The mTOR inhibitor rapamycin alone has been used for contributing to the expansion of pre-existing Treg cells expressing Foxp3 [15, 16]. However, mTor alone (as the other agents disclosed in the prior art) fails to prevent the downregulation of FoxP3 in vitro and maintain the Treg phenotype and suppressive functions. The present invention seeks to overcome these problems.
SUMMARY OF THE INVENTION:
The present invention is based on the discovery that a combination of an inhibitor of mTOR (such as rapamycin), an inhibitor of histone deacetylases (such as vorinostat) and an inhibitor of DNA methylation (such as azacitidine) is efficient for the maintenance of high expression of FoxP3 and suppressive function in human CD4+ T regulatory cells expanded in vitro or ex vivo.
The discovery is all the more unexpected that, as shown in the examples, each molecule and either combinations of two molecules are not efficient for maintaining high expression of FoxP3 and suppressive function in human CD4+ T regulatory cells in vitro.
DETAILED DESCRIPTION OF THE INVENTION:
The present invention arises from the unexpected finding by the inventors that an inhibitor of mTOR, an inhibitor of histone deacetylases and an inhibitor of DNA methylation act synergistically for maintaining high expression of FoxP3 and suppressive function in T regulatory cells in vitro. This combination has proved effective in human CD4+ T regulatory cells in vitro. As a whole, the novel combination is more efficient at maintaining highly FoxP3 expression on expanded cells than either drug alone or either combination of two molecules. As shown in the examples, the combination of the invention enables the expansion of highly pure T regulatory cells displaying high levels of FoxP3.
Method for maintaining FoxP3 expression in expanded Treg cell The present invention concerns a novel method for the stable expression of FoxP3, the signature transcription factor of Tregs, which is maintained when Treg cells are activated in culture medium containing inhibitors of mTOR, inhibitors of histone deacetylases and inhibitors of DNA methylation. According to a first aspect, the present invention provides an in vitro method for maintaining FoxP3 expression in expanded T regulatory cell comprising the steps of:
(i) stimulating Treg cell;
(ii) treating said Treg cell with a combination of at least a m-TOR inhibitor, at least a histone deacetylase inhibitor and at least a DNA methylation inhibitor following or during the stimulation of (i).
In a preferred embodiment the Treg cell stimulated and treated with the combination of the invention is a naive Treg cell.
In a specific embodiment the in vitro method for maintaining Foxp3 expression in T regulatory cell comprises a preliminary step of isolating Treg cells from Peripheral Blood Mononuclear Cells (PBMCs) The term"T regulatory cell" (also called "Tregs" or "Treg cells") means an important component of the healthy immune system. Regulatory T cells are involved in keeping effector T cells in check, and in the prevention of "self recognition" which can be a major factor in autoimmune disease.
Tregs have numerous acknowledged biomarkers known in the art. These include CD4+, CD25+, and Foxp3+. In particular, according to the present invention a T regulatory cell shows Foxp3 expression (Foxp3+).
A Treg cell must preferably display regulatory function. The demonstration of regulatory function may be determined by any suitable method known in the art. In particular, examples of such tests are set out in the example section. Specifically, the tests embodied in example and figures 4 are regarded as standards in vitro tests for the assessment of regulatory T cell function.
Treg cells according to the present invention are mammalian Treg cells, most preferably human Treg cells.
By "na'ive Treg cell" (also called "NTreg cell"), is meant T cells which express the following markers as CD4+CD45RA+CD25+. FoxP3+ regulatory T cells comprise two subsets that distinguish each other by the expression of CD45RA defined as na'ive Tregs that express FOXP3 and CD45RA and effector Tregs that also express FOXP3 and not CD45RA. Cells that are suitable for expansion are naive Tregs since they are highly proliferative under stimulation conditions and in the presence of IL-2 while effector Tregs are poorly proliferative under such conditions. Thus, the preferred target cells to expand are naive Tregs that can be separated as either CD4+CD25+CD45RA+ cells or CD4+CD25+CD45RO- cells.
"Naive Treg cells" may be isolated from PBMCs by any suitable method known in the art. Examples of such method are set out in the example section.
By "stimulating Treg cells" is meant using biological means to obtain physiological activation and expansion of Treg cells using a) lymphokine and b) TCR stimulation.
Stimulation may be performed by any suitable means which renders the T cell sensistive to the action of m-TOR inhibitors histone, deacetylase inhibitors and DNA methylation inhibitors for the maintenance of the Treg phenotype as described herein.
a) Lymphokine
It is widely believed that in vivo T regulatory cells need additional factors such as IL-2 and/or TGFbeta, for their survival and/or the maintenance of their regulatory functions. Thus, the invention relates to the maintenance of Foxp3 expression in Treg cells together with contacting those Treg cells with said lyrnphokines.
