WO2015097210A1 - Immunosuppressive foxa1-expressing t cells - Google Patents

Immunosuppressive foxa1-expressing t cells Download PDF

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WO2015097210A1
WO2015097210A1 PCT/EP2014/079128 EP2014079128W WO2015097210A1 WO 2015097210 A1 WO2015097210 A1 WO 2015097210A1 EP 2014079128 W EP2014079128 W EP 2014079128W WO 2015097210 A1 WO2015097210 A1 WO 2015097210A1
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cells
foxa1
ifn
immunosuppressive
bioactive agent
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Shohreh ISSAZADEH-NAVIKAS
Yawei Liu
Robert Carlsson
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Issazadeh-Navikas Shohreh
Yawei Liu
Robert Carlsson
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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
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or 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
    • A61K39/461—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
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]

Definitions

  • the present invention relates to the identification of FoxA1 as a lineage-specification factor that defines a novel population of immunosuppressive T cells (FoxA1 + T cells), and the use of FoxA1 and FoxA1 + T cells in the treatment of inflammatory disorders.
  • Immune function that preserves tolerance while retaining antimicrobial function is imperative for preventing chronic inflammation and autoimmunity. Regulation of inflammation is critical for the disease management in tissue-specific chronic inflammatory diseases. This is naturally achieved when inflammatory counteracting, functionally intact immune cells are generated.
  • the immune system in vertebrates is composed of the innate immune system and the adaptive immune system.
  • the cells of the adaptive immune system are special types of leukocytes, called lymphocytes.
  • B cells and T cells are the major types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow. B cells are involved in the humoral immune response, whereas T cells are involved in cell-mediated immune response. Subpopulations of T cells with distinct functions and expression patterns exist, including helper T cells, cytotoxic T cells and regulatory T cells.
  • Inflammatory abnormalities are a large group of disorders which underlie a vast variety of human diseases.
  • the immune system is often involved with inflammatory disorders, demonstrated in both allergic reactions and some myopathies, with many immune system disorders resulting in abnormal inflammation.
  • Inflammation is a localized defensive response of the body against pathogens and injury. Immune cells and soluble factors take part in this process to neutralize the injurious agent and initiate tissue repair to restore homeostasis. Loss of regulation of these mechanisms can prevent the final resolution of the inflammatory process, leading to chronic inflammation. Chronic inflammation is extremely relevant in today's modern medicine, as it contributes to the pathogenesis of the most important diseases of the industrialized societies including atherosclerosis, acute and chronic heart failure, cancer, diabetes, and obesity-associated diseases.
  • MS Multiple sclerosis
  • T cells are a highly heterogeneous population comprised of multiple cell subtypes which mediate both adaptive immunity and specific tolerance. Much has been learned about the molecular signals that induce T cell activation and differentiation, and several effective treatments for MS act by altering these activation and differentiation pathways.
  • FoxA1 is an epigenetic-modifying pioneer transcription factor important in embryonic development, stem cell differentiation, hepatocyte development, and cancer epigenetics. FoxA1 modulates postembryonic tissue functions, including the brain. FoxA1 is necessary for epithelial cells, and mediates lineage specification. FoxA1 overexpression has been associated with tumorigenesis at least in prostate cancer, esophageal and lung adenocarcinomas, invasive bladder cancer and breast cancer. However, no definite function has previously been reported for FoxA1 with respect to T cells.
  • FoxA1 is a lineage-specification factor that defines a subpopulation of immunosuppressive T cells (denoted FoxA1 + T cells herein, identical to T RFOX AI cells as used in the figure legends and the examples) and direct function of these novel T cells. FoxA1 thus has a specialized role in supporting the differentiation and the suppressive function of FoxA1 + T cells.
  • FoxA1 -overexpression in T-cells confers suppressive functions and a distinct FoxA1 + T cell phenotype.
  • the FoxA1 + T cells are in one embodiment negative for FoxP3, CTLA4, TGF- ⁇ , IL-10 and IL-35 commonly associated with regulatory T cells, T reg s, and in addition to expressing FoxA1 , in one embodiment they are CD4 hl , PD-L1 hl and CD47 + .
  • mice lacking Ifnb and Ifna/b-receptor genes develop chronic inflammatory and demyelinating experimental autoimmune encephalomyelitis EAE (Ifnb ' ' ' mice), a mouse model for MS.
  • the FoxA1 + T cells were identified in wild type mice but absent in Ifnb ' ' ' mice.
  • Ifnb ' ' ' mice adoptive transfer of stable FoxA1 + T cells was shown to inhibit CNS inflammation and EAE.
  • IFN- ⁇ can induce FoxA1 and FoxA1 - mediated PD-L1 , leading to generation of the FoxA1 + T cells, and as shown herein in patients with multiple sclerosis, response to IFN- ⁇ treatment was associated with expansion of the suppressive FoxA1 + T cells.
  • the present inventors have shown that FoxA1 + T cells can suppress T cell activation, inhibit proliferation and induce cell death in activated T cells (in vitro). Also, an inhibitory effect is shown not only in a MS model but also for inflammatory bowel disease (IBD), delayed type hypersensitivity (DTH) and glioblastoma multiforme.
  • IBD inflammatory bowel disease
  • DTH delayed type hypersensitivity
  • glioblastoma multiforme glioblastoma multiforme.
  • immunosuppressive FoxA1 + T cells inhibit T cell activation and reduce inflammation in various models thereof, they have the potential to treat several inflammatory diseases and disorders. It is an aspect of the present invention to provide immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment of FoxA1 (FoxA1 + T cells). Said immunosuppressive T cells may further be positive for ( + or hl staining) one or more of CD4, PD-L1 , PD-1 , CD47, CD69, CD25, CD45Rb and TCRa3, and negative for FoxP3.
  • a recombinant cell such as a recombinant T cell, comprising a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof.
  • a further aspect of the present invention is to provide a method of making the immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment of FoxA1 , said method comprising one or more steps of
  • the present invention also relates to a method of treating an inflammatory disease or disorder, said method comprising administering to an individual in need thereof an effective amount of a bioactive agent selected from the group consisting of
  • FoxA1 protein or a functional fragment or functional variant thereof
  • nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof;
  • immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof.
  • T cells from an individual having an inflammatory disease or disorder
  • An inflammatory disease or disorder to be treated according to the present invention is a disease or disorder having an inflammatory component, an inflammatory abnormality and/or an etiological origin in inflammatory processes. They comprise at least autoimmune disorders, multiple sclerosis, CNS disorders with an inflammatory component including glioblastoma multiforme, inflammatory diseases of the
  • DTH delayed type hypersensitivity
  • Yet another aspect is the provision of a method of distinguishing an IFN- ⁇ non- responding multiple sclerosis patient (MS-NR) from an IFN- ⁇ responding multiple sclerosis patient (MS-R), said method comprising one or more steps of
  • IFN- ⁇ treatment may be discontinued if said MS patient is characterized as an IFN- ⁇ non-responding multiple sclerosis.
  • T-cells were harvested from draining lymph nodes (LN) and spleen (SP) of WT mice after EAE induction.
  • T RFOX AI have a distinct transcription profile with FoxA1 + and suppress skin and CNS inflammation
  • T RFnX A is suppress activated T-cells in vitro.
  • Purified CD4 + T cells were labeled with CFSE and activated for 24 h (responder T cells/Res), then co-cultured with purified CGN-induced T RFoxA1s .
  • (j-k) Data are mean ⁇ SD from three independent experiments, *** P ⁇ 0.001 , unpaired two-tailed Student's i-test was used.
  • (l-m) TggnxA s suppress tissue inflammation in vivo.
  • IFN- ⁇ induces suppressive T RFo xAis that require IFN- ⁇ signaling.
  • MOG 35 - 55 -EAE is associated with generation of T RFOX AI cells in the CNS of C57BL/6 WT but not Ifnb ' ' ' mice. Ifnb ' ' ' mice treated with recombinant mlFN- ⁇ (5000 U/ml x 3 times) generated T RFOX AI S in the CNS.
  • FACS dot plots from three different experiments shows mlFN- ⁇ inducing T RFo xAis ( ⁇ + ⁇ 4 + ⁇ - ⁇ _1 hi T-cells).
  • e FACS histogram from three different experiments shows FoxA1 + mlFN ⁇ -induced T RFo xAis compared to nontreated cells
  • f Quantification of FoxA1 + CD4 + T cells upon mlFN ⁇ -stimulation. Data are mean ⁇ SD from three different experiments.
  • EncT cells are compiled to determine T RFOX AI S ' heatmap profile, (z) Heatmap of genes commonly regulated by iT RFo xAi and nT RFoX Ai vs. EncT-cells that are involved in commonly regulated pathways determined by GSEA. Data are from triplicates.
  • FoxA1 is essential for generation and function of suppressive T RFo xAis- FoxA1 silencing prevents IFN-p-induced PD-L1 hl expression
  • (a-b) Data are representative of three individual experiments.
  • (c) In vitro suppressive function of IFN-p-induced TgFnxA s is FoxA1 dependent. Percent suppression as "Res+CD4 + T (ctrl siRNA)+mlFN-p" or "Res+CD4 + T
  • NOG mice were i.v. injected with 1 x 10 6 FoxA1 siRNA KD- or CtrlsiRNA-CD4 + T-cells with or without mlFN- ⁇ (100 U/ml).
  • Control CD4 + T-cells (CtrlsiRNA) but not CD4 + T cells lacking FoxA1 (FoxAl siRNA KD) generated T RFOX AI S after in vivo mlFN- ⁇ . Bars, mean ⁇ SD, N 3 mice/group.
  • FoxA1 and PD-L1 are essential for suppressive function of TgFnxA s to prevent EAE (i- m).
  • Adoptive EAE was established in irradiated /fnt) "A -C57BL/6 mice by adoptive transfer of 5-10x10 6 splenocytes from MOG 35 - 55 immunized mice, day 0 and 14. Groups received either equal number of CFSE-labeled Ctrl activated T cells
  • mice receiving iT RFo xAis IFN- ⁇ treated for 48 hours, with ctrl siRNA
  • mice receiving RF OX A-I S had significantly less EAE compared to its control group.
  • Ectopic FoxA1 generates suppressive T RFOX AI S that induce T-cell death via PD-L1 and caspase signaling.
  • TRF OX A-I S were co-cultured with responder T-cells for 24 h with PD-L1 antibodies, PD-1 (5 ⁇ g ml), B7.1 , B7.2 (10 ⁇ g ml), or an isotype control (10 ⁇ g ml). Bars, percent 7AAD + cells gated on responder T-cells (CFSE). Data are mean ⁇ SD of three independent experiments, *** P ⁇ 0.001 by Student's unpaired i-tests.
  • Human TRF OX AI S induce suppression and cell-death of activated T cells through PD-1 -PD-L1 .
  • CFSE-labeled purified human CD4 + T-cells from healthy donors were transfected with control siRNA (UNC) or PD-1 siRNA (PD-1 KD) for 3 days, then activated with plate-coated anti-CD3 for 24 h (responder T-cells/Res).
  • Responder T-cells were co-cultured with purified hlFN-p-induced T RFo xAis from the same donor for 24 h.
  • FoxA1 directly binds labeled PD-L1-B, but not PD-L1-A.
  • EMSA on nuclear extracts from 3T3-FoxA1 cells with labeled PD-L1-A and PD-L1-B probes. Nuclear extract nonspecifically shifted PD- L1-A probe, different from the positive control, which was not supershifted with a FoxA1 -specific antibody.
  • PD-L1-B shifted similar to the positive control, and
  • T RFo xAis in PBMCs showing gating strategy. FACS of T RFo xAis on gated live cells, excluding duplet cells, then gated on surface expression of
  • TCRap + CD47 + CD4 hi PD-L1 hi T-cells Percent CD4 + CD47 + PD-L1 hi T RFoxA is by FACS (b) at baseline, (c) after 24 months IFN- ⁇ . Dots are Percent (with mean) of T RFoxA1s .
  • N 10, 9 and 16 per group, ⁇ P ⁇ 0.05, non-parametric Mann-Whitney test, (d) Percent R Fo xAis before and after IFN- ⁇ treatment in RRMS-NR and RRMS-R patients, sampling blood from non-treated healthy controls at indicated time points served as control.
  • N 9 and 16 per group respectively, ⁇ P ⁇ 0.05, non-parametric Mann-Whitney test, ** P ⁇ 0.01 Student's paired i-test to compare IFN- ⁇ treatment effect in groups at baseline versus 24 months of treatment.
  • Representative FACS micrographs show gating strategy for T RFoxA i purification
  • Graphs are mean ⁇ SD, representative of 3 individual patients. ** P ⁇ 0.01 , *** P ⁇ 0.001 by Student's unpaired i-test.
  • mice developed different rate number of relapses, with different duration of remission in between.
  • the mean score per group from these five selected individuals with EAE is also shown for the respective groups to demonstrate that due to variability in disease development, the mean score at each time point becomes relatively low, although all five clearly suffer from severe EAE.
  • WT heterozygous mice
  • Tregs are not defective in Ifnb-/- mice capable of suppressing EAE.
  • N 3-5 mice per group, (b) Percent of CD4 + Foxp3 + Treg cells in the CNS of C57BL/B10.RIII and C57BL/6 mice during peak of diseases after induction of EAE with MBP 8 9-ioi and MOG35-55 respectively, (c) FACS dot plots show gating strategy for CD4 + CD25 + T cells purification, which are also mainly Foxp3 + T cells, (d) CFSE-labeled purified murine CD4 + T cells were activated with plate-coated anti-CD3 antibody for 24 hours that served as responder T cells, then co-cultured with purified CD4 + CD25 + T cells from WT and Ifnb ' ' ' mice for an additional 24 hours.
  • Transcription Factor profile Plot of transcription factor genes differentially regulated in nT RF oxAi vs. their progenitor EncT-cells. Y-axis is log 2 of fold change. The only relevant transcription factors known to regulate T reg signature (Fu et al. 2012 Nat Imm) were found to be downregulated in ⁇ ⁇ ⁇ indicated in red. (b) Heatmap of ⁇ ⁇ ⁇ gene profile. Genes on the y-axis correspond to nT RFoX Ai gene profile, which is based on differentially regulated genes between nT RFoX Ai cells and EncT-cells.
  • TRFoxAls have distinct gene signature and surface markers compared to Exhausted T cells, (a) Work flow used to analyze gene expression profiles of the available data-set for exhausted T cells (ExhT-cells) (Wherry et. al, Immunity 2007, 27, 670-84) to compare with T RFoxA i s - (b) Venn diagram of genes differentially regulated in T RFoxA i and ExhT cells.
  • T RFoxA i s "signature” consists of 1498 genes (21 12 probe sets, non-assigned probes and probes matching to multiple genes included), and ExhCD8 + T cell "signature” consists of 455 genes (504 probes).
  • TRF OX AI cells were purified on the basis of their surface expression of CD4 PD- L1 hi by FACSAria from OVA-activated OT-II cells co-cultured with CGNs.
  • Purified CD4 + T cells were labeled with CFSE and activated with anti-CD3/anti-CD28 for 24 hours (responder T cells), before co-culturing with purified ⁇ ⁇ ⁇ cells for an additional 24 hours.
  • recombinant IL-2 (2.4 pg/ml) was added to the co-cultures.
  • FACS histograms show proliferative (CFSE) or dead (7AAD) responder T cells alone and after co-culture with T RFOX AI cells (with or without IL-2).
  • IL-2 addition rescues T RFoxA i-inhibition of cell proliferation, while there is no effect on their capacity to induce cell death of activated responder T cells. Representative FACS data are from two independent experiments.
  • Purified CD4 + T cells from healthy blood donors were treated in vitro with or without recombinant hlFN- ⁇ (1000U/ml) for 3 days, (a) FACS dot plots show no differences in the FoxP3 expression with or without hlFN- ⁇ treatment, (b) FoxP3 expression do not differ in purified iT RFo xAis (R1 -gated) compared to non-T RFo xAis (R2-gated cells), (c) Histograms of IL-35 expression are shown in gated iT RFo xAis in comparison with non-T RFoxA is. (d) FACS histogram shows PD-1 expression after different treatments. Representative FACS data are showing from three independent experiments. Figure 14.
  • iT RFo xAi cells suppress effector function ofAPCs by inhibiting their pro-inflammatory cytokines production.
  • MACS purified APCs from healthy blood donors were treated with LPS for 2 days prior to co-culture with iT RFoxA i cells. After 24 hours of co-culture of APCs with iT RFoxA i cells, intracellular ⁇ stained cytokines were gated on APCs (HLA-DR + TCR " ), supernatant were analyzed for different cytokines using ELISA.
  • APCs HLA-DR + TCR "
  • Figure 15 Rescue of TRFoxAI phenotype by ectopic expression of FoxA1 confirms specificity of siRNAs targeting, (a) Scheme for gene targeting and rescue strategy depicts pcDNA3.1 foxal , foxal siRNAs and amplified regions by indicated PCR primers, (b) Murine purified CD4+ T cells were transfected with siRNAs. Efficiency of FoxA 1 KD was confirmed by qPCR after 72 h of transfection, using 4 different siRNAs and a smartpool foxal siRNA (4 siRNA pooled), (c) Purified CD4+ T cells were transfected by pcD N A3.1 foxal . pcDNA3.1 vector was used as a control.
  • pcDNA3.1foxa1 is rescuing FoxA1 determined by positive ORF primer readout but not 3'UTR primers. Data are meansiSD from duplicates.
  • iT RFo xA suppress responder T cells via PD-L1-PD-1 mediated inhibition ofpAKT, pP38 and upregulation of cleaved caspase3.
  • Purified CD4 + T cells from healthy blood donors were treated in vitro with hlFN- ⁇ 1000U/ml for 2 days and ITRF OX AI cells were purified.
  • CFSE-labeled purified CD4 + T cells from the same healthy blood donors were transfected either with control siRNA (UNC) or PD-1 siRNA (PD-1 KD) for 3 days, then activated with plate-coated anti-CD3 antibody for 24 h (responder T cells).
  • Control siRNA and PD-1 siRNA silenced responder T cells were co-cultured with purified iT RFo xAi for additional 24 h.
  • a representative CSFE FACS histogram shows that iT RFo xAi inhibits proliferation of (a) phosphorylated AKT and (b) cleaved caspase 3.
  • FIG. 17 Changes on EDSS scores over time in RRMS patients treated with IFN- ⁇ .
  • N 9-15 per group.
  • One-way ANOVA with repeated measures € €P ⁇ 0.01 , € € €P ⁇ 0.001
  • post-test Dunnett's Multiple Comparison * P ⁇ 0.05, ** P ⁇ 0.01
  • linear trend tests ###P ⁇ 0.001
  • the linear trend test revealed a P ⁇ 0.001 with positive slope (increasing EDSS) in the RRMS-NR and P ⁇ 0.001 with a negative slope (decreasing EDSS) in the RRMS-R group.