In a preferred embodiment the IL-2 lymphokine is used to enhance Treg cell expansion.
b) TCR stimulation
In the method of the present invention stimulation of Treg cell is also performed by stimulating the TCR receptor. In particular embodiments, stimulation is a persistent or sustained stimulation, which means that the stimulation is not necessarily withdrawn at the time of action of m-TOR, histone deacetylases and DNA methylation inhibitors. Preferably a stimulated Treg cell is a Treg cell which has been stimulated via the T cell receptor (TCR).
Preferably the Treg cells are also stimulated via the TCR or the TCR-associated CD3 complex of signalling proteins. In practice, TCR signal is in fact transmitted via the associated proteins. Thus, to stimulate via the TCR it is possible to target the actual TCR itself (e.g. antigen specific TCR targeting) or to target CD3 (e.g the TCR associated protein(s)).
In a preferred embodiment, TCR stimulation is polyclonal and may be done via CD3.
Most preferably a costimulatory receptor such as CD28 is also stimulated. Preferably CD28 is stimulated simultaneously with the stimulation of CD3 receptor(s). A preferred embodiment stimulation is via simultaneous stimulation of TCR and/or CD3, and of CD28. In another embodiment stimulation of CD2 can be added to CD3 and CD28 stimulation to strengthen the stimulation.
Preferably stimulating said Treg cell comprises contacting said Treg cell with anti- TC or anti-CD3 antibody. Preferably stimulating said Treg cell further comprises contacting said Treg cell with anti-CD28 antibody, preferably contacting said T cell with anti-TCR or anti-CD3 antibody, and with anti-CD28 antibody simultaneously. The mode of presentation of such antibodies may be chosen by the operator, for example this may be accomplished using plate-bound antibodies or by using beads coated with antibodies such as anti-CD3/anti- CD28 antibodies, or by any other means of presentation known to the operator.
In another embodiment, TCR activation is performed by adding specifc antigen or by contacting Treg cell with activated Antigen Presenting cell (APC).
In the most preferred embodiment, stimulating Treg cell comprises stimulating NTreg cell with Interleukine 2, anti CD3 antibody and anti CD28 antibody.
The Treg cell expanded by the method of the invention may be cultured between one and four weeks and most preferably during 2 weeks.
It should to be noted that the major advantage of the present invention is the sustained expression of Foxp3. This is in contrast with the prior art where the major effect shown to date is the expansion of Foxp3 expressing T cells in a first time but followed by the loss of FoxP3 expression after polyclonal stimulation [see 17] .
The opportunity to actively maintain elevates Foxp3 expression in expanded Treg cells is a significant advantage of the present invention.
By "FoxP3" (also called "forkhead box P3"), is meant a protein involved in responses of the immune system. A member of the FOX protein family, FoxP3 appears to function as a master regulator in the development and function of regulatory T cells.
Foxp3 expression has been associated with regulatory T cell function, in certain settings, Foxp3 expression has been shown to be necessary and sufficient to induce regulatory T cell function. Indeed, Foxp3 expression has in some circumstances been regarded as an indicator or identifier of regulatory T cells as compared to other sub- populations of T cells, in the prior art, the only known way of inducing Foxp3 expression pharmacologically has been by treating a population of T cells with the bioactive peptide TGF-β associated with IL-2. By "m-TOR inhibitor", it meant a class of compounds that interfere with the function of the mammalian target of rapamycin (mTOR) also known as mechanistic target of rapamycin or FK506 binding protein 12-rapamycin associated protein 1 (FRAP1). m-TOR is a protein which in humans is encoded by the FRAP 1 gene. mTOR is a serine/threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis and transcription. mTOR belongs to the phosphatidylinositol 3-kinase-related kinase protein family. The m-TOR inhibitor in the frame of the present invention may be any known m-TOR inhibitor such as rapamycin (sirolimus), or an analogue thereof such as RADOOl (everolimus), CCI-779 (temsirolimus), AP23573, The m-TOR inhibitor also may be wortmannin, and synthetic compounds such as PIK-90.
Preferably said m-TOR inhibitor is rapamycin. The dose used for Rapamicin is between 0.5 and 5 Microg/mL, preferably 1 Microg/mL (i.e. 100 nM).
By "histone deacetylase inhibitor" (also called HDI), it meant a class of compounds that interfere with the function of histone deacetylase (or HDAC) (EC number 3.5.1). Histone deacetylase are a class of enzymes that remove acetyl groups (0=C-CH3) from an ε-Ν-acetyl lysine amino acid on a histone.. HDAC proteins are now also called lysine deacetylases (KDAC), to describe their function rather than their target, which also includes non-histone proteins.
In a preferred embodiment of the present invention, the histone deacetylase inhibitor is selected from the group consisting of
1) classical HDIs : hydro xamic acids (or hydro amates), such as trichostatin A; cyclic tetrapeptides (such as trapoxin B), and depsipeptides; benzamides; electrophilic ketones; and aliphatic acid compounds such as phenylbutyrate and valproic acid.