  • IFN- ⁇ induced FoxA1 + Treg cells kill glioma tumor cells.
  • Purified CD4 + T cells from C57BL/6 mice spleens were treated with murine IFN- ⁇ (100U/ml) for 48 hours.
  • IFN- ⁇ treated CD4 + T cells were sorted by CD4 + PD-L1 high gated cells, served as iFoxAI +T cells.
  • GL261 mouse Glioma tumor cell line
  • FoxA1 + T cells were co-cultured with GL261 cells at 10:1 (iFoxAUreg : GL261 ) ratio. After 24 h, GL261 cells (CFSE + ) cell death (7AAD + ) was analysed by FACS. Results indicate that FoxA1 + T cells are capable of killing cancer cells i.e. 37.2% dead tumor cells compared to original 7.9%.
  • FoxA1 + Treg cells are significantly lacking in the gut environment (i.e. colon and small intestine) of ifnb ⁇ ' ⁇ mice associated with gut inflammation.
  • Lymphocytes were purified from thymus, spleens, colons, mesenteric lymph nodes (MLN) and small intestines (BR10III wild type/WT mice and ifnb '1' mice). Single cells were prepared and stained with antibodies against CD4-APC and PD-L1 -PE. Next stained lymphocytes driven from different tissues were analyzed using FACS.
  • a “treatment effect” or “therapeutic effect” is manifested if there is a change in the condition being treated, as measured by the criteria constituting the definition of the terms “treating” and “treatment.”
  • There is a “change” in the condition being treated if there is at least 5% improvement, preferably 10% improvement, more preferably at least 25%, even more preferably at least 50%, such as at least 75%, and most preferably at least 100% improvement.
  • the change can be based on improvements in the severity of the treated condition in an individual, or on a difference in the frequency of improved conditions in populations of individuals with and without treatment with the bioactive agent, or with the bioactive agent in combination with a pharmaceutical composition of the present invention.
  • “Pharmacologically effective amount” "pharmaceutically effective amount” or
  • physiologically effective amount of a bioactive agent is the amount of an active agent present in a pharmaceutical composition as described herein that is needed to provide a desired level of active agent in the bloodstream or at the site of action in an individual (e.g. the lungs, the gastric system, the colorectal system, prostate, etc.) to be treated to give an anticipated physiological response when such composition is administered.
  • the precise amount will depend upon numerous factors, e.g., the active agent, the activity of the composition, the delivery device employed, the physical characteristics of the composition, intended patient use (i.e. the number of doses administered per day), patient considerations, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein.
  • an “effective amount” of a bioactive agent can be administered in one administration, or through multiple administrations of an amount that total an effective amount. It can be determined using standard clinical procedures for determining appropriate amounts and timing of administration. It is understood that the "effective amount" can be the result of empirical and/or
  • polypeptide is a polymer of amino acid residues preferably joined exclusively by peptide bonds, whether produced naturally or synthetically.
  • polypeptide as used herein covers proteins, peptides and polypeptides, wherein said proteins, peptides or polypeptides may or may not have been post-translationally modified.
  • a peptide is usually shorter in length than a protein.
  • isolated polypeptide is a polypeptide separated and/or recovered from a component of their natural, typically cellular, environment, that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature.
  • a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure.
  • amino acid residue can be a natural or non-natural amino acid residue linked peptide bonds or bonds different from peptide bonds.
  • the amino acid residues can be in D-configuration or L-configuration.
  • An amino acid residue comprises an amino terminal part (NH 2 ) and a carboxy terminal part (COOH) separated by a central part comprising a carbon atom, or a chain of carbon atoms, at least one of which comprises at least one side chain or functional group.
  • NH 2 refers to the amino group present at the amino terminal end of an amino acid or peptide
  • COOH refers to the carboxy group present at the carboxy terminal end of an amino acid or peptide.
  • the generic term amino acid comprises both natural and non-natural amino acids. Natural amino acids are Y, G, F, M, A, S, I, L, T, V, P, K, H, Q, E, W, R, D, N and C. Non-natural amino acids are those not listed here.
  • non-natural amino acid residues include, but are not limited to, modified amino acid residues, L-amino acid residues, and stereoisomers of D-amino acid residues. Where the L or D form (optical isomers) has not been specified it is to be understood that the amino acid in question has the natural L form.
  • an “equivalent amino acid residue” refers to an amino acid residue capable of replacing another amino acid residue in a polypeptide without substantially altering the structure and/or functionality of the polypeptide. Equivalent amino acids thus have similar properties such as bulkiness of the side-chain, side chain polarity (polar or non-polar), hydrophobicity (hydrophobic or hydrophilic), pH (acidic, neutral or basic) and side chain organization of carbon molecules (aromatic/aliphatic). As such, “equivalent amino acid residues” can be regarded as "conservative amino acid substitutions”.
  • the classification of equivalent amino acids refers in one embodiment to the following classes: 1 ) HRK, 2) DENQ, 3) C, 4) STPAG, 5) MILV and 6) FYW.
  • one amino acid may be substituted for another, in one embodiment, within the groups of amino acids indicated herein below: i) Amino acids having polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys,) ii) Amino acids having non-polar side chains (Gly, Ala, Val, Leu, lie, Phe, Trp, Pro, and Met)
  • Bioactive agent i. e., biologically active substance/agent
  • a FoxA1 peptide according to the present invention, compounds or compositions comprising these and nucleic acid constructs encoding said peptides, as well as the immunosuppressive T cells expressing FoxA1 (FoxA1 + T cells).
  • this term further includes any physiologically or pharmacologically active substance that produces a localized or systemic effect in an individual.
  • bioactive agents include, but are not limited to, agents comprising or consisting of an
  • oligosaccharide agents comprising or consisting of a polysaccharide, agents comprising or consisting of an optionally glycosylated peptide, agents comprising or consisting of an optionally glycosylated polypeptide, agents comprising or consisting of a nucleic acid, agents comprising or consisting of an oligonucleotide, agents comprising or consisting of a polynucleotide, agents comprising or consisting of a lipid, agents comprising or consisting of a fatty acid, agents comprising or consisting of a fatty acid ester and agents comprising or consisting of secondary metabolites. It may be used either prophylactically, therapeutically, in connection with treatment of an individual, such as a human or any other animal.
  • Immune function that preserves tolerance to self, while retaining antimicrobial function, is imperative for preventing chronic inflammation and autoimmunity. Regulation of inflammation is critical for the disease management in tissue-specific chronic inflammatory diseases, including multiple sclerosis (MS). This is naturally achieved when inflammatory counteracting, functionally intact immune cells are generated.
  • MS multiple sclerosis
  • T reg regulatory T-cell
  • EAE encephalomyelitis
  • MS multiple sclerosis
  • CNS central nervous system
  • Mice lacking Ifnb and Ifna/b-receptor genes develop chronic inflammatory and demyelinating EAE (Ifnb ' ' ' mice).
  • the present inventors have found no defects associated with Foxp3 + T regs in the inflamed CNS of Ifnb ' ' ' mice; however, the inventors did identify a novel population of T-cells in wildtype mice that was absent in Ifnb ' ' ' mice.
  • These newly identified immunosuppressive T cells denoted herein FoxA1 + T-cells (or TRF OX AI in the figure legends and the examples), are largely defined by expression of the transcription factor FoxA1.
  • FoxA1 + T-cells suppress T cell activation, inhibit proliferation and induce cell death in activated T cells in vitro, which effect translates in vivo into an inhibition of CNS inflammation and RR-EAE in Ifnb ' ' ' mice.
  • the immunosuppressive properties of FoxA1 in T cells makes FoxA1 and the immunosuppressive FoxA1 + T-cells potentially useful in the treatment of a range of disorders where immunosuppression is desired, such as diseases having an inflammatory component including inflammatory disorders.
  • the immune system
  • the immune system is a system of biological structures and processes within an organism that protects against disease. To function properly, an immune system must detect a wide variety of agents, from bacteria and viruses to parasitic worms, and distinguish them from the organism's own healthy tissue.
  • the immune system protects organisms from infection with layered defenses of increasing specificity.
  • physical barriers prevent pathogens such as bacteria and viruses from entering the organism. If a pathogen breaches these barriers, the innate immune system provides an immediate, but non-specific response. If pathogens successfully evade the innate response, vertebrates possess a second layer of protection, the adaptive immune system, which is activated by the innate response.
  • the immune system adapts its response during an infection to improve its recognition of the pathogen. This improved response is then retained after the pathogen has been eliminated, in the form of an immunological memory, and allows the adaptive immune system to mount faster and stronger attacks each time this pathogen is encountered.
  • the immune system is thus tightly controlled system, with many inhibitory and stimulatory effectors coordinating the final outcome.
  • the innate immune system The innate immune system
  • the innate immune system comprises inflammation, the complement system, cellular barriers and natural killer cells (or NK cells). Inflammation is one of the first responses of the immune system to infection. The symptoms of inflammation are redness, swelling, heat, and pain, which are caused by increased blood flow into tissue. Inflammation is produced by eicosanoids and cytokines, which are released by injured or infected cells.
  • Eicosanoids include prostaglandins that produce fever and the dilation of blood vessels associated with inflammation, and leukotrienes that attract certain white blood cells (leukocytes).
  • cytokines include interleukins that are responsible for communication between white blood cells; chemokines that promote chemotaxis; and interferons that have anti-viral effects, such as shutting down protein synthesis in the host cell. Growth factors and cytotoxic factors may also be released. These cytokines and other chemicals recruit immune cells to the site of infection and promote healing of any damaged tissue following the removal of pathogens.
  • the complement system is a biochemical cascade that attacks the surfaces of foreign cells. It contains over 20 different proteins and is named for its ability to "complement” the killing of pathogens by antibodies. Complement is the major humoral component of the innate immune response. In humans, this response is activated by complement binding to antibodies that have attached to these microbes or the binding of complement proteins to carbohydrates on the surfaces of microbes. This recognition signal triggers a rapid killing response and signal amplification.
  • Leukocytes white blood cells
  • the innate leukocytes include the phagocytes (macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. These cells identify and eliminate pathogens, either by attacking larger pathogens through contact or by engulfing and then killing microorganisms. Innate cells are also important mediators in the activation of the adaptive immune system.
  • the adaptive immune system evolved in early vertebrates and allows for a stronger immune response as well as immunological memory, where each pathogen is
  • the adaptive immune response is antigen- specific and requires the recognition of specific "non-self antigens during a process called antigen presentation. Antigen specificity allows for the generation of responses that are tailored to specific pathogens or pathogen-infected cells. The ability to mount these tailored responses is maintained in the body by "memory cells”. Should a pathogen infect the body more than once, these specific memory cells are used to quickly eliminate it.
  • the cells of the adaptive immune system are special types of leukocytes, called lymphocytes. B cells and T cells are the major types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow.
  • T cells are involved in the humoral immune response, whereas T cells are involved in cell-mediated immune response. Both B cells and T cells carry receptor molecules that recognize specific targets. T cells recognize a "non-self target, such as a pathogen, only after antigens (small fragments of the pathogen) have been processed and presented in combination with a major histocompatibility complex (MHC) molecule. T-cells can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T- cell receptor (TCR) on the cell surface. There are two major subtypes of T cells: the killer T cell and the helper T cell.
  • MHC major histocompatibility complex
  • Killer T cells only recognize antigens coupled to Class I MHC molecules, while helper T cells only recognize antigens coupled to Class II MHC molecules. These two mechanisms of antigen presentation reflect the different roles of the two types of T cell.
  • a third, minor subtype are the ⁇ T cells that recognize intact antigens that are not bound to MHC receptors.
  • the B cell antigen-specific receptor is an antibody molecule on the B cell surface, and recognizes whole pathogens without any need for antigen processing. Each lineage of B cell expresses a different antibody, so the complete set of B cell antigen receptors represent all the antibodies that the body can manufacture.
  • Killer T cells (CD8+) (or cytotoxic T cells, T c cells, CTL) are activated when their T cell receptor (TCR) binds to this specific antigen in a complex with the MHC Class I receptor of another cell. Recognition of this MHC:antigen complex is aided by a co- receptor on the T cell, called CD8. The T cell then travels throughout the body in search of cells where the MHC I receptors bear this antigen. When an activated T cell contacts such cells, it releases cytotoxins. T cell killing of host cells is particularly important in preventing the replication of viruses. T cell activation is tightly controlled and generally requires a very strong MHC/antigen activation signal, or additional activation signals provided by helper T cells
  • Helper T cells regulate both the innate and adaptive immune responses and help determine which immune responses the body makes to a particular pathogen. These cells have no cytotoxic activity and do not kill infected cells or clear pathogens directly. They instead control the immune response by directing other cells to perform these tasks.
  • Helper T cells express T cell receptors (TCR) that recognize antigen bound to Class II MHC molecules.
  • TCR T cell receptors
  • the MHC:antigen complex is also recognized by the helper cell's CD4 co-receptor, which recruits molecules inside the T cell that are responsible for the T cell's activation. The activation of a resting helper T cell causes it to release cytokines that influence the activity of many cell types.
  • Cytokine signals produced by helper T cells enhance the microbicidal function of macrophages and the activity of killer T cells
  • Tregs are a subpopulation of T cells (CD4+) which modulate the immune system, maintain tolerance to self-antigens, and abrogate autoimmune disease.
  • Mouse models have suggested that modulation of Tregs can treat
  • T regulatory cells are a component of the immune system that suppresses immune responses of other cells in order to prevent excessive reactions. Regulatory T cells come in many forms with the most well-understood being those that express CD4, CD25, and Foxp3. CD4 + Foxp3 + regulatory T cells have been called “naturally- occurring" regulatory T cells (nTreg). An additional regulatory T cell subset, denoted induced regulatory T cells (iTreg) (also CD4 + CD25 + Foxp3 + ), are needed for tolerance and suppression. Regulatory T cells are defined by expression of the forkhead family transcription factor FOXP3 (forkhead box p3). Thus, FOXP3 can be used as a good marker for CD4 + CD25 + T cells, although also expressed in CD4 + CD25 " T cells and conventional T-cells.
  • FOXP3 forkhead box p3
  • Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with "memory" against past infections.
  • Memory T cells comprise three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (T E M cells and T E MRA cells).
  • TCM cells central memory T cells
  • T E M cells T E M cells
  • T E MRA cells T E M cells
  • Memory cells may be either CD4 + or CD8 + .
  • Memory T cells typically express the cell surface protein CD45RO.
  • Natural killer T cells bridge the adaptive immune system with the innate immune system.
  • NKT cells recognize glycolipid antigen presented by a molecule called CD1 d. Once activated, these cells can perform functions ascribed to both T h and T c cells (i.e., cytokine production and release of cytolytic/cell killing molecules). They are also able to recognize and eliminate some tumor cells and cells infected with herpes viruses.
  • FoxA1 or forkhead box A1 is a transcription factor of 472 amino acids (aa) in length (UniProt accession number P55317 (FOXA1_HUMAN); NCBI
  • NM_004496 An often used synonym is hepatocyte nuclear factor 3, alpha (HNF-3- alpha).
  • the protein sequence of FoxA1 is (SEQ ID NO:1 ):
  • the DNA binding region (Fork-head) has been identified as aa 169-206, namely the
  • the FOXA1 gene comprises a DNA sequence of 3396 bp (SEQ ID NO:3):
  • FoxA1 is a transcription factor involved in embryonic development, establishment of tissue-specific gene expression and regulation of gene expression in differentiated tissues. Originally described as a transcription activator for a number of liver genes such as AFP, albumin, tyrosine aminotransferase, PEPCK, etc. Is thought to act as a 'pioneer' factor opening the compacted chromatin for other proteins through interactions with nucleosomal core histones and thereby replacing linker histones at target enhancer and/or promoter sites. Also proposed to play a role in translating the epigenetic signatures into cell type-specific enhancer-driven transcriptional programs. FoxA1 is involved in the development of multiple endoderm-derived organ systems such as liver, pancreas, lung and prostate.
  • FoxA1 modulates the transcriptional activity of nuclear hormone receptors (oestrogen receptor-a (ER) and androgen receptor (AR)) and is involved in ESR1 -mediated transcription in breast cancer cells. It is also involved in regulation of apoptosis by inhibiting the expression of BCL2, and in cell cycle regulation by activating expression of CDKN1 B, alone or in conjunction with BRCA1.
  • nuclear hormone receptors oestrogen receptor-a (ER) and androgen receptor (AR)
  • FoxA1 expression has been positively associated with tumorigenesis / tumour progression at least in prostate cancer, esophageal and lung adenocarcinomas, invasive bladder cancer and breast cancer.
  • FoxA1 or a functional variant or functional fragment of FoxA1 ; either as comprised or expressed in T cells, as a protein or as DNA.
  • DNA When in the form of DNA it is preferably comprised in a nucleic acid construct, as defined herein below.
  • FoxA1 as defined herein above may be a functional variant or functional fragment of said FoxA1.
  • Variants and fragment according to the present invention are meant to be the functional equivalents of FoxA1 , i.e. retaining the same biological activity or capabilities as the sequence from which it is derived.
  • a functional variant or functional fragment of FoxA1 is able to render T cells immunosuppressive, such as being able to suppress activated T cells.
  • said FoxA1 is in the form of a protein (SEQ ID NO:1 ), or a functional variant or functional fragment thereof (of SEQ ID NO:1 ).
  • said FoxA1 is in the form of DNA (SEQ ID NO:3), or a functional variant or functional fragment thereof (of SEQ ID NO:3). It is understood that a functional variant or functional fragment of FoxA1 is meant to include also a functional variant of a fragment of FoxA1 (i.e. a fragment also being a variant).
  • a functional variant or functional fragment of FoxA1 is able to suppress production of one or more pro-inflammatory cytokines, such as pro-inflammatory cytokines selected from the group consisting of IL- 12 and IL-17 in APCs; ii) do not induce production of IL-10 in APCs, iii) induce expression in T cells of one or more of CD4, PD-L1 , PD-1 , CD47, CD69, CD25, CD45Rb and TCRa3 + ; iv) is able to suppress activated T cells, such as inhibit proliferation and/or induce cell death of activated T cells; v) is able to down-regulate c- Fos expression in T cells; and/or vi) is able to reduce nuclear pc-Fos in T cells.