2) "Second-generation" HDIs include the hydroxamic acids vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat (LBH 89); and the benzamides : entinostat (MS-275), CI994, and mocetinostat (MGCD0103).
Vorinostat is preferred histone deacetylase inhibitor in the frame of the present invention. The dose used for Vorinostat are between 0,1 and 5 microM, preferably 0.5 microM or 1 microM. By "DNA methylation inhibitor" also called "Demethylating agents", is meant a class of compounds that interfere with DNA methylation which is the addition of a methyl group to the 5- position of the cytosine pyrimidine ring or the nitrogen in position 6 of the adenine purine ring. DNA methylation stably alters the gene expression pattern in cells i.e. decrease gene expression
Demethylating agents are compounds that can inhibit methylation, resulting in the expression of the previously hypermethylated silenced genes. Cytidine analogs such as 5- azacytidine (azacitidine) and 5-azadeoxycytidine (decitabine) are the most commonly used demethylating agents . These compounds work by binding to the enzymes that catalyse the methylation reaction, i.e. DNA methyltransferases.
Azacitidine is preferred DNA methylation inhibitor that can be used in the frame of the present invention. The dose used for Azacitidine is between 1 and 10 microM, preferably 2.5 microM or 5 micro M.
Another aspect of the invention is an in vitro method of maintaining Foxp3 expression in a previously stimulated Treg cell comprising treating said Treg cell with a combination of at least a m-TOR inhibitor, at least a histone deacetylase inhibitor and at least a DNA methylation inhibitor.
Another aspect of the invention is the in vitro and/or ex-vivo use of mTOR inhibitor, histone deacetylase inhibitor and DNA methylation inhibitor in order to maintain the differentiation of expanded regulatory T cell.
Culture Medium, Kit and Method for expending Treg cells
The present invention also relates to a culture medium comprising Treg cell stimulant- TOR inhibitor and histone deacetylase inhibitor and DNA methylation inhibitor.
The culture medium of the present invention is suitable for use to expand regulatory T cells for maintaining high expression of FoxP3 and suppressive function in Treg cells in vitro The term "culture medium" as used herein refers to a liquid medium suitable for the in vitro culture of Treg cells, preferably manufactured at clinical grade. Typically, the culture medium of the invention contains:
- a source of carbon as energy substrate, such as glucose, galactose or sodium pyruvate;
- essential amino-acids;
- vitamins, such as biotin, folic acid, B 12... ;
- at least a purine and a pyrrolidine as nucleic acid precursors;
- inorganic salts;
The culture medium may also contain pH buffers in order to maintain the pH of the medium at a value suitable for cell growth
The culture medium of the invention may be based on a commercially available medium such as X-VIVO 15 or MACOPHARMA HP01.
The term "Treg cell stimulant" means any mean previously describe in the "stimulation of Treg cell". Most preferably the Treg cell stimulants are Interleukine 2 and anti-CD28 and anti-CD3 antibodies.
Another aspect of the invention relates to an in vitro method for expanding Treg cell wherein said method comprises the step of culturing Treg cells with the culture medium as described above.
The step of culturing Treg cells (preferably na'ive Treg cell) with the culture medium of the invention shall be carried out for the necessary time required for the production of functional Treg cell. Typically, the culture of Treg cells with the medium of the invention shall be carried out for at least 7 days, preferably at least 10 days, even more preferably at least 14 days.
If necessary, the culture medium of the invention can be renewed, partly or totally, at regular intervals. Typically, the culture medium of the invention can be replaced with fresh culture medium of the invention every other day, for 14 days.
Another aspect of the invention is a kit comprising: (i) a Treg cell stimulant; and (ii) combination of at least a mTOR inhibitor, at least a histone deacetylase inhibitor and at least a DNA methylation inhibitor. In a preferred embodiment, the m-TOR inhibitor is Rapamycin the histone deacetylase inhibitor is Voronistat and the DNA methylation inhibitor is Azacitidin.
Method of treatment
A further aspect of the invention relates to a method of treating a subject in need of regulatory T cell(s) comprising
(i) removing a sample comprising a Treg cell from a subject
(ii) stimulating said Treg cell
(iii) treating said Treg cell with a combination of at least a m-TOR inhibitor and at least a histone deacetylase inhibitor and at least a DNA methylation inhibitor; and
(iv) reintroducing said Treg cell to said subject
In a preferred embodiment the Treg cell removed and stimulated are naive Treg cell.
As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a subject according to the invention is a human.
The method and combination for uses according to the invention find use in a patient in need of regulatory T cell(s) which means for the treatment of autoimmune diseases (such as systemic lupus erythematosus, multiple sclerosis, vasculitites etc. ), allergic diseases and in transplantation (Graft-versus-host (GVH) disease, ....).
As shown in the examples, the combination of the invention enables the expansion of highly pure human T regulatory cells with sustained high expression of FoxP3 said Treg cells having a high suppression activities in vitro (in cell culture) and in vivo (in a mice model of Graft-versus-host (GVH) disease)
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES: FIGURE 1 Rapamycin increases FoxP3 levels in expanded nTregs but does not completely prevent the downregulation of FoxP3 expression
nTreg sorted as shown in ref 13 from a healthy donor PBMCs were cultured for 7 days in the presence of IL-2 and anti-CD3/CD28 beads 300 IL-2 with rapamycin (right) or without rapamycin (left). FoxP3 and Ki-67 levels were analyzed by FACS. Data are representative of 10 independent experiments
FIGURE 2 Azacitidine and Vorinostat increase FoxP3 levels upon activation
Whole PBMCs from a healthy donor were cultured in the presence of IL-2, anti- CD3/CD28 beads, 300 IU/mL IL-2 and of Azacytidine (A) or Vorinostat (B) using various concentration to determine optimal concentration for the enhancement of FoxP3 levels. FoxP3 and Ki-67 levels were analyzed by FACS after 48-72 hours of culture. Data are representative of 3 independent experiments. FIGURE 3 Combination of rapamycin with Azacitidine and Vorinostat enables the expansion of highly pure Treg cells displaying high levels of FoxP3
nTregs isolated from a healthy donor PBMCs following the gating strategy shown in ref [13] were cultured for 14 days in the presence of anti-CD3/CD28 beads, 3001 U/mL IL-2 and of rapamycin (R), Azacytidine (A), Vorinostat (B) alone or in the presence of a combination of R with A, R with B or A with B, or in the presence of the 3 molecules. CD4+CD25-CDR45RA+ T cells were also cultured in order to set the threshold for FoxP3 expression (bottom FACS plot). Ki-67 and FoxP3 expression were analyzed by FACS at day 14-16 of culture. Data representative of 8 independent experiments. FIGURE 4 Combination of Rapamycin (R) with Azacitidine (A) and Vorinostat
(B) enables the expansion of Tregs with highly potent suppressive capacity.
nTreg cultured for 14 days in conditions described in Figure 3 were cocultured with autologous CD4+CD25-CD45RA+ responder cells labeled with CFSE in the presence of plate bound anti-CD3 (0.5 microg/mL) and autologous irradiated B cells and monocytes. CFSE dilution in responder cells was analyzed after 96 hours of culture on gated CD4+CFSE+ T cells. % suppression are indicated. Data representative of 4 independent experiments.
FIGURE 5 Azacytidin/vorinostat/rapamycin combination preserves suppressive nTreg function in a xenoGVH model in NSG mice Treg cells (106 cells) cultured for 14 days with RAV combination were co -transferred with 2xl06 autologous PBMCs in NSG mice (n=3). Weight and survival of these mice (dot lines) were compared to NSG mice injected with 2x10A6 PBMCs alone (black lines, n=5). Comparison was made using a Log-rank (Mantel-Cox) Test, p value < 0.05 is considered significant (B).
EXAMPLE:
Methods
Isolation of live naive Treg cells
PBMCs were isolated through Ficoll gradient separation from freshly drawn blood. CD4+T cells were first magnetically isolated using a CD4 T cell separation kit (Miltenyi) and subsequently surface stained using a combination of flurochrome conjugated mAbs: anti-CD4-PErCP 5.5, anti-CD25-PE, anti-CD 127- AF647 and anti-CD45RA-FITC obtained from BD bioscience.
Na'ive CD25++CD45RA+ Treg cells (NTreg cells) were isolated from PBMCs following flow isolation according to the gating strategy we validated (see ref.[13]).
Isolated na'ive Treg cells were immediately distributed into U bottom well for culture and expansion. To analyze the effect of tested molecules or combination of molecules, 10 to 30 * 103 nTreg cells were collected per U bottom
Expansion strategy
NTreg cells were first polyclonally stimulated using anti-CD3/anti-CD28 beads (concentration: 40 000 beads/microL; 2 microL/well) or anti-CD2/anti-CD3/anti-CD28 beads in the presence of 300 IU/mL IL-2 in culture media in the presence of a combination of Rapamycin diluted in culture medium (1 microg/mL), Azacytidine first diluted in PBS and then in culture medium (5 microM) and Vorinostat first diluted in DMSO and then in culture medium (1 microM). 300 IU/mL IL-2 was added every 3-4 days and cells were split every week and restimulated by additional anti-CD3/anti-CD28 beads every week.
Cells were cultured for 1 to 4 weeks.
Expanded cells were counted and flow analyzed every week for the expression of FoxP3 and Ki-67 after fixation and permeabilization. Because FoxP3 expression cutoff that differentiates positive cells from negative cells is different in cultured cells compared to ex vivo isolated cells, we also cultured as control cells CD4+CD25-CD45RA+ cells. The expression of FoxP3 in these cells enabled us to set the right threshold for FoxP3 expression in expanded nTreg cells.