  • pro-inflammatory cytokines such as pro-inflammatory cytokines selected from the group consisting of IL- 12 and IL-17 in APCs
  • do not induce production of IL-10 in APCs iii
  • a functional variant or functional fragment of FoxA1 according to the present invention has between 60-99.9% sequence identity, such as between 60- 70%, for example 70-75%, such as 75-80%, for example 80-85%, such as 85-90%, for example 90-95%, such as 95-96%, for example 96-97%, such as 97-98%, for example 98-99%, such as 99-99.9% sequence identity to SEQ ID NO:1 (protein) or SEQ ID NO:3 (DNA).
  • a functional variant or functional fragment of FoxA1 has at least 60% sequence identity, such as at least 65% sequence identity, for example at least 70% sequence identity, such as at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity to SEQ ID NO:1
  • a functional variant or functional fragment of FoxA1 comprises from 10 to 471 consecutive amino acids of SEQ ID NO: 1 , such as from 10-25, 25-50, 50-75, 75-100, 100-125, 125-150, 150-175, 175-200, 200-250, 250-300, 300-350, 350-400, 400-450 or from 450-471 consecutive amino acids of SEQ ID NO:1.
  • a functional variant or functional fragment of FoxA1 comprises the FoxA1 Fork-head DNA-binding region (aa 169- 260) (SEQ ID NO:2).
  • a functional variant or functional fragment of FoxA1 comprises the naturally occurring variants of FoxA1 , including but not limited to VAR_015183, VAR_013457, VAR_055835, VAR_015184,
  • a functional variant or functional fragment of FoxA1 is a sequence wherein any amino acid specified in SEQ ID NO:1 is changed to a different amino acid, provided that no more than 20 of the amino acid residues in the sequence are so changed, such as provided than no more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 of the amino acid residues in the sequence are so changed.
  • the 1 -20 amino acids so changed are changed to equivalent amino acids.
  • a Clustal W alignment of the FoxA1 protein sequence between different species can be used to predict which amino acid residues can be substituted without substantially affecting the biological activity of the protein; and which are deemed conservative or semi-conservative.
  • a consensus sequence for mature FoxA1 protein can be derived, which consensus sequences may be regarded as conserved domains.
  • the conserved and/or semi- conserved amino acid residues must be located at corresponding positions in a variant.
  • the FoxA1 protein and its use according to the present invention may be further modified or optimized in order to obtain favorable properties with respect to e.g.
  • the FoxA1 protein as defined herein may be linked covalently or non-covalently with a carrier molecule, with a protective molecule, with a vehicle or delivery molecule or with a localization molecule known to the skilled person.
  • PTDs Protein transduction domains
  • CPP cell permeable proteins
  • MTS membrane translocating sequences
  • PEGylation defines the modification of a protein by the linking of one or more polyethylene glycol (PEG) chains.
  • PEG polyethylene glycol
  • This polymer is non-toxic, non-immunogenic, non- antigenic, highly soluble in water and FDA approved.
  • the PEG-drug conjugates have several advantages: a prolonged residence in body, a decreased degradation by metabolic enzymes and a reduction or elimination of protein immunogenicity.
  • the FoxA1 protein of the present invention having sequence SEQ ID NO:1 , or a functional variant or functional fragment thereof, further comprises one or more protein transduction domains (PTDs).
  • PTDs protein transduction domains
  • the FoxA1 protein of the present invention having sequence SEQ ID NO:1 , or a functional variant or functional fragment thereof, further comprises one or more polyethylene glycol (PEG) chains
  • TRF OX AI immunosuppressive FoxA1 + T cells, or simply FoxA1 + T cells. These cells are referred to as "TRF OX AI" in the figure legends and examples.
  • an isolated population of immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment of FoxA1 .
  • an in vitro cell culture comprising the immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment of FoxA1 .
  • said in vitro cell culture further comprises a cell culture medium.
  • said immunosuppressive FoxA1 + T cells are derived from a population of T cells selected from the group consisting of Regulatory T cells (Tregs), Helper T cells (T h cells), Cytotoxic T cells (T c cells, TCL), Natural killer T cells (NKT) and Memory T cells.
  • said immunosuppressive FoxA1 + T cells further comprise or express, or are positive for, one or more of the group consisting of CD4, PD-L1 , PD-1 , CD47, CD69, CD25, CD45Rb and TCRa3.
  • said immunosuppressive T cells are FoxA1 +, CD4+, PD-L1 +, PD- 1 +, CD47+, CD69+, CD25+, CD45Rb+ and/or TCRa3+.
  • '-' e.g. FoxP3-
  • '+' e.g. CD4+ and FoxA1 +
  • expression is detectable, and/or have higher intensity of staining than '-' cells and possibly higher than intermediate staining populations.
  • 'hi' cells e.g. PD-L1 hl
  • '+' cells have higher expression and/or intensity of staining than the '+' cells (e.g. PD-L1 + ) cells.
  • determination of expression or intensity of staining is detected by the FACS technique, wherein the intensity of staining correlates to degree expression.
  • determination of expression is determined by gene expression analysis (gene array, western blot, ELISA, and any technique known to the skilled person).
  • said immunosuppressive T cells do not comprise or express, or are negative for, one or more of FoxP3, TGF- ⁇ , IFN-gamma, TNF-alpha, CTLA-4, FAS, FASL, CD8, IL-17, IL-4, IL-10 and IL-13. In one embodiment, said immunosuppressive T cells do not comprise or express, or are negative for, FoxP3 (are FoxP3 " ).
  • 'do not comprise or express' is meant to comprise no or essentially no expression or content, or undetectable expression or content, such as identified by (essentially) no staining by FACS. In this respect they may be classified as 'negative' (i.e. FoxP3 negative). '+' and/or 'hi' means the cells may be classified as 'positive' (e.g. CD4 + or CD4 hl ). In one embodiment 'do not comprise or express' or being classified as 'negative' for the marker, means that the cells express less than those that are classified as 'positive' ( + or hl ).
  • said immunosuppressive T cells further comprise or express CD4, or are CD4 positive. In one embodiment, said immunosuppressive T cells are CD4 + and/or CD4 hi (CD4 +/hi ).
  • said immunosuppressive T cells further comprise or express PD- L1 , or are PD-L1 positive. In one embodiment, said immunosuppressive T cells are PD- L1 + and/or PD-L1 hi . In one embodiment, said immunosuppressive T cells further comprise or express both CD4 and PD-L1 , or are CD4 and PD-L1 positive (i.e. CD4 + and PD-L1 + ; CD4 + and PD- L 1 hi. CD4 hi and P D . L1 + . or CD4 hi and PD-L1 hi ).
  • said immunosuppressive T cells further comprise or express one or more of CD4, PD-L1 , PD-1 , CD47 and CD69; or are positive for ( + or hi ) one or more of CD4, PD-L1 , PD-1 , CD47 and CD69.
  • the immunosuppressive T cells according to the present invention are FoxA1 + CD4 + PD-L1 + T cells, FoxA1 + CD4 hi PD-L1 hi T cells or FoxA1 + CD4 + PD-L1 hi T cells.
  • the immunosuppressive T cells according to the present invention are FoxA1 + CD4 + PD-L1 + FoxP3 " T cells.
  • the immunosuppressive T cells according to the present invention are FoxA1 + CD4 hi PD-L1 hi FoxP3 " T cells. In one embodiment, the immunosuppressive T cells according to the present invention are FoxA1 + CD4 + PD-L1 hi FoxP3 " T cells.
  • the immunosuppressive T cells according to the present invention are FoxA1 + CD4 +/hi PD-L1 hi TCRa3 + T cells.
  • the immunosuppressive T cells according to the present invention are FoxA1 + CD4 +/hi PD-L1 hi TCRa3 + FoxP3 " T cells.
  • the immunosuppressive T cells according to the present invention are FoxA1 + CD4 +/hi PD-L1 hi CD47 + T-cells.
  • the immunosuppressive T cells according to the present invention are FoxA1 + CD4 +/hi PD-L1 hi CD47 + FoxP3 " T-cells.
  • the immunosuppressive T cells according to the present invention are FoxA1 + CD4 +/hi PD-L1 hi CD47 + CD69 + T-cells.
  • the immunosuppressive T cells according to the present invention are FoxA1 + CD4 +/hi PD-L1 hi CD47 + CD69 + FoxP3 " T-cells.
  • the immunosuppressive T cells according to the present invention are FoxA1 + CD4 +/hi PD-L1 hi CD47 + CD69 + TCRa3 + T-cells.
  • the immunosuppressive T cells according to the present invention are FoxA1 + CD4 +/hi PD-L1 hi CD47 + CD69 + TCRa3 + FoxP3 " T-cells.
  • said immunosuppressive T cells have reduced expression of one or more of the group consisting of TNF-alpha, IL-1 a (interleukin l alpha), I L-1 b, IL-4, IL- 5, IL-7r (IL-7 receptor), IL-10, IL-13, IL-16, IL-17rd (receptor D), IL-17, IL-18 (receptor 1 ), Cerebral endothelial cell adhesion molecule 1 (Cercaml ), Procollagen-lysine, 2- oxoglutarate 5-dioxygenase 3 (Plod3), Procollagen-proline, 2-oxoglutarate 4- dioxygenase (proline 4-hydroxylase), alpha 1 polypeptide (P4ha1 ), at least
  • said immunosuppressive T cells have reduced expression of one or more of the group consisting of CD47 and CD60, at least as compared to
  • said immunosuppressive T cells further comprise or express one or more transcription factors selected from the group consisting of Tcf7l2, Spic, Pou3f1 , Nfib, Mafb, Mef2c, Zbtb16, Tcf7l2, Bach2, Esr1 , Mef2c, Klf2, Aff3, Spib, Tcf7, Rorc, BarhH , Hoxb13, Zic3, Six3, Trp73, Hoxa13, Rora, Foxb2, PrrxH , Neurog3 and Zfp369.
  • said immunosuppressive T cells are non-proliferative.
  • c-Fos is down-regulated in said immunosuppressive T cells.
  • nuclear pc-Fos phosphorylated c-Fos
  • the immunosuppressive T cells according to the present invention i) are able to suppress production of one or more pro-inflammatory cytokines, such as pro-inflammatory cytokines selected from the group consisting of IL-12 and IL-17 in APCs; ii) do not induce production of IL-10 in APCs, and/or iii) suppress activated T cells, such as inhibit proliferation and/or induce cell death of activated T cells.
  • pro-inflammatory cytokines such as pro-inflammatory cytokines selected from the group consisting of IL-12 and IL-17 in APCs
  • ii) do not induce production of IL-10 in APCs
  • activated T cells such as inhibit proliferation and/or induce cell death of activated T cells.
  • the method of making the immunosuppressive T cells comprises one or more steps of introducing into T cells FoxA1 protein, or a functional variant or functional fragment thereof, and treating said T cells with an effective amount of IFN- ⁇ .
  • the method of making the immunosuppressive T cells comprises one or more steps of introducing into T cells a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof, and treating said T cells with an effective amount of IFN- ⁇ .
  • the method of making the immunosuppressive T cells comprises introducing into T cells FoxA1 protein, or a functional variant or functional fragment thereof.
  • the method of making the immunosuppressive T cells comprises introducing into T cells a nucleic acid construct encoding FoxA1 or a functional variant or functional fragment thereof. In one embodiment the method of making the immunosuppressive T cells comprises treating T cells with an effective amount of IFN- ⁇ .
  • an effective amount of IFN- ⁇ is an amount sufficient to induce expression of FoxA1 in said T cells.
  • a nucleic acid construct encoding FoxA1 or a functional variant or functional fragment thereof will in one embodiment comprise all or part of the FOXA1 gene (SEQ ID NO:3).
  • introducing FoxA1 into T cells may be by any technical means known to the skilled person.
  • said T cells are cultured animal cells.
  • said T cells are extracted from an individual, such as an individual having an inflammatory disease or disorder as specified herein.
  • said T cells are selected from the group consisting of Regulatory T cells (Tregs), Helper T cells (T h cells), Cytotoxic T cells (T c cells, TCL), Natural killer T cells (NKT) and Memory T cells.
  • said method of making the immunosuppressive T cells of the present invention is performed in vitro or ex vivo. In another embodiment, said method of making the immunosuppressive T cells of the present invention is performed in vivo.
  • introducing into T cells FoxA1 protein, or a functional variant or functional fragment thereof comprise one or more steps of simply adding protein in solution (such as media) to a T cell culture; microinjection of protein into T cells; one or more membrane-vesicle methods or one or more physical methods.
  • microinjection is usually associated with direct pressure injection of proteins or other molecules into cells through glass microcapillaries ('needle microinjection'). This is one of the simplest microinjection procedures, and the one most frequently used, but the term is commonly applied to other methods of introducing
  • membrane-vesicle methods in which pre-loaded membrane vesicles (erythrocyte ghosts, liposomes, protoplasts) are caused to fuse with cultured cells and release their contents into the cytoplasm; and (2) physical methods, which rely on macromolecules entering cells by diffusion through holes transiently introduced in their plasma membranes by mechanical means (scraping from substratum, agitating with glass beads).
  • introducing into T cells the FoxA1 gene or a functional variant or functional fragment thereof; or introducing a nucleic acid construct encoding FoxA1 or a functional variant or functional fragment thereof comprise one or more steps of transfection.
  • Transfection is the process of deliberately introducing nucleic acids into cells. The term is often used for non-viral methods in eukaryotic cells.
  • Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane, to allow the uptake of material.
  • Transfection can be chemical-based (carried out using e.g. calcium phosphate, dendrimers or cationic polymers); by non- chemical methods such as electroporation, sonoporation or optical transfection; by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell membrane and deposit their cargo inside. Liposome transfection is termed lipofection. Also particle-based methods and viral methods may be employed. Medical treatment
  • Such methods according to the present invention in one embodiment comprise one or more steps of administration or release of an effective amount of a bioactive agent (FoxA1 protein, nucleic acid construct comprising FoxA1 or FoxA1 + T cells, as defined herein) according to the present invention, or a pharmaceutical composition comprising one or more such bioactive agents, to an individual in need thereof.
  • a bioactive agent FoxA1 protein, nucleic acid construct comprising FoxA1 or FoxA1 + T cells, as defined herein
  • a pharmaceutical composition comprising one or more such bioactive agents
  • An individual in need as referred to herein is in one embodiment an individual that benefits from the administration of a bioactive agent according to the present invention. Such an individual in one embodiment suffers from an inflammatory condition in the tissue of one or more organs, or is at risk of suffering therefrom.
  • the term "Individual” refers preferably to vertebrates, particular members of the mammalian species, preferably primates including humans. The individual is in one embodiment any human being, male or female, infant, middle-aged or old.
  • the disorder to be treated or prevented in the individual in one embodiment relates to the age of the individual, the general health of the individual, the medications used for treating the individual and whether or not the individual has a prior history of suffering from diseases or disorders that may have or have induced ischemic and/or inflammatory conditions in the individual.
  • treatment refers to the management and care of a patient for the purpose of combating a condition, disease or disorder.
  • the term is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of the bioactive agent for the purpose of: alleviating or relieving symptoms or complications; delaying the progression of the condition, partially arresting the clinical manifestations, disease or disorder; curing or eliminating the condition, disease or disorder; and/or preventing or reducing the risk of acquiring the condition, disease or disorder, wherein "preventing” or “prevention” is to be understood to refer to the management and care of a patient for the purpose of hindering the development of the condition, disease or disorder, and includes the administration of the active compounds to prevent or reduce the risk of the onset of symptoms or complications.
  • the patient to be treated is preferably a mammal, in particular a human being.
  • mice such as mice, rats, dogs, cats, cows, horses, sheep and pigs
  • animals such as mice, rats, dogs, cats, cows, horses, sheep and pigs
  • the patients to be treated according to the present invention can be of various ages, for example, adults, children, children under 16, children age 6-16, children age 2-16, children age 2 months to 6 years or children age 2 months to 5 years.
  • the invention is thus, in one embodiment, directed to a bioactive agent selected from the group consisting of FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined herein above; for use as a medicament.
  • a bioactive agent selected from the group consisting of FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined herein above; for use as a medicament.
  • the invention relates to a bioactive agent selected from the group consisting of FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined herein above, for use in the treatment of an inflammatory disease or disorder.
  • a bioactive agent selected from the group consisting of FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined herein above, for use in the treatment of an inflammatory disease or disorder.
  • the present invention relates to use of a bioactive agent selected from the group consisting of FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined herein above, for the manufacture of a medicament for the treatment of an inflammatory disease or disorder.
  • a bioactive agent selected from the group consisting of FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined herein above, for the manufacture of a medicament for the treatment of an inflammatory disease or disorder.
  • Also provided is a method of treating an inflammatory disease or disorder comprising administering to an individual in need thereof an effective amount of one or more bioactive agents selected from FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined herein.
  • said treatment is prophylactic, ameliorative and/or curative.
  • said mammal is a human (homo sapiens). Said treatment may be initiated prior to symptom onset or after symptom onset.
  • the present invention relates to FoxA1 protein, or a functional fragment or functional variant thereof; for use as a medicament or for use in the treatment of an inflammatory disease or disorder.
  • the present invention relates to use of FoxA1 protein, or a functional fragment or functional variant thereof, for the manufacture of a medicament for the treatment of an inflammatory disease or disorder.
  • the present invention relates to a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; for use as a medicament or for use in the treatment of an inflammatory disease or disorder.
  • the present invention relates to use of a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; for the manufacture of a medicament for the treatment of an inflammatory disease or disorder.
  • the inflammatory disease or disorder is an inflammatory condition in the tissue of one or more organs, such as one or more organs selected from the group consisting of kidney, liver, brain, heart, muscles, bone marrow, skin, skeleton, lungs, the respiratory tract, spleen, exocrine glands, bladder, endocrine glands, reproduction organs including the phallopian tubes, eye, ear, vascular system, the gastroinstestinal tract including small intestines, colon, rectum, canalis analis and the prostate gland.
  • organs such as one or more organs selected from the group consisting of kidney, liver, brain, heart, muscles, bone marrow, skin, skeleton, lungs, the respiratory tract, spleen, exocrine glands, bladder, endocrine glands, reproduction organs including the phallopian tubes, eye, ear, vascular system, the gastroinstestinal tract including small intestines, colon, rectum, canalis analis and the prostate gland.
  • the inflammatory disease to be treated according to the present invention is an autoimmune disorder.
  • Autoimmune disorders are characterized by an overactive immune response.
  • the immune system fails to properly distinguish between self and non-self, and attacks part of the body.
  • the present invention relates to immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells); for use as a medicament or for use in the treatment of an inflammatory disease or disorder.
  • the present invention relates to use of immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells); for the manufacture of a medicament for the treatment of an inflammatory disease or disorder.
  • T cells from an individual having an inflammatory disease
  • said collected T cells are transformed into immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), by introducing into said collected T cells i) FoxA1 protein, or a functional fragment or functional variant thereof; or ii) a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof.