In vitro Suppressive function
Expanded cells were assessed for suppressive function between 10 and 14 days of culture. 1 x 104 CFSE-labeled responder CD25-CD45RA+CD4+ T cells were cocultured with 1 x 104 unlabeled cells assessed for their suppressive capacity and 1 x 105 irradiated autologous accessory cells and were stimulated with 0.5 μg mL plate-bound anti-CD3 (OKT3 mAb) in 96-well round-bottom plate in supplemented RPMI medium. Proliferation of CFSE- labeled cells was assessed by flow cytometry after 84-90 hr of culture. Percent suppression was calculated as follows: 100 - (number of proliferating CFSE-diluting responder cells in the presence of suppressor cells at a 1 to 1 ratio / number of proliferating responder cells when cultured alone) and multiplied by 100. Mice
NOD.Cg-Prkdcscld H2rgtmlwjl/SzJ (NSG) (stock ≠ 005557, The Jackson Laboratory) mice were bred in our own animal facilities in Specific Pathogen-Free conditions (accreditation number from the Veterinary services: A75-13-10). with an enriched fat regime and addition of Bactrim in drinking water every other week. The colony was regularly checked for γ-c deficiency by PCR according to the Jackson Laboratory protocol. All procedures were approved by the Regional Ethical Commitee on Animal Experimentation.
Induction of xeno-Graft vs. Host Disease in NSG mice
Youg adult NSG female mice (11 weeks old) were irradiated (2 Gy) and received 2x106 PBMCs from a healthy donor with or without 106 autologous Treg cells cultured in IL- 2 + RAV conditions for 14 days. Animals were euthanasied if their weight dropped below 80% of their initial weight.
Results
CD45RA+ Treg cells are the adequate population to isolate for expansion [9].
However, even with high doses IL-2, these cells lose FoxP3 expression after polyclonal stimulation [10]. Treg cells require high levels of FoxP3 to be efficiently suppressive [11]. Thus, it is necessary to add molecules that prevent the downregulation of FoxP3 in vitro. Given that inhibitors of mTOR such as rapamycin enables the preferential expansion of FoxP3 expressing cells by eliminating other effector cells that do not express FoxP3 primarily [12], we evaluated the effect of rapamycin (at commonly reported concentration that is 1 microg/mL) in the maintenance of FoxP3 expression and noticed, although FoxP3negative cells were indeed less prevalent, that FoxP3 losing CD4+ T cells were still detectable upon culture (FIGURE 1).
We have evaluated inhibitor of DNA methylation Azacitine (A) and inhibitor of histone deacetylase Vorinostat (B) for their capacities to enhance FoxP3 expression in PBMCs after polyclonal stimulation and could observe that both molecules enhanced FoxP3 expression. We could determine the optimal concentration of Azacitidine and Vorinostat that maintained cell viability and gave highest proportions of FoxP3 expressing cells and brightest expression of FoxP3 as 5 microM for Azacytidine and 1 microM for vorinostat (Figure 2).
We thus tested each Azacitidine and Vorinostat in their capacities to maintain FoxP3 expression in expanded nTreg cells and noticed that they did not give rise to increased expression of FoxP3 compared to Rapamycin. We thus tested several associations that combined 2 molecules or all molecules together. Strikingly, only the combination of the 3 molecules together maintained highly pure FoxP3bright cells (>95 % of cells) at day 14 with 16-20 fold expansion (Figure 3).
We thus believe that the combination of rapamycin (1 microg/mL), Azacitidine (at 5 microM in these experiments) and Vorinostat (1 microM in theses experiments) when included in the expansion culture media containing anti-CD3/anti-CD28 beads and high dose IL-2 (300 IU/mL) enables the expansion of pure FoxP3high Treg cells.
Analysis of their suppressive capacities in vitro indicated that nTreg cells expanded with Rapamycin, Azacitidine and Vorinostat together were highly suppressive (figure 4). Of note, nTregs expanded in the presence of each molecule alone or in the presence of RA, VR or VA combinations showed less potent suppression than nTreg cells expanded under the three inhibitor combination regimen
Therefore, optimized expansion of nTreg cells requires the addition of these 3 molecules in commonly accepted Treg expanding conditions.
Finally, we evaluated the suppressive capacity of nTreg cells expanded in the presence of RAV combination in vivo as their capacity to delay xeno GVHD (Graft vs. Host Disease) in NOD common gamma chain (-/-) SCID mice (NSG) mice. Irradiated NSG mice develop severe GVH disease within 3 weeks after the transfer of human PBMCs. In comparison, when the same amount of human PBMCs was co-transferred with 1 million nTreg cells expanded for 14 days under RAV condition in vitro, we observed that GVHD was significantly delayed (FIGURE 5).