  • said collected T cells are transformed into immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), by treating said collected T cells with an effective amount of IFN- ⁇ . It is understood that an effective amount of IFN- ⁇ corresponds to a dosage sufficient to transform the T cells into immunosuppressive T cells expressing FoxA1 (FoxA1 + T cells).
  • said collected T cells are transformed into immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), by introducing into said collected T cells i) FoxA1 protein, or a functional fragment or functional variant thereof; or ii) a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof, and also treating said collected T cells with an effective amount of IFN- ⁇ .
  • said T cells are collected from the blood of an individual, from the cerebrospinal fluid (CSF) of an individual and/or from the lymph of an individual.
  • CSF cerebrospinal fluid
  • said T cells are re-introduced into the blood of an individual, into the cerebrospinal fluid (CSF) of an individual, and/or into the lymph of an individual.
  • CSF cerebrospinal fluid
  • said T cells are collected from the same individual that receives them after transformation into FoxA1 + T cells.
  • one or more subpopulations of T cells are specifically collected and re-introduced.
  • one or more subpopulations of T cells selected from the group consisting of Regulatory T cells (Tregs), Helper T cells (T h cells), Cytotoxic T cells (T c cells, TCL), Natural killer T cells (NKT) and Memory T cells, are specifically collected and re-introduced.
  • Tregs Regulatory T cells
  • T h cells Helper T cells
  • T c cells Cytotoxic T cells
  • TCL Natural killer T cells
  • Memory T cells are specifically collected and re-introduced.
  • Inflammatory abnormalities are a large group of disorders which underlie a vast variety of human diseases.
  • the immune system is often involved with inflammatory disorders, demonstrated in both allergic reactions and some myopathies, with many immune system disorders resulting in abnormal inflammation.
  • Non-immune diseases with etiological origins in inflammatory processes include cancer, atherosclerosis, and ischaemic heart disease.
  • An inflammatory disease or disorder may be defined as a disorder having an inflammatory component, an inflammatory abnormality and/or an etiological origin in inflammatory processes.
  • Inflammation is a localized defensive response of the body against pathogens and injury. Immune cells and soluble factors take part in this process to neutralize the injurious agent and initiate tissue repair to restore homeostasis. Loss of regulation of these mechanisms can prevent the final resolution of the inflammatory process, leading to chronic inflammation. Chronic inflammation is extremely relevant in today's modern medicine, as it contributes to the pathogenesis of the most important diseases of the industrialized societies including atherosclerosis, acute and chronic heart failure, cancer, diabetes, and obesity-associated diseases.
  • Arthritis is a form of joint disorder that involves inflammation of one or more joints.
  • arthritis There are over 100 different forms of arthritis.
  • the most common form, osteoarthritis (degenerative joint disease) is a result of trauma to the joint, infection of the joint, or age.
  • Other arthritis forms are rheumatoid arthritis, psoriatic arthritis, and related autoimmune diseases.
  • Septic arthritis is caused by joint infection
  • RA rheumatoid arthritis
  • gout a type of arthritis
  • the inflammatory disease to be treated according to the present invention is an inflammatory disease selected from the group consisting of arthritis, an arthropathy (a disease of a joint, Arthritis (including diseases associated with arthritis), osteoartritis, rheumatoid arthritis; spondylarthropathies (e.g.
  • ankylosing spondilitis reactive arthritis (including arthritis following rheumatic fever), Henoch-Schonlein purpura, Reiter's disease, Juvenile Chronic arthritis including Still 's disease, juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, psoriasis, osteoarthritis, osteoarthritis secondary to hypermobilty, congenital dysplasias, slipped femoral epiphysis, Perthes' disease, intra-articular fractures, meniscectomy, obesity, recurrent dislocation, repetitive actions, crystal depositions and diseases and metabolic abnormalities of cartilage including pyrophosphate arthropathy, ochronosis, haemochromatosis, avascular necrosis including Sickle Cell disease, therapy with corticoids or other drugs, Caisson disease, septic or infectious arthitis (including tuberculous arthritis, meningococcal arthritis, gonococcal arthritis, salmonella arthritis), infective endocarditis,
  • said inflammatory disease is a connective tissue disorder; in one embodiment selected from the group consisting of systemic lupus erythematosus, polymyositis/dermatomyositis, systemic sclerosis, mixed connective tissue disease, sarcoidosis and primary Sjogrens syndrome including keratoconjunctivitis sicca, polymyalgia rheumatica, and other types of vasculitis.
  • said inflammatory disease is a soft-tissue rheumatism including bursitis, tenosynovitis or peritendonitis, enthesitis, nerve compression, periarthritis or capsulitis, muscle tension and muscle dysfunction.
  • said inflammatory disease is selected from the group consisting of vasculitis including vasculitis secondary to rheumatoid arthritis, infective vasculitis due to infections with bacterial species including spirochaetal diseses as Lyme disease, syphilis, rickettsial and mycobacterial infections, fungal, viral or protozoal infections, non-infective vasculitis secondary to hypersensibility and leucocytoplastic vasculitis including Serum Sickness and Henoch-Schonlein purpura, Drug induced vasculitis, essential mixed cryoglobulinaemia, hypocomplentaemia, Vasculitis associated with other kinds of malignancy, non-infective vascultitis including Takayasu's
  • said inflammatory disease is an inflammatory disease of the gastrointestinal system.
  • Said inflammatory disease of the gastrointestinal system is in one embodiment selected from the group consisting of inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behget's disease, indeterminate colitis,
  • coeliac disease coeliac disease, gluten sensitive enteropathy, eosinophilic gastroenteritis, intestinal lympangiectasia, diverticular disease of the colon, radiation enteritis, irritable bowel syndrome, Whipple 's diease, stomatitis of all kinds, salivary gland diseases (such as sarcoidosis, salivary duct obstruction and Sjogrens syndrome), inflammaton of the oesophagus (e.g. due to gastro- oesophagel reflux or infections with Candida species, herpes simplex and cytomegalus virus), inflammatory diseases of the stomach (including acute and chronic gastritis, helicobacter pylori infection and Mentriers disease), and inflammation of the small intestine.
  • salivary gland diseases such as sarcoidosis, salivary duct obstruction and Sjogrens syndrome
  • inflammaton of the oesophagus e.g. due to gastro- oesophagel reflux or infections with
  • the inflammatory disease to be treated is an inflammatory bowel disease, such as an inflammatory bowel disease selected from the group consisting of Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behget's disease and indeterminate colitis.
  • an inflammatory bowel disease selected from the group consisting of Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behget's disease and indeterminate colitis.
  • said inflammatory disease is selected from the group consisting of dermatitis, pemfigus, bulloid pemphigoid, benign mucous membrane pemphigoid, dermatitis herpitiformis, tropical sprue, systemic amyloidosis, primary biliary cirrhosis, Goodpasture syndrome, all kinds of deposition diseases as Gout, pyrophosphate arthopathy and acute calcific periarthritis, pancreatitis, septic discitis, tuberculosis, malignancies (such as metastases, myeloma and others), spinal tumours, ancylosing spondylitis, acute disc prolapse, chronic disc disease/osteoarthritis, osteoporosis, and osteomalacia, Pagets disease, hyperparathyroidism, renal osteodystrophy,
  • said inflammatory disease is selected from the group consisting of upper and lower airway diseases such as chronic obstructive pulmonary diseases (COPD), allergic and non-allergic asthma, allergic rhinitis, allergic and non-allergic conjunctivitis, allergic and non-allergic dermatitis and lung inflammation.
  • COPD chronic obstructive pulmonary diseases
  • allergic and non-allergic asthma allergic rhinitis
  • allergic and non-allergic conjunctivitis allergic and non-allergic dermatitis and lung inflammation.
  • the inflammatory disease or disorder to be treated is a CNS disorder, such as a CNS disorder having an inflammatory component.
  • the CNS disorder having an inflammatory component is a cancer of the CNS, a neurodegenerative disorder, a stroke, or trauma to the head.
  • a stroke sometimes referred to as a cerebrovascular accident (CVA)
  • CVA cerebrovascular accident
  • said inflammatory disease is a neurodegenerative disease, such as a neurodegenerative disease having an inflammatory component.
  • Neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS (Amyotrophic lateral sclerosis), Polyglutamine (PolyQ) Diseases (These include Huntington's disease, spinocerebellar ataxias, DRPLA (Dentatorubropallidoluysian atrophy) and SBMA (Spinobulbar muscular atrophy or Kennedy disease)) and Non-Polyglutamine Diseases.
  • PolyQ Polyglutamine
  • Gliomas are tumours arising from glial cells and may occur in the spinal cord or the brain, the latter being more common. Gliomas are the most common type of brain tumour and can be either supratentorial or infratentorial. There are four main types of glioma:
  • Ependymomas (ependymal cells).
  • astrocytes Astrocytomas (astrocytes), of which glioblastoma multiforme (GBM) is the most common.
  • GBM glioblastoma multiforme
  • Oligodendrogliomas oligodendrocytes
  • Glioblastoma multiforme is the most common and most aggressive type of primary brain tumour. It involves glial cells and has small areas of necrotising tissue surrounded by anaplastic cells. There are also hyperplastic blood vessels.
  • the inflammatory disease or disorder to be treated is a cancer of the CNS, for example glioma, including ependymomas, astrocytomas, oligodendrogliomas and mixed gliomas.
  • the inflammatory disease or disorder to be treated is glioblastoma multiforme.
  • the inflammatory disease or disorder to be treated is cancer. Multiple sclerosis
  • MS Multiple sclerosis
  • disseminated sclerosis also known as disseminated sclerosis or encephalomyelitis disseminata
  • MS is an inflammatory disease in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged, leading to demyelination and scarring as well as a broad spectrum of signs and symptoms.
  • MS Middlemanosus sarcoma . Almost any neurological symptom can appear with the disease, and often progresses to physical and cognitive disability. MS takes several forms, with new symptoms occurring either in discrete attacks (relapsing forms) or slowly accumulating over time (progressive forms). Between attacks, symptoms may go away completely, but permanent neurological problems often occur, especially as the disease advances.
  • RRMS relapsing-remitting MS
  • PPMS primary progressive MS
  • RRMS relapsing-remitting multiple sclerosis
  • Natalizumab reduces the relapse rate more than first-line agents; however, due to issues of adverse effects is a second-line agent reserved for those who do not respond to other treatments or with severe disease.
  • Mitoxantrone whose use is limited by severe adverse effects, is a third-line option for those who do not respond to other medications.
  • the disease-modifying treatments have several adverse effects.
  • an autologous attenuated T-cell vaccine (Tovaxin®) is proposed for treatment of MS.
  • Interferon beta 1 a is sold under the trade names Avonex (Biogen pout) and Rebif (Merck Serono), (Pfizer); CinnoVex (CinnaGen) is biosimilar.
  • Interferon beta 1 b is marketed only by Bayer in the US as Betaseron and outside the US as Betaferon.
  • the present inventors have shown that IFN- ⁇ can induce FoxA1 and FoxA1 -mediated PD-L1 , leading to the generation of the FoxA1 + T cells; and furthermore that the immunosuppressive FoxA1 + T cells are generated in IFN-p-responsive relapsing- remitting MS (RRMS-R) patients.
  • FoxA1 + T cells have also been identified in PPMS IFN-p-responders (PPMS-R).
  • PPMS-R PPMS IFN-p-responders
  • a bioactive agent as defined herein whether in the form of FoxA1 protein, DNA or FoxA1 + T cells, can potentially increase the treatment effect of IFN- ⁇ administered in RRMS-R and/or PPMS-R patients; and be a co-treatment or an alternative treatment to IFN- ⁇ in IFN-p-non-responder RRMS patients (RRMS-NR) and/or IFN-p-non-responder PPMS patients (PPMS-NR).
  • the invention relates to a bioactive agent selected from the group consisting of FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), for use in the treatment of multiple sclerosis.
  • a bioactive agent selected from the group consisting of FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), for use in the treatment of multiple sclerosis.
  • the invention relates to a method of treating multiple sclerosis comprising administering to an individual in need thereof an effective amount of a bioactive agent selected from the group consisting of FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells).
  • a bioactive agent selected from the group consisting of FoxA1 protein, or a functional fragment or functional variant thereof
  • a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof
  • immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells).
  • said multiple sclerosis is IFN-p-responding MS (MS-R).
  • said multiple sclerosis is IFN-p-non-responding MS (MS-NR).
  • said multiple sclerosis is selected from the group consisting of relapse-remittent MS and primary progressive MS. In one embodiment, said multiple sclerosis is relapse-remittent MS (RRMS).
  • said multiple sclerosis is IFN-p-responding relapse-remittent MS (RRMS-R).
  • said multiple sclerosis is IFN-p-non-responding relapse-remittent MS (RRMS-NR).
  • said multiple sclerosis is primary progressive MS (PPMS).
  • said multiple sclerosis is IFN-p-responding primary progressive
  • said multiple sclerosis is IFN-p-non-responding primary progressive MS (PPMS-NR).
  • PPMS-NR primary progressive MS
  • the bioactive agent for use in the treatment of multiple sclerosis according to the present invention is administered in combination with IFN- ⁇
  • a bioactive agent according to the present invention for the treatment of IFN-p-non-responding relapse-remittent MS (RRMS-NR).
  • said bioactive agent is administered to said RRMS-NR alone or at least without co-administration of IFNB.
  • a bioactive agent according to the present invention for the treatment of IFN-p-non-responding primary progressive MS (PPMS- NR).
  • said bioactive agent is administered to said PPMS-NR alone or at least without co-administration of IFNB.
  • a bioactive agent according to the present invention for the treatment of IFN-p-responding relapse-remittent MS (RRMS-R), wherein said bioactive agent is administered in combination with IFN- ⁇ .
  • RRMS-R relapse-remittent MS
  • PPMS-R primary progressive MS
  • MS-NR IFN- ⁇ non-responding multiple sclerosis
  • MS-R IFN- ⁇ responding multiple sclerosis
  • said IFN- ⁇ non-responding multiple sclerosis (MS-NR) patient comprises IFN- ⁇ non-responding relapse-remittent multiple sclerosis (RRMS-NR) patients and/or IFN- ⁇ non-responding primary progressive multiple sclerosis (PPMS- NR) patients.
  • RRMS-NR IFN- ⁇ non-responding relapse-remittent multiple sclerosis
  • PPMS- NR primary progressive multiple sclerosis
  • said IFN- ⁇ responding multiple sclerosis (MS-R) patient comprises IFN- ⁇ responding relapse-remittent multiple sclerosis (RRMS-R) patients and/or IFN- ⁇ responding primary progressive multiple sclerosis (PPMS-R) patients.
  • MS-R IFN- ⁇ responding multiple sclerosis
  • RRMS-R IFN- ⁇ responding relapse-remittent multiple sclerosis
  • PPMS-R IFN- ⁇ responding primary progressive multiple sclerosis
  • a method of distinguishing an IFN- ⁇ non-responding multiple sclerosis patient from an IFN- ⁇ responding multiple sclerosis patient is equivalent to a method for identifying an IFN- ⁇ non-responding multiple sclerosis patient and a method for identifying an IFN- ⁇ responding multiple sclerosis patient.
  • a method of distinguishing is equivalent to a method for separating, discriminating or differentiating MS-R and MS-NR.
  • said sample is a blood sample.
  • said blood sample is whole blood, optionally treated with an anticoagulant.
  • said blood sample is blood plasma.
  • said blood sample is a buffy coat.
  • the lymphocytes from said blood sample are separated or isolated. It is understood that said IFN- ⁇ treatment may be according to a conventional IFN- ⁇ treatment scheme for MS patients.
  • Said sample is preferably collected from said MS patient when IFN- ⁇ has been administered at a sufficient dosage and for a sufficient time.
  • said sample is collected from said MS patient 2-48 hours after IFN- ⁇ treatment, such as 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-18, 18-20, 20-22, 22-24, 24-26, 26-28, 28-30, 30-32, 32-34 or 34-36 hours after IFN- ⁇ treatment.
  • the sample is collected 12-48 hours after treatment, such as 24-36 hours, for example 24-48 hours after treatment.
  • the presence of FoxA1 + T cells in the sample is detected by FACS analysis.
  • the presence of FoxA1 + T cells in the sample is detected by expression analysis.
  • IFN- ⁇ treatment is discontinued if said MS patient is characterized as an IFN- ⁇ non-responding multiple sclerosis (MS-NR) patient.
  • MS-NR patient is treated instead with a bioactive agent according to the present invention and/or other known treatment options for MS including but not limited to disease-modifying treatments.
  • IFN- ⁇ treatment is continued if said MS patient is characterized as an IFN- ⁇ responding relapse-remittent multiple sclerosis (MS-R) patient.
  • MS-R relapse-remittent multiple sclerosis
  • Hypersensitivity is an immune response that damages the body's own tissues. They are divided into four classes (Type I - IV) based on the mechanisms involved and the time course of the hypersensitive reaction.
  • Type I hypersensitivity is an immediate or anaphylactic reaction, often associated with allergy. Symptoms can range from mild discomfort to death.
  • Type I hypersensitivity is mediated by IgE, which triggers degranulation of mast cells and basophils when cross-linked by antigen.
  • Type II hypersensitivity occurs when antibodies bind to antigens on the patient's own cells, marking them for destruction. This is also called antibody-dependent (or cytotoxic) hypersensitivity, and is mediated by IgG and IgM antibodies.
  • Type IV hypersensitivity also known as cell-mediated or delayed type hypersensitivity
  • Type IV reactions usually takes between two and three days to develop.
  • Type IV reactions are involved in many autoimmune and infectious diseases, but may also involve contact dermatitis (poison ivy). These reactions are mediated by T cells, monocytes, and macrophages.
  • DTH Delayed type hypersensitivity
  • CD4+ helper T cells recognize antigen in a complex with Class 2 major
  • the antigen-presenting cells in this case are macrophages that secrete IL-12, which stimulates the proliferation of further CD4+ T h 1 cells.
  • CD4+ T cells secrete IL-2 and interferon gamma, further inducing the release of other T h 1 cytokines, thus mediating the immune response.
  • Activated CD8+ T cells destroy target cells on contact, whereas activated macrophages produce hydrolytic enzymes and, on presentation with certain intracellular pathogens, transform into multinucleated giant cells.
  • the invention relates to FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined herein above, for use in the treatment of one or more of hypersensitivity, a disease associated with hypersensitivity, delayed type hypersensitivity or a disease associated with delayed type hypersensitivity.
  • a disease associated with delayed type hypersensitivity or diseases with a DTH-component comprise diabetes mellitus type I, multiple sclerosis, rheumatoid arthritis, some peripheral neuropathies, Hashimoto's thyroiditis, Crohn's disease, allergic contact dermatitis, psoriasis, temporal or giant-cell arteritis (GCA), symptoms of leprosy, symptoms of tuberculosis, coeliac disease, graft-versus host disease and chronic transplant rejection.