These results indicate that a 10 to 14 days culture with high dose IL-2 and the combination of mTOR inhibitor such as rapamycin, histone deacetylase inhibitor such as vorinostat and DNA methyltransferase inhibitor such as azacytdin is optimal for the expansion of human CD45RA+ naive Treg cells with sustained high expression of FoxP3 and potent suppression capacities in vitro and in vivo. REFERENCES:
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.
1. Miyara M, Wing K, Sakaguchi S (2009) Therapeutic approaches to allergy and autoimmunity based on FoxP3+ regulatory T-cell activation and expansion. J Allergy Clin Immunol 123: 749-755; quiz 756-747.
2. Billiard F, Litvinova E, Saadoun D, Djelti F, Klatzmann D, et al. (2006) Regulatory and effector T cell activation levels are prime determinants of in vivo immune regulation. J
Immunol 177: 2167-2174.
3. Bluestone JA, Thomson AW, Shevach EM, Weiner HL (2007) What does the future hold for cell-based tolerogenic therapy? Nat Rev Immunol 7: 650-654.
4. Uhlig HH, Coombes J, Mottet C, Izcue A, Thompson C, et al. (2006) Characterization of Foxp3+CD4+CD25+ and IL-10-secreting CD4+CD25+ T cells during cure of colitis. J Immunol 177: 5852-5860.
5. Sakaguchi S, Yamaguchi T, Nomura T, Ono M (2008) Regulatory T cells and immune tolerance. Cell 133: 775-787.
6. Mozes E, Sharabi A (2010) A novel tolerogenic peptide, hCDRl, for the specific treatment of systemic lupus erythematosus. Autoimmun Rev 10: 22-26.
7. Zhou X, Bailey-Bucktrout SL, Jeker LT, Penaranda C, Martinez-Llordella M, et al. (2009) Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nat Immunol 10: 1000-1007. 8. Fujii H, Arakawa A, Kitoh A, Miyara M, Kato M, et al. (2010) Perturbations of both nonregulatory and regulatory FOXP3+ T cells in patients with malignant melanoma. Br J Dermatol.
9. Hoffmann P, Eder R, Boeld TJ, Doser K, Piseshka B, et al. (2006) Only the CD45RA+ subpopulation of CD4+CD25high T cells gives rise to homogeneous regulatory T- cell lines upon in vitro expansion. Blood 108: 4260-4267.
10. Hoffmann P, Boeld TJ, Eder R, Huehn J, Floess S, et al. (2009) Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation. Eur J Immunol 39: 1088-1097.
11. Allan SE, Passerini L, Bacchetta R, Crellin N, Dai M, et al. (2005) The role of 2
FOXP3 iso forms in the generation of human CD4+ Tregs. J Clin Invest 115: 3276-3284.
12. Battaglia M, Stabilini A, Migliavacca B, Horejs-Hoeck J, aupper T, et al. (2006) Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients. J Immunol 177: 8338-8347.
13. Miyara M, Yoshioka Y, Kitoh A, Shima T, Wing K, et al. (2009) Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor. Immunity 30: 899-911.
14. Chen W. W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM. (2003) Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J. Exp. Med. vol 198 (12), ppl875-1886
15 Zheng XX, Sanchez-Fueyo A, Sho M, Domenig C, Sayegh MH, Strom TB, (2003) Favorably tipping the balance between cytopathic and regulatory T cells to create transplantation tolerance. Immunity vol 19 (4) p503-514
16 Battaglia M, Stabilini A, Roncarolo MG. (2005) Rapamycin selectively expands
CD4+CD25+FoxP3+ regulatory T cells. Blood vol 105 (12) p4743-48.
17 Petra Hoffman, Eur J Immunol. 2009 Apr;39(4): 1088-97)

Claims

CLAIMS;
1. An in vitro method of maintaining Foxp3 expression in expanded T regulatory cell comprising :
(i) stimulating a Treg cell
(ii) treating said Treg cell with a combination of at least m-TOR inhibitor and at least histone deacetylase inhibitor and at least DNA methylation inhibitor following or during the stimulation of (i).
2. An in vitro method according to claim 1 comprising a preliminary step of isolating Treg cell from Peripheral Blood Mononuclear Cells (PBMCs).
3. An in vitro method of maintaining Foxp3 expression in a previously stimulated Treg cell comprising treating said Treg cell with a combination of at least a m-TOR inhibitor, at least a histone deacetylase inhibitor and at least a DNA methylation inhibitor.
4. An in vitro method according to any of claims 1 to 3 wherein the Treg cell is a na'ive Treg cell.
5. An in vitro method according to any preceding claim further wherein said m-TOR inhibitor is rapamycin, said histone deacetylase inhibitor is Vorinostat and DNA methylation inhibitor is Azacitidin.
6. An in vitro method according to any preceding claim wherein stimulating said Treg cell comprises stimulating said Treg cell with Interleukine 2, anti CD3 antibody and anti CD28 antibody.
7. In vitro use of mTOR inhibitor, histone deacetylase inhibitor and DNA methyl ation inhibitor to maintain differentiation of expanded regulatory T cell.
8. A culture medium comprising Treg cell stimulant and the combination of mTOR inhibitor, histone deacetylase inhibitor and DNA methylation inhibitor.
9. A culture medium according to claim 8 wherein said m-TOR inhibitor is rapamycin said histone deacetylase inhibitor is Voronistat and said DNA methylation inhibitor is Azacitidin.
10. A culture medium according to claim 8 or 9 wherein said Treg stimulant comprises, Interleukine 2, anti-CD3 antibody and anti-CD28 antibody.
11. An in vitro method for expanding Treg cell wherein said method comprises the step of culturing Treg cells with the culture medium as defined in any one of claims 8 or 10.
12. A kit comprising: (i) a Treg stimulant; and (ii) combination of mTOR inhibitor, histone deacetylase inhibitor and DNA methylation inhibitor.
13. A kit according to claim 12 wherein said Treg stimulant comprises, Interleukine 2, anti-CD3 antibody and anti-CD28 antibody. A kit according to claim 12 wherein said m-TOR inhibitor is Rapamycin said histone deacetylase inhibitor is Voronistat and said DNA methylation inhibitor is Azacitidin.
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015160869A1 (en) 2014-04-16 2015-10-22 Signal Pharmaceuticals, Llc Methods for treating cancer using tor kinase inhibitor combination therapy
JP2016525124A (en) * 2013-07-18 2016-08-22 ベイラー カレッジ オブ メディスンＢａｙｌｏｒ Ｃｏｌｌｅｇｅ Ｏｆ Ｍｅｄｉｃｉｎｅ Methods for enhancing the capacity of immune cells
WO2017105265A1 (en) 2015-12-17 2017-06-22 Gdański Uniwersytet Medyczny Method for ex vivo expansion of regulatory t cells
WO2018024896A1 (en) * 2016-08-05 2018-02-08 INSERM (Institut National de la Santé et de la Recherche Médicale) EX VIVO GENERATION OF γδ FOXP3+ REGULATORY T CELLS AND THERAPEUTIC USES THEREOF
WO2018024894A1 (en) * 2016-08-05 2018-02-08 INSERM (Institut National de la Santé et de la Recherche Médicale) Ex vivo generation of mhcii restricted cd4+foxp3+ regulatory t cells and therapeutic uses thereof
JP2019530470A (en) * 2016-08-05 2019-10-24 アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル Ex vivo generation of γδ Foxp3 + regulatory T cells and their therapeutic use
WO2019234221A1 (en) 2018-06-08 2019-12-12 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for stratification and treatment of a patient suffering from chronic lymphocytic leukemia
US10905706B2 (en) 2015-01-08 2021-02-02 The Johns Hopkins University Compositions and methods to accelerate resolution of acute lung inflammation
US11365392B2 (en) 2016-08-05 2022-06-21 INSERM (Institut National de la Santé et de la Recherche Médicale) Ex vivo generation of MHCII restricted CD4+ FOXP3+ regulatory T cells and therapeutic uses thereof
US11384336B2 (en) 2016-12-07 2022-07-12 East Carolina University Compositions and methods for in vitro cultivation and/or expansion of regulatory T cells

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008071974A2 (en) 2006-12-14 2008-06-19 Medical Research Council Use of pi3k m-tor and akt inhibitors to induce f0xp3 expression and generate regulatory t cells
WO2009155477A1 (en) * 2008-06-19 2009-12-23 The Trustees Of The University Of Pennsylvania Inducible regulatory t-cell generation for hematopoietic transplants

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008071974A2 (en) 2006-12-14 2008-06-19 Medical Research Council Use of pi3k m-tor and akt inhibitors to induce f0xp3 expression and generate regulatory t cells
WO2009155477A1 (en) * 2008-06-19 2009-12-23 The Trustees Of The University Of Pennsylvania Inducible regulatory t-cell generation for hematopoietic transplants

Non-Patent Citations (20)

* Cited by examiner, † Cited by third party
Title
ALLAN SE; PASSERINI L; BACCHETTA R; CRELLIN N; DAI M ET AL.: "The role of 2 FOXP3 isoforms in the generation of human CD4+ Tregs", J CLIN INVEST, vol. 115, 2005, pages 3276 - 3284, XP002439087, DOI: doi:10.1172/JCI24685