  • diabetes mellitus type I multiple sclerosis, rheumatoid arthritis, some peripheral neuropathies, Hashimoto's thyroiditis, Crohn's disease, allergic contact dermatitis, psoriasis, temporal or giant-cell arteritis (GCA), symptoms of leprosy, symptoms of tuberculosis, coeliac disease, graft-versus host disease and chronic transplant rejection.
  • GCA giant-cell arteritis
  • Diabetes mellitus type 1 is a form of diabetes mellitus that results from autoimmune destruction of insulin-producing beta cells of the pancreas. The subsequent lack of insulin leads to increased blood and urine glucose causing classical symptoms of polyuria, polydipsia, polyphagia, and weight loss.
  • Glutamic acid decarboxylase autoantibodies GADA
  • ICA islet cell autoantibodies
  • IA-2 insulinoma-associated
  • ZnT8 zinc transporter autoantibodies
  • the invention relates to FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined herein above, for use in the treatment of diabetes mellitus type 1 .
  • Psoriasis a functional fragment or functional variant thereof.
  • Psoriasis is a common, chronic immune-mediated skin disease which may also affect the joints.
  • the invention relates to FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined herein above, for use in the treatment of psoriasis.
  • FoxA1 may be administered in the form of a protein or in the form of a gene (DNA; nucleic acid construct), by gene therapy.
  • Gene therapy is the use of DNA as a pharmaceutical agent to treat disease.
  • a common form of gene therapy involves using DNA that encodes a functional, therapeutic gene to replace a mutated gene.
  • Other forms involve directly correcting a mutation, or using DNA that encodes a therapeutic protein drug (rather than a natural human gene) to provide treatment.
  • DNA that encodes a therapeutic protein is packaged within a vector from which, once inside cells, the DNA becomes expressed by the cell machinery, resulting in the production of the therapeutic protein.
  • Glybera became the first gene therapy treatment to be approved for clinical use.
  • nucleic acid construct encoding for and/or being capable of expressing a FoxA1 peptide according to the present invention comprises all or part of SEQ ID NO:3 (FOXA1 gene).
  • nucleic acid construct will be able to continuously express a peptide according to the present invention for a prolonged period of time, such as at least 1 month, for example at least 2 months, such as at least 3 months, for example at least 4 months, such as at least 5 months, for example at least 6 months, such as at least 7 months, for example at least 8 months, such as at least 9 months, for example at least 12 months.
  • nucleic acid construct encoding for and being capable of expressing FoxA1 , or a functional variant or functional fragment thereof.
  • the encoded FoxA1 of the nucleic acid construct is a functional variant having at least 60% sequence identity, such as at least 65% sequence identity, for example at least 70% sequence identity, such as at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity to SEQ ID NO:1 (protein) or SEQ ID NO:3 (DNA).
  • the encoded FoxA1 of the nucleic acid construct is a functional variant having from 60 to 65% sequence identity, for example from 65 to 70% sequence identity, such as from 70 to 75% sequence identity, for example from 75 to 80% sequence identity, such as from 80 to 85% sequence identity, for example from 85 to 90% sequence identity, such as from 90 to 95% sequence identity, for example from 95 to 99% sequence identity, such as 99 to 99.9 sequence identity to SEQ ID NO:1 (protein) or SEQ ID NO:3 (DNA).
  • the encoded FoxA1 of the nucleic acid construct is a functional fragment of SEQ ID NO: 1 comprising from 10 to 471 consecutive amino acids of SEQ ID NO:1 , such as from 10-25, 25-50, 50-75, 75-100, 100-125, 125-150, 150-175, 175- 200, 200-250, 250-300, 300-350, 350-400, 400-450 or from 450-471 consecutive amino acids of SEQ ID NO:1.
  • nucleic acid construct is understood a genetically engineered nucleic acid.
  • the nucleic acid construct may be a non-replicating and linear nucleic acid, a circular expression vector or an autonomously replicating plasmid.
  • a nucleic acid construct may comprise several elements such as, but not limited to genes or fragments of same, promoters, enhancers, terminators, poly-A tails, linkers, polylinkers, operative linkers, multiple cloning sites (MCS), markers, STOP codons, internal ribosomal entry sites (IRES) and host homologous sequences for integration or other defined elements. It is to be understood that the nucleic acid construct according to the present invention may comprise all or a subset of any combination of the above-mentioned elements.
  • nucleic acid constructs are well known in the art (see, e.g., Molecular Cloning: A Laboratory Manual, Sambrook et al., eds., Cold Spring Harbor Laboratory, 2nd Edition, Cold Spring Harbor, N.Y., 1989). Further, nucleic acid constructs according to the present invention may be synthesized without template, and may be obtained from various commercial suppliers (e.g. Genscript Corporation).
  • the nucleic acid construct are naked DNA constructs comprising sequences encoding the peptide of the invention.
  • An expression cassette is a part of a vector DNA used for cloning and transformation.
  • An expression cassette is composed of one or more genes and the sequences controlling their expression. Three components comprise an expression cassette: a promoter sequence, an open reading frame, and a 3' untranslated region that, in eukaryotes, usually contains a polyadenylation site.
  • Different expression cassettes can be transformed into different organisms including bacteria, yeast, plants, and mammalian cells as long as the correct regulatory sequences are used.
  • the present invention also provides the nucleic acid construct as described herein above comprised within a delivery vehicle.
  • a delivery vehicle is an entity whereby a nucleotide sequence or polypeptide or both can be transported from at least one media to another. Delivery vehicles are generally used for expression of the sequences encoded within the nucleic acid construct and/or for the intracellular delivery of the construct or the polypeptide encoded therein.
  • a delivery vehicle comprising the nucleic acid construct according to the present invention.
  • a delivery vehicle may be selected from the group consisting of: plasmid vectors, RNA based vehicles, DNA based vehicles/ vectors, lipid based vehicles (such as a liposome), polymer based vehicles (such as a cationic polymer DNA carrier), colloidal gold particles (coating) and virally derived DNA or RNA vehicles or vectors.
  • Methods of non-viral delivery include physical (carrier-free delivery) and chemical approaches (synthetic vector-based delivery).
  • electroporation, ultrasound, and hydrodynamic delivery employ a physical force that permeates the cell membrane and facilitates intracellular gene transfer.
  • Said physical force may be electrical or mechanical.
  • chemical delivery vehicles include, but are not limited to: biodegradable polymer microspheres, lipid based formulations such as liposome carriers, cationically charged molecules such as liposomes, calcium salts or dendrimers,
  • lipopolysaccharides polypeptides and polysaccharides.
  • a vector which herein is denoted a viral vector (i.e. not a virus) as a delivery vehicle.
  • Viral vectors according to the present invention are made from a modified viral genome, i.e. the actual DNA or RNA forming the viral genome, and introduced in naked form. Thus, any coat structures surrounding the viral genome made from viral or non-viral proteins are not part of the viral vector according to the present invention.
  • the virus from which the viral vector is derived is selected from the non-exhaustive group consisting of: adenoviruses, retroviruses, lentiviruses, adeno- associated viruses, herpesviruses, vaccinia viruses, foamy viruses, cytomegaloviruses, Semliki forest virus, poxviruses, RNA virus vector and DNA virus vector.
  • adenoviruses retroviruses
  • lentiviruses lentiviruses
  • adeno-associated viruses adeno-associated viruses
  • herpesviruses vaccinia viruses
  • foamy viruses cytomegaloviruses
  • Semliki forest virus poxviruses
  • RNA virus vector RNA virus vector and DNA virus vector.
  • said viral vector is selected from the group consisting of adenoviruses, lentiviruses, adeno-associated viruses (AAV) and recombinant adeno- associated viruses (rAAV). In one embodiment, said viral vector is a therapeutic rAAV vector.
  • An adenovirus is a group of double-stranded DNA containing viruses.
  • Adenoviruses can be genetically modified making them replication incompetent or conditionally replication incompetent.
  • adenoviral constructs or adenovectors they can be used as gene delivery vehicles for vaccination or gene therapy.
  • AAV vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell. These features make AAV a very attractive candidate for creating viral vectors for gene therapy. To date, AAV vectors have been used in over 80 clinical trials worldwide.
  • the nucleic acid construct and/or the delivery vehicle is designed in order to achieve T-cell specific delivery and/or expression, i.e. the construct is delivered and/or expressed exclusively, predominantly or mainly in T cells.
  • T cells may be any given T cell, such as one or more subpopulations selected from the group consisting of Regulatory T cells (Tregs), Helper T cells (T h cells), Cytotoxic T cells (T c cells, TCL), Natural killer T cells (NKT) and Memory T cells.
  • a viral vector comprising the nucleic acid construct according to the present invention, wherein said viral vector specifically targets T cells.
  • a nanoparticle comprising the protein or nucleic acid construct according to the present invention, wherein said nanoparticle specifically targets T cells. This may be achieved by providing a nanoparticle with a T cell selective and/or specific surface.
  • the nucleic acid construct and/or the delivery vehicle according to the present invention is CD4+ and/or CD8+ specific.
  • said nucleic acid construct comprises a T-cell specific promoter or T cell specific expression cassette.
  • An expression cassette is composed of one or more genes and the sequences controlling their expression. Three components comprise an expression cassette: a promoter sequence, an open reading frame, and a 3' untranslated region that, in eukaryotes, usually contains a polyadenylation site.
  • An aspect of the present invention relates to a cell comprising the nucleic acid construct according to the present invention.
  • a recombinant cell can be used a tool for in vitro research, as a delivery vehicle for the nucleic acid construct or as part of a gene-therapy regime.
  • the nucleic acid construct according to the invention can be introduced into cells by techniques well known in the art and which include
  • Suitable cells include
  • autologous and non-autologous cells may include xenogenic cells.
  • a recombinant cell such as a recombinant T cell, comprising a nucleic acid construct comprising FoxA1 , or a functional variant or functional fragment thereof.
  • a FoxA1 peptide or a nucleic acid construct encoding said peptide, or FoxA1 + T cells, or a composition comprising the same is to be administered to an individual in need of treatment in pharmaceutically effective doses or a therapeutically effective amount.
  • the dosage requirements will vary with the particular drug composition employed, the route of administration and the particular subject being treated, which depend on the severity and the sort of the disorder as well as on the weight and general state of the subject. It will also be recognized by one skilled in the art that the optimal quantity and spacing of individual dosages will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optima can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of a compound given per day for a defined number of days, can be ascertained using conventional course of treatment determination tests.
  • a therapeutically effective amount according to the present invention is in one embodiment an amount sufficient to cure, prevent, reduce the risk of, alleviate or partially arrest the clinical manifestations of a given disease or disorder and its complications.
  • the amount that is effective for a particular therapeutic purpose will depend on the severity and the sort of the disorder as well as on the weight and general state of the subject. An amount adequate to accomplish this is defined as a "therapeutically effective amount”.
  • a 'bioactive agent' will be used to denote collectively a peptide, a nucleic acid construct encoding said peptide, FoxA1 + T cells, and a composition comprising the same, according to the present invention.
  • the bioactive agent is administered in doses of from 1 ⁇ g day to 100 mg/day; such as from 1 ⁇ g/day to 10 Mg/day, such as 10 g/day to 100 Mg/day, such as 100 Mg/day to 250 Mg/day, such as 250 Mg/day to 500 Mg/day, such as 500 Mg/day to 750 Mg/day, such as 750 Mg/day to 1 mg/day, such as 1 mg/day to 2 mg/day, such as 2 mg/day to 5 mg/day, or such as 5 mg/day to 10 mg/day, such as 10 mg/day to 20 mg/day, such as 20 mg/day to 30 mg/day, such as 30 mg/day to 40 mg/day, such as 40 mg/day to 50 mg/day, such as 50 mg/day to 75 mg/day, or such as 75 mg/day to 100 mg/day.
  • 1 mg/day to 2 mg/day such as 2 mg/day to 5 mg/day
  • 5 mg/day to 10 mg/day such as
  • one single dose of the bioactive agent is administered and may comprise of from 1 Mg/kg body weight to 100 mg/kg body weight; such as from 1 to 10 Mg/kg body weight, such as 10 to 100 Mg/day, such as 100 to 250 Mg/kg body weight, such as 250 to 500 Mg/kg body weight, such as 500 to 750 Mg/kg body weight, such as 750 Mg/kg body weight to 1 mg/kg body weight, such as 1 mg/kg body weight to 2 mg/kg body weight, such as 2 to 5 mg/kg body weight, such as 5 to 10 mg/kg body weight, such as 10 to 20 mg/kg body weight, such as 20 to 30 mg/kg body weight, such as 30 to 40 mg/kg body weight, such as 40 to 50 mg/kg body weight, such as 50 to 75 mg/kg body weight, or such as 75 to 100 mg/kg body weight.
  • 1 Mg/kg body weight to 100 mg/kg body weight such as from 1 to 10 Mg/kg body weight, such as 10 to 100 Mg/day, such as
  • a dose according to the present invention may be administered one or several times per day, such as from 1 to 6 times per day, such as from 1 to 5 times per day, such as from 1 to 4 times per day, such as from 1 to 3 times per day, such as from 1 to 2 times per day, such as from 2 to 4 times per day, such as from 2 to 3 times per day.
  • a dose may also be administered in intermittent intervals, or intervals, whereby a dose is not administered every day. Rather one or more doses may be administered every second day, every third day, every fourth day, every fifth day, every sixth day, every week, every second week, every third week, every fourth week, every fifth week, every sixth week, or intervals within those ranges (such as every 2 to 4 weeks, or 4 to 6 weeks).
  • the preferred route of administration will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated, the location of the tissue to be treated in the body and the active ingredient chosen.
  • the route of administration allows for the bioactive agent to cross the blood-brain barrier.
  • Systemic treatment allows for the bioactive agent to cross the blood-brain barrier.
  • the route of administration is capable of introducing the bioactive agent (a peptide, a nucleic acid construct encoding said peptide, FoxA1 + T cells, and a composition comprising the same, according to the present invention) into the blood stream to ultimately target the sites of desired action.
  • the bioactive agent a peptide, a nucleic acid construct encoding said peptide, FoxA1 + T cells, and a composition comprising the same, according to the present invention
  • Such routes of administration are any suitable routes, such as an enteral route (including the oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal and intraperitoneal administration), and/or a parenteral route (including subcutaneous, intramuscular, intrathecal, intracerebral, intravenous and intradermal administration).
  • enteral route including the oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal and intraperitoneal administration
  • parenteral route including subcutaneous, intramuscular, intrathecal, intracerebral, intravenous and intradermal administration.
  • Appropriate dosage forms for such administration may be prepared by conventional techniques. Parenteral administration
  • Parenteral administration is any administration route not being the oral/enteral route whereby the medicament avoids first-pass degradation in the liver. Accordingly, parenteral administration includes any injections and infusions, for example bolus injection or continuous infusion, such as intravenous administration, intramuscular administration or subcutaneous administration. Furthermore, parenteral administration includes inhalations and topical administration.
  • the bioactive agent may be administered topically to cross any mucosal membrane of an animal to which the biologically active substance is to be given, e.g. in the nose, vagina, eye, mouth, genital tract, lungs, gastrointestinal tract, or rectum, preferably the mucosa of the nose, or mouth, and accordingly, parenteral
  • administration may also include buccal, sublingual, nasal, rectal, vaginal and intraperitoneal administration as well as pulmonal and bronchial administration by inhalation or installation.
  • the agent may be administered topically to cross the skin.
  • the intravenous, subcutaneous and intramuscular forms of parenteral administration are generally preferred.
  • the bioactive agent according to the invention may in one embodiment be used as a local treatment, i.e. be introduced directly to the site(s) of action. Accordingly, the bioactive agent may be applied to the skin or mucosa directly, or the bioactive agent may be injected into the site of action, for example into the diseased tissue or to an end artery leading directly to the diseased tissue.
  • These administration forms preferably avoid the blood brain barrier, and the blood- retina barrier.
  • the bioactive agent according to the present invention is applied or injected directly into the brain, such as into a specific region of the brain.
  • an effect of the bioactive agent may be achieved in the region of the brain where it is mainly required. This may depend on the condition being treated. This may be termed intracerebral administration.
  • the bioactive agent is administered via intrathecal
  • bioactive agent of the present invention a peptide, a nucleic acid construct encoding said peptide, or T cells comprising said peptide, and a composition comprising the same
  • a pharmaceutical formulation may be referred to as a pharmaceutical composition, pharmaceutically acceptable composition or pharmaceutically safe composition.
  • the present invention further provides a pharmaceutical formulation, which comprises a bioactive agent of the present invention, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier, excipient and/or diluent.
  • a pharmaceutical formulation which comprises a bioactive agent of the present invention, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier, excipient and/or diluent.
  • the pharmaceutical formulations may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 2005,
  • the pharmaceutically acceptable carriers can be either solid or liquid.
  • Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • a solid carrier can be one or more excipients which may also act as diluents, flavouring agents, solubilizers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrating agents, or an encapsulating material.
  • solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl ethers of cellulose.
  • liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene, water, saline or a glucose solution.
  • the carrier or diluent may include any sustained release material known in the art, such as glycerol monostearate or glycerol distearate, alone or mixed with a wax.
  • solid form preparations which are intended to be converted, shortly before use, to liquid form preparations.
  • liquid forms include solutions,
  • compositions may contain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
  • bioactive agent of the present invention may be formulated for parenteral administration and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers, optionally with an added
  • compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol.
  • oily or non-aqueous carriers, diluents, solvents or vehicles examples include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.
  • the bioactive agent of the invention may also be formulated for topical delivery.
  • Regions for topical administration include the eye or the cornea, the skin surface and also mucous membrane tissues of the vagina, rectum, nose, mouth, and throat.
  • the topical formulation may include a pharmaceutically acceptable carrier adapted for topical administration.
  • the composition may take the form of a suspension, solution, ointment, lotion, sexual lubricant, cream, foam, aerosol, spray, suppository, implant, inhalant, tablet, capsule, dry powder, syrup, balm or lozenge, for example.
  • Formulations for use in nasal, pulmonary and/or bronchial administration are normally administered as aerosols in order to ensure that the aerosolized dose actually reaches the mucous membranes of the nasal passages, bronchial tract or the lung.
  • aerosols are administered by use of a mechanical device designed for pulmonary and/or bronchial delivery, including but not limited to nebulizers, metered dose inhalers, and powder inhalers.
  • a mechanical device designed for pulmonary and/or bronchial delivery including but not limited to nebulizers, metered dose inhalers, and powder inhalers.
  • any form of aerosolization known in the art including but not limited to spray bottles, nebulization, atomization or pump aerosolization of a liquid formulation, and aerosolization of a dry powder formulation, can be used.
  • Liquid aerosol formulations in general contain a compound of the present invention in a pharmaceutically acceptable diluent.
  • Pharmaceutically acceptable diluents include but are not limited to sterile water, saline, buffered saline, dextrose solution, and the like.
  • Formulations for dispensing from a powder inhaler device will normally comprise a finely divided dry powder containing a pharmaceutical composition of the present invention (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device.
  • Dry powder formulations for inhalation may also be formulated using powder- filled capsules, in particularly capsules the material of which is selected from among the synthetic plastics.
  • the formulations of the present embodiment may also include other agents useful for pH maintenance, solution stabilization, or for the regulation of osmotic pressure.
  • salts of the instant peptide compounds are also intended to be covered by this invention. These salts will be ones which are acceptable in their application to a pharmaceutical use. By that it is meant that the salt will retain the biological activity of the parent compound and the salt will not have untoward or deleterious effects in its application and use in treating diseases.
  • compositions are prepared in a standard manner. If the parent compound is a base it is treated with an excess of an organic or inorganic acid in a suitable solvent. If the parent compound is an acid, it is treated with an inorganic or organic base in a suitable solvent.
  • the compounds of the invention may be administered in the form of an alkali metal or earth alkali metal salt thereof, concurrently, simultaneously, or together with a pharmaceutically acceptable carrier or diluent, especially and preferably in the form of a pharmaceutical composition thereof, whether by oral, rectal, or parenteral (including subcutaneous) route, in an effective amount.
  • Examples of pharmaceutically acceptable acid addition salts for use in the present inventive pharmaceutical composition include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, for example.
  • Second active ingredients such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, for example.
  • the bioactive agent of the present invention may be combined with or comprise one or more second active ingredients which are understood as other therapeutical compounds or pharmaceutically acceptable derivatives thereof.
  • “Co-administering” or “co-administration” of bioactive agents of the invention and state- of-the-art medicaments, as used herein, refers to the administration of one or more bioactive agents of the present invention, or administration of one or more bioactive agents of the present invention and a state-of-the-art pharmaceutical composition within a certain time period or administered together.
  • Methods for treatment according to the present invention may thus further comprise one or more steps of administration of one or more second active ingredients, either concomitantly or sequentially, and in any suitable ratios.
  • ingredients may, for example, be selected from compounds used to treat or prevent inflammatory disorders or symptoms and complications associated with inflammatory disorders.
  • exemplary drugs comprise immunosuppressive drugs, anti-inflammatory drugs, and so forth.
  • Methods of treatment according to the present invention may include a step wherein the bioactive agent as defined herein is administered simultaneously, sequentially or separately in combination with one or more second active ingredients.
  • a drug approved for this specific purpose may be complemented with the peptide, compound or composition according to the present invention to optimise and improve treatment outcome for the patient. This is regardless of whether the approved drug for the specific purpose is prophylactic, ameliorating or curative.
  • the bioactive agent according to the present invention is administered in combination with IFNB (interferon beta, including IFNB 1 a and 1 b).
  • IFNB interferon beta, including IFNB 1 a and 1 b.
  • FoxA1 directs the lineage and immunosuppressive properties of novel T RF OXAI regulatory cells important in EAE and MS
  • Inflammation is a self-destructive process that can lead to irreversible chronic tissue destruction.
  • the defective generation or function of T reg uiatory cells contributes to chronic autoimmune inflammation.
  • FoxA1 as a novel transcription factor in T-cells that conveys suppressive properties in a new T reg population, hereby called ⁇ ⁇ ⁇ (or FoxA1 +Treg or FoxA1 +T-cells). FoxA1 bound to the pdl1 promoter, inducing PD-L1 , which was essential for T RFo xAis to kill activated T-cells.
  • T RFOX AI cells had a distinct transcription profile.
  • T RFo xAis were CD4 hl , PD-L1 hl and CD47 + .
  • ⁇ 1nb ⁇ ' ⁇ and Ifnaf' ' mice were defective in T RFoxA i s -
  • adoptive transfer of stable T RFoxA i inhibited experimental autoimmune encephalomyelitis mediated by functional FoxA1 and PD-L1.
  • response to IFN- ⁇ treatment was associated with expansion of suppressive T RFoxA i cells. Therefore, FoxA1 is a lineage-specification factor with a specialized role in supporting differentiation and the suppressive function of T RFoxA i cells.
  • T reg regulatory T-cell
  • T-cell differentiation into effector or T reg cells is determined by the dynamic action of transcription factors.
  • Tbet determines Th1
  • GAT A3 decides Th2
  • RORyt controls Th17 fate and function.
  • FoxP3 is the cell-lineage commitment factor for natural and induced (n/i)T reg s 1 "3 . In mice and humans, FoxP3 mutations lead to multiorgan failure and systemic autoimmunity 4,5 .
  • T reg defects are reported in experimental autoimmune encephalomyelitis (EAE) 6 , a model for multiple sclerosis (MS), a tissue-specific inflammatory disease affecting the central nervous system (CNS). Neuron-induced, FoxP3-expressing T reg s control CNS-inflammation in EAE 7 .
  • EAE experimental autoimmune encephalomyelitis
  • MS multiple sclerosis
  • CNS central nervous system
  • IFN interferon
  • endogenous IFN- ⁇ does not regulate EAE through differential T-cell priming and/or effector cytokines, T-helper-subset shift, B-cell activation, or antibody production, but through limitation of CNS-inflammation 13,16 .
  • mice Although ⁇ 1nb ⁇ ' ⁇ mice generate T reg s systemically, they might lack the capacity to generate tissue-specific T reg s in the inflamed CNS.
  • T reg s a novel population of T reg s in wildtype mice that was absent in lfnb ' mice.
  • These suppressive cells hereby called T RFOX AI, were generated in IFN-p-responsive relapsing-remitting MS (RRMS) patients.
  • T RFOX AI cells express FoxA1 , an epigenetic-modifying pioneer transcription factor 17 important in embryonic development, stem cell differentiation, hepatocyte development, and cancer epigenetics 18"22 .
  • FoxA1 modulates postembryonic tissue functions, including the brain. FoxA1 is necessary for epithelial cells, and mediates lineage specification 23,24 .
  • FoxA1 is a lineage-specification factor that defines T RFOX AI cells and direct function of these novel T reg cells.
  • the CD4 hi PD-L1 hi T-cells were enriched in the CNS of WT RR-EAE mice compared to spleen or lymph nodes (Fig. 1 g). We hypothesized that these cells affected inflammation control and their absence in Ifnb mice contributed to disease chronicity.
  • FoxA1 is the unique transcription factor of T RFOX AI cells
  • EncT 9-ioi-reactive T-cell line
  • FoxA1 a gene critical for epigenetic reprogramming and cell-lineage commitment 24 was robustly upregulated in CD4 hi PD-L1 hi T-cells (Fig. 2b, Table II). FoxA1 was found as the first ranking regulated transcription factor using Gene
  • nT RFo xAis were nonproliferative measured by Ki-67 + (Fig. 2e).
  • Fig. 2e Ki-67 +
  • RF OX A-I S inhibited proliferation and increased cell-death of responder T-cells (ResT) (Fig. 2j-k).
  • nT RFo xAi cells generated by co-culturing of OVA-activated CD4 + OTII- cells with CGNs also suppressed ResT-cells.
  • IL-2 rescued ResT-cell proliferation but not cell-death (Fig. 1 1 , indicating that T RFo xAis regulated these events independently.
  • nT RFoxA was adoptively transferred to ears, in a murine delayed type hypersensitivity model of tissue inflammation. Ears receiving nT RFoxA is had significantly less inflammation (Fig. 2I). nT RFoX Ais also significantly reduced adoptive-EAE incidence, prevented clinical severity and inhibited CNS-inflammation in lfnb ⁇ ; ⁇ mice (Fig. 2m-n, Table VII). These results supported the in vivo anti-inflammatory and suppressive properties of T RFoxA is- IFN induction of suppressive T requires IFNA-receptor signaling
  • IFN- ⁇ - induced (i)T RFoxA is- Immunocytochemistry revealed nuclear FoxA1 expression after mIFN-p-stimulation (Fig. 3g). Compared to PD-L1 l0 T-cells, only PD-L1 hi T-cells expressed nuclear FoxA1 (Fig. 3h-i).
  • CD4 + T-cells were purified from healthy donors and cultured with or without human (h)IFN-p. Treatment did not induce FoxP3 and IL-35, markers of classical T reg s, or PD-1 hl , an exhausted T-cell phenotype (Fig. 13a-d). However, hlFN- ⁇ significantly induced TCRap + CD4 + PD-L1 hi FoxA1 + iT RFoxA is (Fig. 3j-k). Purified iT RFoxA is (R1 -gated) expressed nuclear FoxA1 compared to PD-L1 l0 T-cells (R2-gated) (Fig. 3I).
  • T r egs and Th2-cells modulate antigen-presenting cells (APCs) 34 and effective IFN-p-treatment of RRMS is associated with APC modulation 35 ' 36 .
  • IFN- ⁇ induces IL-10 in APCs associated with reduced MS-symptoms 37 .
  • iT RFoxA is did not induce IL-10 in APCs.
  • iT RFoxA i s significantly suppressed production of pro-inflammatory cytokines (IL-12 and IL-17) by APCs (Fig. 14).
  • FoxAI KD and CtrlsiRNA T-cells were transferred to chimeric NOG mice populated with preactivated ResT-cells.
  • FoxAI KD CD4 + T-cells did not suppress ResT-cells (Fig. 4e-f), indicating that FoxA1 was required for functional T RFoxA is- T RFoxA i s ectopically overexpressing FoxA1 (Fig. 4g, Fig. 15c) profoundly reduced responder T-cell proliferation (Fig. 4h), suggesting that FoxA1 conveyed the suppressive capacity of T RFoxA is-
  • IFN-p-induced T RFo xAis suppress EAE mediated by FoxA1 and PD-L1
  • T RFoxA To verify the in vivo suppressive function of T RFoxA is, iT RFo xAis were transferred to lfnb ⁇ ; ⁇ mice with adoptive-EAE. While iT RFo xAis suppressed EAE- progression and CNS-inflammation, FoxAI KD eliminated their suppressive function. Rescuing this phenotype by ectopic expression of siRNA-insensitive pcDNA3.1 foxal, expressing ORF sequence of foxal (Fig. 15a, d-e), was sufficient to restore the EAE- suppressive function of iT RFoxA is- PD-L1 KD and anti-PD-L1 prevented the EAE- suppressive capacity of iT RFo xAis (Fig. 4i-j, Table XI). Though the antibody was removed, remaining anti-PD-L1 could have affected encephalitogenicity of cells.
  • iT RFo xA is and all control variants were CFSE-labeled prior to transfer to EAE.
  • Post-EAE analysis of CFSE + T-cells revealed that while iT RFoxA is did not proliferate, the control T-cells and iT RFoxA is with FoxAI KD and PD-L1 KD proliferated in vivo (Fig. 4k-l).
  • iT RFoxA is phenotype was stable in vivo as they maintained FoxA1 and did not gain FoxP3 expression up to 40 days post-transfer (Fig. 4m-n).
  • PD-L1 is involved in negative signaling to T-cells, and cell-cycle arrest 38 .
  • iT RFoxA is inhibited pAKT in ResT-cells. This was dependent on the PD-1 signaling, as PD-1 KD of ResT-cells rescued pAKT (Fig. 5d, Fig. 16a).
  • iT RFo xA is induced PD-1 -dependent caspase3 cleavage in ResT-cells, which triggers an apoptotic caspase chain (Fig. 5e, Fig. 16b).
  • Phosphorylation of P38 (pP38), a mitogen-activated protein kinase (P38MAPK) regulates the cell-cycle and apoptosis.
  • iT RFoxA upregulated cleaved caspase3 in ResT-cells (Fig. 5f), which was diminished by PD-L1 and caspase inhibition (Fig. 5g).
  • iT RFoxA i inhibition of ResT-cells was associated with pAKT reduction since blocking PD-L1 and caspases restored pAKT in ResT-cells (Fig. 5h).
  • FoxA1 was found necessary for the generation and function of T RFoxA i s and FoxA1 was sufficient to activate PD-L1 in T RFoxA i s - T RFoxA i s killing of ResT-cells was mediated by PD-L1 -PD-1 -signaling that inhibited pAKT and triggered caspase- mediated apoptotic pathway.
  • FoxA1 binds the pd-11 promoter and regulates PD-L1
  • FoxA1 binds an enhancer sequence in cancer cells 17 and controls TTR transcription, suggesting that in addition to enhancer activity, FoxA1 controls promoters 40 .
  • FoxA1 bound to the Pdl1-B promoter site compared to a housekeeping gene (Fig. 5I).
  • a housekeeping gene Fig. 5I.
  • mTTR FoxA1 -binding electromobility shift assays with a probe containing the FoxA1 -binding site but lacking the c-Fos-binding site of the control TTR promoter 40 (mTTR). Labeled mTTR probe was incubated alone or with unlabeled PD-L1-A and PD-L1-B probes. The PD-L1-B but not the PD-L1-A probe competed for FoxA1 -binding to the mTTR probe (Fig. 5m).
  • T RFo xAis were significantly increased in the IFN-p-responsive group, compared to baseline and the RRMS-NRs (Fig. 6a-d). No expansion of T RFo xAis was detected in the RRMS-NRs (Fig. 6b-d). Only gated TCRaP + CD4 + PD-L1 hi T-cells from RRMS-Rs were positive for nuclear FoxA1 ; with a significant FoxA1 increase after 24 months of treatment (Fig. 6e-f).
  • T RFoxA i s from IFN-p-responder RRMS-R patients suppressed activated T-cells by inhibiting proliferation and exerting killing.
  • Significant elevation of T RFoxA i s in RRMS-R patients could be associated with clinical benefit of IFN- ⁇ therapy.
  • PD-L1 - mediated suppression might be a similarity between the T RFOX AI S and T re g S .
  • PdIT A and WT mice were shown to have similar numbers of T re gs(CD4 + CD25 hi CD45RB l0 ) 44 , but PD-L1 's role in generation and function of T reg s is now reported 45"47 .
  • PD-L1 - mediated induction of T reg development is associated with downregulation of pAkt/mTOR signaling and ERK2 but not P38MAPK 47 .
  • the signaling molecules attenuated in activated T-cells via PD-L1 -PD-1 are unclear.
  • T RFoxA In T RFoxA is, PD-L1 was required for suppression via inhibition of pAKT and pP38 and induction of caspase3- associated T-cell killing.
  • T RFOX AI S are negative for FoxP3, CTLA4, TGF- ⁇ , IL-10 and IL-35 49 ' 50 , commonly associated with Tregs-
  • the genetic signature of T RFOX AI S is distinct from their T-cell progenitors, neuron- induced T r egs, n/iTregs 28"31 and exhausted T-cells 32 .
  • FoxA1 a 'pioneer' factor binds to chromatinized DNA directly, opens the chromatin and regulate its target genes but can also enhance binding of other co- factors to their target genes 48 , such as GATA3 and Tbet 49 .
  • Additional FoxA1 activity as an activator or repressor, its T-cell target genes, and interaction with other factors remain to be determined.
  • IFN- ⁇ was sufficient to induce FoxA1 and FoxA1 -mediated PD-L1 , leading to TRF OX AI generation.
  • nT RFo xAis and iT RFo xAis shared homology in their gene-profile with
  • T RFOX AI cell-profile was stable in vivo leading to EAE-prevention. Additional pathways for IFNp-IFNAR-mediated FoxA1 regulation could include activation of STAT molecules.
  • STAT3 binds near the pdl1-B site in tolerogenic APCs 50 , hence FoxA1 and STAT3 could interact to direct pdl1 transcription in T-cells.
  • T RFoxA Understanding novel functions of T RFoxA is in inflammatory diseases could be promising for designing new therapies.
  • mice in C57BL/10.RIII or C57BL/6 were bred and kept at conventional animal facilities at the University of Copenhagen. Ifnbaf ⁇ -mlce were from B&K Universal, UK. NOG
  • mice were from Taconic and The Jackson Laboratroy, respectively. Experiments were performed in accordance with the ethical committees in Copenhagen, Denmark and approved by the respective Institutional Review Boards, approval number
  • mice were irradiated
  • mice received co-transfer of either 2x10 6 in 300 ⁇ of PBS of control T cells, or purified T RFOX AI S - At day 0 and 2, each animal was given an i.p. injection of 500 ng of pertussis toxin. Active MOG35. 55 .EAE in C57BL/6-/fnt) "/" and WT were induced as previously described 7 . Mice received i.p. injection of mouse recombinatantlFN- ⁇
  • mice aged 8-15 weeks were immunized with 250 ⁇ g of MBP 8 9-ioi emulsified in 50 ⁇ of PBS and 50 ⁇ of CFA.
  • mice were injected with 100 ⁇ g of MBP 89- 10 i (in PBS) + TRF OX A-I S (3X1 O 4 cells/ear) in the right ear or 100 ⁇ g of MBP 89-10 i + control T-cells (3x10 4 cells/ear) in the left ear.
  • Control mice received an injection of 100 ⁇ g of MBP 89-10 i in the left ear and PBS + control T cells (3x10 4 cells/ear) in the right ear.
  • DTH response was measured as the difference in thickness (mm) of the right and left ears.
  • Data for the control T-cell group are presented as: (ear thickness after injection with MBP 89-10 i + control T cells) - (ear thickness after injection with MBP 89-10 i ).
  • Data for the TRF OX A-I S group are presented as: (ear thickness injected with MBP 89-10 i + T RFOX AI S ) - (ear thickness after injection with MBP 89-10 i ).
  • brains and spinal cords were dissected and infiltrating cells isolated as described 7 .
  • FoxA1 was synthesized by Geneart into pMA with 5' Hind III and 3' Not I sites. FoxA1 was transferred to the mammalian expression vector pCDNA3.1 (Invitrogen) by standard cloning techniques.
  • Purified CD4 + T-cells were transfected with pcDNA3.1foxa1 or controlpcDNA3.1 using the Amaxa mouse T-cells Nucleofector Kit (DPA-1007) (program X-001 ) according to the manufacturer's instructions. The transfection efficiency was evaluated by FoxA1 staining and FACS analysis.
  • Proteins were extracted from pcDNA3.1foxa1 transfected T RFo xAis and pcDNA3.1 transfected non-T RFo xAis- Standard procedures were followed 7 .
  • Brains and Spinal cords of mice with EAE were dissected and stained as described 13 . Slides were visualized under light microscopy.