BATTAGLIA M; STABILINI A; MIGLIAVACCA B; HOREJS-HOECK J; KAUPPER T ET AL.: "Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients", J IMMUNOL, vol. 177, 2006, pages 8338 - 8347
BATTAGLIA M; STABILINI A; RONCAROLO MG: "Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells", BLOOD, vol. 105, no. 12, 2005, pages 4743 - 48, XP003004231, DOI: doi:10.1182/blood-2004-10-3932
BATTAGLIA MANUELA ET AL: "Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells.", BLOOD 15 JUN 2005 LNKD- PUBMED:15746082, vol. 105, no. 12, 15 June 2005 (2005-06-15), pages 4743 - 4748, XP003004231, ISSN: 0006-4971 *
BILLIARD F; LITVINOVA E; SAADOUN D; DJELTI F; KLATZMANN D ET AL.: "Regulatory and effector T cell activation levels are prime determinants of in vivo immune regulation", J IMMUNOL, vol. 177, 2006, pages 2167 - 2174
BLUESTONE JA; THOMSON AW; SHEVACH EM; WEINER HL: "What does the future hold for cell-based tolerogenic therapy?", NAT REV IMMUNOL, vol. 7, 2007, pages 650 - 654, XP002521314, DOI: doi:10.1038/nri2137
CHEN W. W; JIN W; HARDEGEN N; LEI KJ; LI L; MARINOS N; MCGRADY G; WAHL SM.: "Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3", J. EXP. MED., vol. 198, no. 12, 2003, pages 1875 - 1886, XP003010702, DOI: doi:10.1084/jem.20030152
FUJII H; ARAKAWA A; KITOH A; MIYARA M; KATO M ET AL.: "2010) Perturbations of both nonregulatory and regulatory FOXP3+ T cells in patients with malignant melanoma", BR J DERMATOL
HOFFMANN P; BOELD TJ; EDER R; HUEHN J; FLOESS S ET AL.: "Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation", EUR J IMMUNOL, vol. 39, 2009, pages 1088 - 1097, XP002639445, DOI: doi:10.1002/EJI.200838904
HOFFMANN P; EDER R; BOELD TJ; DOSER K; PISESHKA B ET AL.: "Only the CD45RA+ subpopulation of CD4+CD25high T cells gives rise to homogeneous regulatory T-cell lines upon in vitro expansion", BLOOD, vol. 108, 2006, pages 4260 - 4267
LUCAS JULIE L ET AL: "Induction of Foxp3+ regulatory T cells with histone deacetylase inhibitors.", CELLULAR IMMUNOLOGY 2009 LNKD- PUBMED:19358983, vol. 257, no. 1-2, 2009, pages 97 - 104, XP026057557, ISSN: 1090-2163 *
MIYARA M; WING K; SAKAGUCHI S: "Therapeutic approaches to allergy and autoimmunity based on FoxP3+ regulatory T-cell activation and expansion", J ALLERGY CLIN IMMUNOL, vol. 123, 2009, pages 749 - 755, XP026445394, DOI: doi:10.1016/j.jaci.2009.03.001
MIYARA M; YOSHIOKA Y; KITOH A; SHIMA T; WING K ET AL.: "Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor", IMMUNITY, vol. 30, 2009, pages 899 - 911
MOZES E; SHARABI A: "A novel tolerogenic peptide, hCDRl, for the specific treatment of systemic lupus erythematosus", AUTOIMMUN REV, vol. 10, 2010, pages 22 - 26, XP027477251, DOI: doi:10.1016/j.autrev.2010.07.004
PETRA HOFFMAN, EUR J IMMUNOL., vol. 39, no. 4, April 2009 (2009-04-01), pages 1088 - 97
POLANSKY JULIA K ET AL: "DNA methylation controls Foxp3 gene expression.", EUROPEAN JOURNAL OF IMMUNOLOGY JUN 2008 LNKD- PUBMED:18493985, vol. 38, no. 6, June 2008 (2008-06-01), pages 1654 - 1663, XP002639446, ISSN: 0014-2980 *
SAKAGUCHI S; YAMAGUCHI T; NOMURA T; ONO M: "Regulatory T cells and immune tolerance", CELL, vol. 133, 2008, pages 775 - 787
UHLIG HH; COOMBES J; MOTTET C; IZCUE A; THOMPSON C ET AL.: "Characterization of Foxp3+CD4+CD25+ and IL-10-secreting CD4+CD25+ T cells during cure of colitis", J IMMUNOL, vol. 177, 2006, pages 5852 - 5860
ZHENG XX; SANCHEZ-FUEYO A; SHO M; DOMENIG C; SAYEGH MH; STROM TB: "Favorably tipping the balance between cytopathic and regulatory T cells to create transplantation tolerance", IMMUNITY, vol. 19, no. 4, 2003, pages 503 - 514, XP002517162, DOI: doi:10.1016/S1074-7613(03)00259-0
ZHOU X; BAILEY-BUCKTROUT SL; JEKER LT; PENARANDA C; MARTINEZ-LLORDELLA M ET AL.: "Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo", NAT IMMUNOL, vol. 10, 2009, pages 1000 - 1007, XP055048032, DOI: doi:10.1038/ni.1774

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US11072779B2 (en) 2015-12-17 2021-07-27 Gdanski Uniwersytet Medyczny Method for ex vivo expansion of regulatory T cells
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