  • Chromatin ImmunoPrecipitation was performed essentially as described 51 with the following modifications. Sonications were performed on a Biruptor Next Gen (Diagenode) set for 30s on, 30s off for 12 cycles. Goat IgG (Sigma) was used as a negative control for FoxA1 antibody (Abeam, ab5089).
  • the ChlPed DNA was purified on QIAquick PCR purification kit (Qiagen cat no 28104) and qPCR was performed with primers (table V) with the Lightcycle 480 DNA SYBR Green I Master Mix (Roche). Primers for the selected sequences were designed using Primer3 (v. 0.4.0).
  • FoxA1 -transduced 3T3L1 -cells were used to extract nuclear fraction 52 .
  • Pdl1-B was identified from ChlP-seq peak data in ZR751 cells 17 . The precise location of FoxA1 binding sequence was predicted using Clover and ContraV2.
  • Pdl1-A and Pdl1-B EMSA probes sequence were selected from genomic mm9 assembly (UCSC) (Table VI) and are within the amplicon of the Pdl1-A and Pdl1-B primer pairs used in the ChIP assay. EMSA was run as described previously . FoxA1 antibodies were (2F83) (Millipore, 05-
  • RRMS-NR 7 females/2 males; mean age: 37.1 years [8.6]
  • Patients with RRMS were classified as good responders to IFN- ⁇ based on the absence of relapses and no progression on the EDSS score during the first two years of treatment 41 .
  • PBMC peripheral blood was collected by standard venipuncture into vacuum tubes with EDTA.
  • PBMC peripheral blood cells were isolated by Ficoll-lsopaque density gradient centrifugation (Gibco-BRL) and freshly used or stored in liquid nitrogen until used.
  • PBMC were collected at baseline and 24 mo after IFN ⁇ -treatment.
  • Purified CD4 + T cells from healthy donors were transfected with 100 nM of a PD- I siRNA or a control-siRNA using the Amaxa Human T-cells Nucleofector Kit (VPA- 1002) (program U-014) according to the manufacturer's instructions.
  • VPA- 1002 Amaxa Human T-cells Nucleofector Kit
  • siRNA Accell SMART pool small-interfering RNA (siRNA, combines four different siRNAs) (Foxal -poolsiRNA; cat:B-005000-100, nontargeting controlsiRNA; cat:D- 001910-01 -05, Dharmacon, Thermo Scientific) was introduced into purified CD4 + T-cells according to manufacturer's protocol. Delivery efficiency and siRNA specificity were examined by intracellular staining of FoxA1.
  • PBMC peripheral blood mononuclear cells
  • IFN- ⁇ human recombinant IFN- ⁇
  • PBMC or from in vitro IFN-p-induced, lymphocytes were purified with CD4 + T Cell Isolation Kit II (Miltenyi Biotec, Cat. 130-091 -155), stained with anti-CD4, anti-TCR and anti-PD-L1 Abs for 20 min at 4°C in the dark.
  • T RFoxA is (TCRap + CD4 + PD-L1 high ) were purified utilizing a FACSAria sorting program.
  • TRF OX A-I S and non-T RF oxAis were generated utilizing purified CD4 + T-cells treated with mlFN- ⁇ (100U/ml) or hlFN- ⁇ (1000U/ml) for 48h, then sorted with FACSaria and applied to slides, and subsequently stained with antibodies.
  • Murine T RFo xAis obtained either from mlFN- ⁇ (100U/ml) treated CD4 + T-cells (for 48 h), or purified CD4 + T-cells from WT or Ifnaf' ' mice, or purified CD4 + T-cells were transfected with pcDNA3.1 FoxA1 or its controlpcDNA3.1 , or with foxal siRNA or its controlsiRNA.
  • Human T RFo xAis obtained from hlFN- ⁇ (1000U/ml) treated CD4 + T-cells (for 48 h) or purified from RRMS-R patients treated with IFN- ⁇ . ResponderT-cells
  • T-cells from either mouse spleens or corresponding human peripheral blood
  • CFSE CFSE
  • plate-bound anti-CD3/anti-CD28 for 24 h.
  • T RF oxAis were purified by FACSAria, labeled with Texas-red tracker (Genovis) for some experiments.
  • Suppressor and responderT-cells were co-cultured in a new culture plate without any antibody at a 1 :1 ratio. After 24h, cells were stained with violet dead cell marker (Invitrogen) or 7AAD and analyzed by FACS.
  • mice were generated by transferring OTII-responderT-cells to NOG mice. 24h later mice received foxal siRNA, controlsiRNA tranfected, WT or Ifnar' CD4 + T-cells, with or without an in vivo injection of mlFN- ⁇ . 24h later the suppression was assayed.
  • mice develop more severe relapsing EAE than WT mice.
  • Relapsing frequency Number of relapsing mice are calculated from total number of mice.
  • Recovery frequency Number of recovered mice per total number of mice.
  • Mean cumulative CS (clinical score): was calculated by summing up each individual score registered during the follow-up period till day 51 divided by the number of mice per group.
  • Table II Differential expression of an array of selected genes in CD4 'PD-Ll 1 T cells versus encephalitogenic (EncT cells/MBPj loi-specific) progenitor CD4 + T cells.
  • Table III Top Canonical Pathways overrepresented in nT RFO xAi versus EncT cells.
  • GSEA analysis was quantile normalized and summarized for each comparison using justPlier implementation of Plier algorithm in R.
  • ES enrichment score
  • NES normalized enrichment score
  • NOM p-val nominal p value
  • FDR q-val False Discovery Rate
  • FWER p-val family- wise error rate
  • RANK AT MAX position ' the ranked list at which the maximum enrichment score occurred.
  • Table V FACS characterization of murine nT RFO xAi cells.
  • Mean cumulative CS (clinical score): was calculated by summing up each individual score registered during the follow-up period until day 24 divided by the number of mice per group.
  • Table IX Top Canonical Pathways OTerrep resented in HTRF OS AI among commonly regulated genes by different TRF 0I AI cells.
  • Table X Top Canonical Pathways overrep resented In IFNP-lnduced/lTRFoiAi among commonly regulated genes by different TEFOIAI cells.
  • Table XL T RF O XAI cells suppress adoptive EAE in Ifnb " mice mediated by FoxAl and PD-L1 signaling.
  • Control CD3 activated T cells, with ctrl siRNA+pcDNA3.1), ITRF OXA I (IFN- ⁇ treated 48 hours, with ctrl siRNA), iTRp oxA1 (sifoxal KD for 24 hour + IFN- ⁇ treated 48 hours), iTRF 0xA1 (sipdll KD for 24 hour + IFN- ⁇ treated 48 hours) and TRP oxA1 (foxal siRNA+pcDNA3.1 foxal) cells were co-transferred each time with EncT cells. The results are sum of 3 independent experiments.
  • Mean cumulative CS (clinical score): was calculated by summing up each individual score registered during the follow-up period until day 40 divided by the number of mice per group.
  • Forkhead-box protein A1 a marker of luminal A type breast cancer, parallels low Oncotype DX 21 -gene recurrence scores. Mod Pathol 23, 270-275 (2010).
  • Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses. Immunity 27, 1 1 1 -122 (2007).

Abstract

The present invention relates to the identification of the transcription factor FoxA1 as a lineage-specification factor that defines a novel population of immunosuppressive T cells (FoxA+ T cells), and the use of FoxA1 and FoxA+ T cells in the treatment of inflammatory diseases or disorders.

Description

Immunosuppressive FoxA1 -expressing T cells Field of invention
The present invention relates to the identification of FoxA1 as a lineage-specification factor that defines a novel population of immunosuppressive T cells (FoxA1 + T cells), and the use of FoxA1 and FoxA1 + T cells in the treatment of inflammatory disorders.
Background of invention
Immune function that preserves tolerance while retaining antimicrobial function is imperative for preventing chronic inflammation and autoimmunity. Regulation of inflammation is critical for the disease management in tissue-specific chronic inflammatory diseases. This is naturally achieved when inflammatory counteracting, functionally intact immune cells are generated. The immune system in vertebrates is composed of the innate immune system and the adaptive immune system. The cells of the adaptive immune system are special types of leukocytes, called lymphocytes. B cells and T cells are the major types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow. B cells are involved in the humoral immune response, whereas T cells are involved in cell-mediated immune response. Subpopulations of T cells with distinct functions and expression patterns exist, including helper T cells, cytotoxic T cells and regulatory T cells.
Inflammatory abnormalities are a large group of disorders which underlie a vast variety of human diseases. The immune system is often involved with inflammatory disorders, demonstrated in both allergic reactions and some myopathies, with many immune system disorders resulting in abnormal inflammation.
Inflammation is a localized defensive response of the body against pathogens and injury. Immune cells and soluble factors take part in this process to neutralize the injurious agent and initiate tissue repair to restore homeostasis. Loss of regulation of these mechanisms can prevent the final resolution of the inflammatory process, leading to chronic inflammation. Chronic inflammation is extremely relevant in today's modern medicine, as it contributes to the pathogenesis of the most important diseases of the industrialized societies including atherosclerosis, acute and chronic heart failure, cancer, diabetes, and obesity-associated diseases.
Multiple sclerosis (MS) is a multifocal demyelinating disease of the central nervous system pathologically characterized by lesions of infiltrating macrophages and T cells. Multiple lines of evidence implicate that T cells play a central role in both mediating and regulating MS pathophysiology, and efforts to develop rational therapeutic strategies for MS have focused on understanding factors which control T cell function. T cells are a highly heterogeneous population comprised of multiple cell subtypes which mediate both adaptive immunity and specific tolerance. Much has been learned about the molecular signals that induce T cell activation and differentiation, and several effective treatments for MS act by altering these activation and differentiation pathways. In recent years, increasing recognition has been given to T cell subsets which serve immunosuppressive or regulatory functions, and it has been proposed that patients with MS have a functional defect in these cells. Current work is beginning to shed light on interactions of pathogenic and regulatory T cells with the intrinsic cells of the CNS to provide a more comprehensive picture of MS pathogenesis (see e.g. Severson & Hafler, Results Probl Cell Differ. 2010;51 :75-98). While there is no cure for MS, current treatments comprise disease-modifying agents, with interferon beta and glatiramer acetate are first-line treatments with modest effects in reducing attacks but to some extent reduce relapses. The disease-modifying treatments have several adverse effects. Immunization with irradiated autologous T cells (T cell vaccination) is shown to induce regulatory T cell responses that are poorly understood. FoxP3 is a lineage specification factor for regulatory T cells. Hong et al. 2006 (PNAS vol. 103 no. 13, p.5024-5029) discloses a regulatory T cell subpopulation identified in samples from T cell vaccinated MS patients, characterized as CD4+CD25+Foxp3~ T cells. These cells express IL-10.
FoxA1 is an epigenetic-modifying pioneer transcription factor important in embryonic development, stem cell differentiation, hepatocyte development, and cancer epigenetics. FoxA1 modulates postembryonic tissue functions, including the brain. FoxA1 is necessary for epithelial cells, and mediates lineage specification. FoxA1 overexpression has been associated with tumorigenesis at least in prostate cancer, esophageal and lung adenocarcinomas, invasive bladder cancer and breast cancer. However, no definite function has previously been reported for FoxA1 with respect to T cells.
Summary of invention
The present inventors have shown that FoxA1 is a lineage-specification factor that defines a subpopulation of immunosuppressive T cells (denoted FoxA1 + T cells herein, identical to TRFOXAI cells as used in the figure legends and the examples) and direct function of these novel T cells. FoxA1 thus has a specialized role in supporting the differentiation and the suppressive function of FoxA1 + T cells.
FoxA1 -overexpression in T-cells confers suppressive functions and a distinct FoxA1 + T cell phenotype. The FoxA1 + T cells are in one embodiment negative for FoxP3, CTLA4, TGF-β, IL-10 and IL-35 commonly associated with regulatory T cells, Tregs, and in addition to expressing FoxA1 , in one embodiment they are CD4hl, PD-L1 hl and CD47+.
Mice lacking Ifnb and Ifna/b-receptor genes develop chronic inflammatory and demyelinating experimental autoimmune encephalomyelitis EAE (Ifnb''' mice), a mouse model for MS. The FoxA1 + T cells were identified in wild type mice but absent in Ifnb''' mice. In Ifnb''' mice, adoptive transfer of stable FoxA1 + T cells was shown to inhibit CNS inflammation and EAE. Interestingly, IFN-β can induce FoxA1 and FoxA1 - mediated PD-L1 , leading to generation of the FoxA1 + T cells, and as shown herein in patients with multiple sclerosis, response to IFN-β treatment was associated with expansion of the suppressive FoxA1 + T cells.
The present inventors have shown that FoxA1 + T cells can suppress T cell activation, inhibit proliferation and induce cell death in activated T cells (in vitro). Also, an inhibitory effect is shown not only in a MS model but also for inflammatory bowel disease (IBD), delayed type hypersensitivity (DTH) and glioblastoma multiforme.
As the herein identified immunosuppressive FoxA1 + T cells inhibit T cell activation and reduce inflammation in various models thereof, they have the potential to treat several inflammatory diseases and disorders. It is an aspect of the present invention to provide immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment of FoxA1 (FoxA1 + T cells). Said immunosuppressive T cells may further be positive for (+ or hl staining) one or more of CD4, PD-L1 , PD-1 , CD47, CD69, CD25, CD45Rb and TCRa3, and negative for FoxP3.
Also provided is a recombinant cell, such as a recombinant T cell, comprising a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof. A further aspect of the present invention is to provide a method of making the immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment of FoxA1 , said method comprising one or more steps of
i. Introducing into T cells FoxA1 protein, or a functional variant or functional
fragment thereof,
ii. Introducing into T cells a nucleic acid construct encoding FoxA1 or a functional variant or functional fragment thereof, and/or
iii. treating T cells with an effective amount of IFN-β.
The present invention also relates to a method of treating an inflammatory disease or disorder, said method comprising administering to an individual in need thereof an effective amount of a bioactive agent selected from the group consisting of
i. FoxA1 protein, or a functional fragment or functional variant thereof;
ii. a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or
iii. immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof.
It is also an aspect of the present invention to provide a method of treating an inflammatory disease or disorder, said method comprising
a) collecting T cells from an individual having an inflammatory disease or disorder, and
b) transforming said collected T cells into immunosuppressive T cells comprising or expressing FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells) a. by introducing into said collected T cells FoxA1 protein, or a functional fragment or functional variant thereof; or a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof, and/or
b. by treating said collected T cells with an effective amount of IFN-β; and c) re-introducing said immunosuppressive T cells comprising or expressing
FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), to said individual having an inflammatory disease or disorder. An inflammatory disease or disorder to be treated according to the present invention is a disease or disorder having an inflammatory component, an inflammatory abnormality and/or an etiological origin in inflammatory processes. They comprise at least autoimmune disorders, multiple sclerosis, CNS disorders with an inflammatory component including glioblastoma multiforme, inflammatory diseases of the
gastrointestinal system including inflammatory bowel diseases, delayed type hypersensitivity (DTH)-related disorders such as psoriasis, allergy and diabetes mellitus type I, and cancer.
Yet another aspect is the provision of a method of distinguishing an IFN-β non- responding multiple sclerosis patient (MS-NR) from an IFN-β responding multiple sclerosis patient (MS-R), said method comprising one or more steps of
a. treating a MS patient with an effective amount of IFN-β,
b. collecting a sample from said MS patient, and
c. identifying if immunosuppressive FoxA1 + T cells are present in said
sample,
wherein the positive identification or presence of immunosuppressive FoxA1 + T cells in said sample is indicative of said MS being IFN^-responsive, and negative identification or absence of immunosuppressive FoxA1 + T cells in said sample is indicative of said MS being IFN^-non-responsive. IFN-β treatment may be discontinued if said MS patient is characterized as an IFN-β non-responding multiple sclerosis. Description of Drawings
Figure 1. CD4hlPD-L1hl, a novel population of T cells absent in the inflamed CNS of lfnb'1' mice, (a) Ifnb''' mice develop more severe RR-EAE than WT. EAE was induced with MPB89-ioi - Data are the mean of 20-21 mice per group (N WT = 21 mice, N ifnb-/- = 20 mice) from two independent experiments, **P < 0.01 . One-way ANOVA
Kruskal-Wallis test with multiple comparisons was used, (b) Spinal cord and brain cryo- sections from Ifnb''' and WT mice were stained with anti-TCRp and detected by diaminobenzidine (brown). Hematoxylin (blue) was used for the counterstain.
Micrographs represent 3 individuals in each group. Scale bar = 100 μηη. (c) FACS gating strategy for TCRp+CD4+T cells in d-g. (d) CD4hiPD-L1 hi T-cells, but not Tregs, are severely reduced in Ifnb''' mice. Tregs (CD4+CD25+Foxp3+) and CD4hiPD-L1 hi T-cells from CNS infiltrating T-cells of WT and Ifnb''' mice, 20 days after EAE induction, (e) CNS-infiltrating CD4hiPD-L1 hi T-cells (R1 -gated) are Foxp3\ CD4+PD-L1 '° cells (R2- gated) express Foxp3. Data (c-e) represent three independent experiments, (f) Time- course of CD4hiPD-L1 hi T-cells in WT and Ifnb'1' mice CNS during RR-EAE. (g)
Enrichment of CD4hlPD-L1 hlT cells in inflamed CNS. T-cells were harvested from draining lymph nodes (LN) and spleen (SP) of WT mice after EAE induction. Data (f, g) are mean ± SD from two independent experiments; each sample was pooled from 2 CNS tissues (total 20 mice, samples size=10) for FACS-staining. ***P < 0.001 , Two- way ANOVA with post Tukey's multiple comparisons test.
Figure 2. TRFOXAI have a distinct transcription profile with FoxA1+ and suppress skin and CNS inflammation, (a) Representative FACS dot plots of CD4hiPD-L1 hi T-cell generation upon co-culture of MPB89-ioi -reactive EncT-cells with CGNs (cerebellar granular neurons). Lymph nodes from EAE mice were analyzed directly (ex vivo EncT- cells), or after 48 h with recall antigen (1st stimulation EncT-cells). Activated EncT-cells were restimulated with antigen-loaded APCs for 96 h without CGNs (EncT-cell line), or co-cultured with CGNs (EncT-cell line+N). Data are from four independent
experiments, (b) Signal intensity scatter plots from mouse Affymetrix 430 2.0 arrays hybridized with RNA from EncT-cells, FACSAria-purified CD4hiPD-L1 hi T-cells and Tregs (CD4+CD25+ and membrane-bound TGF-p+) after co-culture with CGNs. Signal intensities (log2) analyzed by unpaired two-tailed Student's i-test for independent triplicates filtered for 95% confidence of differential gene expression (P≤ 0.05).
Differences in Y/X-axis expression: >2-fold, red; 0, yellow; <2-fold, blue; differential expression of 1.5-fold or higher at P≤ 0.05, white, (c) Transcription differences of EncT-progenitors, CD4hlPD-L1 hl T-cells, and TregS. One ChannelGUI was used for analysis of Affymetrix probe sets determined by up- or down-regulation of at least >1.5- fold vs. <0.67-fold at P <0.05. Unpaired two-tailed Student's i-test was used for independent triplicates, (d) Histogram of FACS analysis for CD47, CD69 and nuclear- FoxA1 expression in CD4hiPD-L1 hi (R1 , TRFoxAis) and CD4|0PD-L1 l0 (R2, non-TRFoxAis). Data are representative of three independent experiments, (e) TRFOXAIS (R1 ) compared to controls (R2) are nonproliferating by FACS for Ki-67+. Data are mean ± SD of three independent experiments, ***P < 0.001 . Unpaired two-tailed Student's i-test was used. Ectopic overexpression of FoxA1 in purified CD4+T cells downregulates c-Fos signaling, (f) Real time PCR of c-fos expression in pcDNA3.1foxA1 -transfected murine CD4+T cells (TRFoxA1s). Data are mean ± SD of duplicates. One representative experiment is shown from two independent experiments, (g) Representative Western blot of FoxA1 after transfection are shown from two independent experiments. Total c- Fos after pcDNA3.1foxA1 transfection was reduced compared to vector transfection. (h) Representative fluorescent immunocytochemistry micrographs of overexpressed FoxA1 nuclear localization in TRFoxAis which showed no nuclear pc-Fos, compared to control. Scale bar = 10 μηη. Micrographs represent 4 individuals in each group, (i) FACS of FoxA1 and pc-Fos in pcDNA3.1foxA1 -transfected TRFoxAis compared with vector-transfected CD4+ T-cells. Representative data are from three independent experiments.
(i-k) TRFnXAis suppress activated T-cells in vitro. Purified CD4+T cells were labeled with CFSE and activated for 24 h (responder T cells/Res), then co-cultured with purified CGN-induced TRFoxA1s. (]) Cell proliferation measured by CFSE. (k) Cell death assessed by 7AAD staining, (j-k) Data are mean ± SD from three independent experiments, ***P < 0.001 , unpaired two-tailed Student's i-test was used.
(l-m) TggnxA s suppress tissue inflammation in vivo. (I) DTH measured as ear thickness at 48 h post injection. Data are representative of three independent experiments. Bars show means ± SD of 3 mice. ***P < 0.001 , unpaired two-tailed Student's i-test was used, (m) nTRFoxAis inhibit CNS inflammation and EAE progression. EAE was adoptively transferred to irradiated WT and Ifnb''' C57BL10RIII mice as 2 x 106 MBP89- ioi-specific EncT-cells, with 2 x 106 purified nTRFoxA1s or control T-cells. Data are the mean clinical score from 5 mice. ***P < 0.001 , \fnb''' mice developed significant EAE than WT; ***P < 0.001 , nTRFoxAI of Ifnb''' mice reduced significantly EAE compared to control Ifnb''' mice. One-way ANOVA Kruskal-Wallis test with multiple comparisons was used, (n) Representative micrographs show more infiltrating T-cells (brown) in controls for Ifnb mice in the spinal cord. Micrographs represent 3 individuals in each group. Scale bar = 100 μηι.
Figure 3. IFN-β induces suppressive TRFoxAis that require IFN-β signaling.
IFN-p-treatment of Ifnb''' mice with EAE leads to in vivo generation of TRFOXA S. (a-c)
MOG35-55-EAE is associated with generation of TRFOXAI cells in the CNS of C57BL/6 WT but not Ifnb''' mice. Ifnb''' mice treated with recombinant mlFN-β (5000 U/ml x 3 times) generated TRFOXAIS in the CNS. (a-b) Representative FACS dot plots of CD4+PD-L1 hi and histogram of CD4+FoxA1 + from 5 FACS samples, (c) Quantification of CNS TRFOXAIS- Data are mean ± SD of 5 mice per group, *P < 0.05, **P < 0.01 , ***P < 0.001 using one-way ANOVA with Newman-Keuls post hoc test for multiple comparison correction. IFN-β induces FoxA1 in murine and human CD4+T cells (d-i) Purified murine CD4+T cells with or without recombinant mlFN-β 100 U/ml for 48 h. (d)
Representative FACS dot plots from three different experiments shows mlFN-β inducing TRFoxAis (Τ^αβ+ΰϋ4+ΡΟ-Ι_1 hi T-cells). (e) FACS histogram from three different experiments shows FoxA1 + mlFN^-induced TRFoxAis compared to nontreated cells, (f) Quantification of FoxA1 +CD4+T cells upon mlFN^-stimulation. Data are mean ± SD from three different experiments. *** P≤ 0.001 , unpaired two-tailed Student's t- test was used, (g) Representative fluorescent-immunocytochemistry micrographs of nuclear localization of high FoxA1 expression in mlFN^-induced TRFoxAis VS.
unstimulated cells. Micrographs represent 4 individuals in each group. Scale bar = 10 μηη. (h-i) Representative FACS micrograph and quantification of gated R1 and R2 after mlFN^-stimulation of CD4+T cells for 48 hours. FoxA1 expressed exclusively in R1 (TCRaJ3+CD4+PD-L1 hl) TRFoxAis- Data are from three different experiments, bars are mean ± SD. *P < 0.05 using Student's unpaired i-test. (j-l) Human IFN-β induces IRFOXA-IS in vitro, (j) Purified CD4+ T cells from healthy donors with or without hlFN-β 1000 U/ml for 3 days, (k) Quantification of TCRαβ+CD4+PD-L1 hi TRFoXA1 with or without hlFN-β stimulation. Horizontal lines, mean ± SD. ** P≤ 0.01 , Student's unpaired i-test. N=4. (I) Representative fluorescent-immunocytochemistry micrographs from 4 individuals in each group shows high FoxA1 expression nuclear localization in purified hlFN-p-induced TCRαβ+CD4+PD-L1 hi TRFoXA1s ( 1 ) vs. non-TRFoxAis (R2). Scale bar = 10 μηη. (m-o) mlFN-β induces FoxA1 + in purified CD4+T cells leading to TgfnxA s that require functional IFNA-receptors in vitro and in vivo, (m) mlFN-β induces FoxA1 and RFoxAis in purified WT-CD4+T cells but not purified lfnaf''-CD4+T cells in vitro, (n-o) Purified WT-CD4+ T cells and / harA-CD4+ T cells i.v. transferred to immune-deficient NOG mice with or without mlFN-β. Splenocytes were analyzed after 24 h. mlFN-β directly induced FoxA1 in WT-CD4+T cells in vivo, but not in lfnaf -CD4+ T cells, (o) Percent FoxA1 + cells after mlFN-β. Bars, mean ± SD. N=3 mice/group. **P < 0.01 , using one-way ANOVA with Newman-Keuls post hoc test for multiple comparison correction, (p-w) IFN-p-generated TRFOXA S are suppressive in vitro and in vivo, (p-s) CFSE-labeled purified CD4+ T cells from healthy donors were activated with plate- coated anti-CD3 for 24 h for responder T cells/Res, then co-cultured with purified hlFN- β-induced TRFoxAis (R ) for 24 h. (p) Representative FACS histogram of responder T- cell proliferation. Responder T cells proliferated when co-cultured with control non- RFOXA-IS (purified R2), but not upon co-culture with TRFOXAIS (purified R1 ). Representative histograms are from three individual experiments, (q) Percent suppression as
Res+TRFoxAis/Res+non-TRFoxAis.X100. Bars, mean ± SD. N=3. ***p < 0.001. Unpaired two-tailed Student's i-test was used, (r) Representative FACS histogram of PI staining for cell cycle in responder T-cells. Representative histograms are from three individual experiments, (s) Quantified percent responder T-cells in cell cycle phases. Purified TRFOXA-IS suppress S phase and induce sub-G1. Bars, mean ± SD. N=3. *P < 0.05.
Unpaired two-tailed Student's i-test was used, (t) CFSE-labeled purified murine CD4+ T cells were activated with plate-coated anti-CD3 for 24 h for responder T cells, then co- cultured with purified TRFOXAI cells for 24 h. FACS histogram of responder T-cell proliferation are representative of three individual experiments, (u) Percent gated CFSE with significant suppression of proliferation triggered by TRFOXAI- Bars, mean ± SD. N=3, ***P < 0.001 , using one-way ANOVA with Newman-Keuls post hoc test for multiple comparison correction, (v-w) /fr?arA-CD4+ T-cells do not suppress activated responder T-cells in chimeric mice. NOG mice were i.v. injected with 1 x 106 CFSE- labeled responder T-cells (pre-activated with anti-CD3 1 μg ml and anti-CD28 10 μg ml). After 24 h, 4 groups of chimeric mice received WT or Ifnaf'' CD4+ T-cells with or without mlFN-β (i.v.). Lymphocytes from spleens were analyzed after 24 h. (v)
Representative FACS histograms of showing proliferative cells from activated responder T-cells from chimeric mice receiving 1 x 106 CD4+ T- cells from WT or Ifnaf'' mice, with or without mlFN-β. (w) WT CD4+T-cells with mlFN-β suppressed
proliferation of activated responder T-cells. Bars, mean ± SD. N=3. *P < 0.05 using one-way ANOVA with Newman-Keuls post hoc test for multiple comparison correction, (x-z) Common gene profile of TgFnxAi cells, (x) Venn diagram of overlap between differentially regulated probe sets that are regulated in the same direction in iTRFoXAi and nTRFoxAi vs. EncT-cells. (y) 936 common probe sets of TRFoxAis VS. EncT cells are compiled to determine TRFOXAIS' heatmap profile, (z) Heatmap of genes commonly regulated by iTRFoxAi and nTRFoXAi vs. EncT-cells that are involved in commonly regulated pathways determined by GSEA. Data are from triplicates.
Figure 4. FoxA1 is essential for generation and function of suppressive TRFoxAis- FoxA1 silencing prevents IFN-p-induced PD-L1 hl expression, (a) Representative FACS histogram of FoxAl siRNA KD in murine CD4+T cells after 72 h. (b) Representative FACS histogram of FoxAl siRNA KD effect on IFN-p-induced PD-L1 hi expression on CD4+ T-cells. (a-b) Data are representative of three individual experiments, (c) In vitro suppressive function of IFN-p-induced TgFnxA s is FoxA1 dependent. Percent suppression as "Res+CD4+ T (ctrl siRNA)+mlFN-p" or "Res+CD4+ T
(foxa1 siRNA)+mlFN-p" / "Res+CD4+ T (ctrl)-mlFN-p" X100. Bars, mean ± SD. N=3. **P < 0.01 , ***P < 0.001 by one-way ANOVA with Newman-Keuls post hoc test for multiple comparison correction, (d-f) In vivo IFN-p-induced TgFnxA s require FoxAL
NOG mice were i.v. injected with 1 x 106 FoxA1 siRNA KD- or CtrlsiRNA-CD4+ T-cells with or without mlFN-β (100 U/ml). (d) Control CD4+ T-cells (CtrlsiRNA) but not CD4+T cells lacking FoxA1 (FoxAl siRNA KD) generated TRFOXAIS after in vivo mlFN-β. Bars, mean ± SD, N=3 mice/group. **P < 0.01 , ***P < 0.001 by one-way ANOVA with Newman-Keuls post hoc test for multiple comparison correction, (e) CFSE FACS profile of NOG mice receiving CtrlsiRNA CD4+ T-cells but not FoxAl siRNA CD4+ T-cells suppressing responder T-cell proliferation after mlFN-β treatment, (f) Percent of proliferative cells are shown as mean ± SD. N=3. ** P < 0.01 by one-way ANOVA with Newman-Keuls post hoc test for multiple comparison correction. Ectopic
overexpression of FoxA1 alone is sufficient to generate suppressive TgFnxA s. (g)
Representative FACS histogram of FoxA1 expression and transfection efficiency after 96 h. Murine CD4+ T-cells transfected with pcDNA3.1foxA1 or empty plasmid at FoxA1 transfection efficiency of 70.4% ± 5.3% from 4 individual experiments, (h) Purified murine CD4+ T-cells labeled with CFSE were activated for 24 h, and co-cultured with pcDNA3.1foxA1 -transfected TRFOXAIS or control T-cells for 24 h. Percent gated CFSE shows significant proliferation suppression by pcDNA3.1 FoxA1 -transfected TRFoxAis- Bars, means ± SD. ***P < 0.001 by Student's unpaired i-test, N = 3.
FoxA1 and PD-L1 are essential for suppressive function of TgFnxA s to prevent EAE (i- m). (i) Adoptive EAE was established in irradiated /fnt)"A-C57BL/6 mice by adoptive transfer of 5-10x106 splenocytes from MOG35-55 immunized mice, day 0 and 14. Groups received either equal number of CFSE-labeled Ctrl activated T cells
(ctrlsiRNA+pcDNA3.1 ), ίΤΚίοχΑι (ctrlsiRNA), iTRfoxA1 (foxal siRNA), ίΤΚίοχΑι
(pdl1 siRNA+antiPDL1 ), or TRf0xAi (pcDNA3.1foxa1 ). Data are the mean from three independent experiments. N=22, 9, 13, 8 and 10 mice in respective groups. ***P < 0.001 , mice receiving iTRFoxAis (IFN-β treated for 48 hours, with ctrl siRNA) had significantly less EAE compared to all control groups. ***P < 0.001 , mice receiving RFOXA-IS (foxal siRNA+pcDNA3.1 foxal) had significantly less EAE compared to its control group. One-way ANOVA Kruskal-Wallis test with multiple comparisons was used, (j) One representative micrograph of H&E staining per group (3-6/group) illustrates prevention of inflammatory cell infiltrates (arrows) in spinal cords of mice treated with TRFOXAIS, day 40 post adoptive EAE. Scale bar = 100 μηι. (k) One representative histogram of FACS shows proliferative cells in CFSE+ gated splenocytes from different groups. (I) Percent proliferative cells after 40 days induction of EAE were found in splenocytes. Bars, mean ± SD. N=5 mice/group. **P < 0.01 , ***P < 0.001. One-way ANOVA with Newman Keuls post-test was used. A representative gated CFSE+ (m) iTRf0XAi (ctrlsiRNA) and (n) TRFoxAis (foxal siRNA+pcDNA3.1 foxal ) show stable profile by positive expression of FoxA1 but not FoxP3, 40 days post adoptive in vivo transfer. Data are representative from 6 FACS samples (N=3 mice, duplicates).
Figure 5. Ectopic FoxA1 generates suppressive TRFOXAIS that induce T-cell death via PD-L1 and caspase signaling.
(a-b) Murine TgFnxA s suppress T-cell proliferation and induce cell-death via PD-1 -PD- L1 . (a) CFSE-labeled activated responder T-cells were co-cultured with purified murine TRFOXA-IS with or without anti-PD-L1 (5 μg ml). FACS histograms represent three experiments. Percent proliferative (CFSE) or dead (LIVE/DEAD) responder T-cells; error bars, SD; *P < 0.05, **P < 0.01 , ***P < 0.001 by Student's unpaired i-tests. (b) TRFOXA-IS were co-cultured with responder T-cells for 24 h with PD-L1 antibodies, PD-1 (5 μg ml), B7.1 , B7.2 (10 μg ml), or an isotype control (10 μg ml). Bars, percent 7AAD+ cells gated on responder T-cells (CFSE). Data are mean ± SD of three independent experiments, ***P < 0.001 by Student's unpaired i-tests. (c-e) Human TRFOXAIS induce suppression and cell-death of activated T cells through PD-1 -PD-L1 . CFSE-labeled purified human CD4+ T-cells from healthy donors were transfected with control siRNA (UNC) or PD-1 siRNA (PD-1 KD) for 3 days, then activated with plate-coated anti-CD3 for 24 h (responder T-cells/Res). Responder T-cells were co-cultured with purified hlFN-p-induced TRFoxAis from the same donor for 24 h. (c) Representative CFSE-FACS histogram of hTRFoxAis inhibiting responder T-cell proliferation. PD-1 silencing in responder T-cells abrogated suppression by hTRFoxAis- (d) Percent pAKT and (e) cleaved caspase3 in responder T-cells. Percent positive expression in control was 1. pAKT and cleaved caspase3 expression are % relative to control. Bars, mean ± SD. *P < 0.05, ***P < 0.001 by Student's unpaired i-tests, N=3. (f-h) Ectopic
overexpression of FoxA1 in murine CD4+ T-cells generated suppressive TgFnxA s that required PD-L1 -PD-1 signaling for suppression, (f) Representative FACS dot plots of cleaved caspase3 in responder T cells co-cultured with pcDNA3.1foxa1 -transfected TRFOXA-IS compared with responder only. FACS of (g) ratio of cleaved caspase3 expression and (h) ratio of pAKT in responder T cells co-cultured with pcDNA3.1foxa1 - transfected TRFOXAIS with PD-L1 antibody (10 μg ml) or caspase inhibitor Z-VAD-FMK (4 μΜ). Bars, mean ± SD. *P < 0.05, **P < 0.01 , ***P < 0.001 by Student's unpaired t- tests, N=3. (i-o) FoxA1 binds the pdl1 promoter and regulates the pdl1 gene and PD- L1 expression, (i) Real time PCR of pdl1 mRNA expression in murine pcDNA3.1foxa1 - transfected TRFOXAIS compared to pc.DNA3.1 control-transfected cells. Data are mean ± SD from duplicates, (j) Representative FI-IC micrographs of PD-L1 in murine pcDNA3.1foxa1 -transfected TRFoXAis and control vector-transfected cells. Micrographs represent 4 individuals in each group. Scale bar = 10 μηη. (k) pdl1 locus. Chromosome 19 upstream of pdl1 in the mouse genome (mm9) with PD-L1 -A and PD-L1 -B EMSA probes and ChIP amplicons, FoxA1 ChlP-seq peak in ZR751 cells, converted to mm9 assembly using LiftOver in UCSC from hg18 assembly. FoxA1 binding sites selected using Clover. (I) Purified murine CD4+ T-cells transfected with pCDNA3.foxa1 or control (plasmid) analyzed by ChlP-qPCR of FoxA1 -occupied DNA. Results are percentage of input normalized for FoxA1 enrichment to IgG control. Data are one of three
comparable experiments. Bars are mean ± SD from duplicates, (m) FoxA1 binding to positive control is dose-dependently competed by PD-L1-B but not PD-L1-A. EMSA on nuclear extracts from FoxA1 -transfected 3T3 cells using labeled mTTR positive control probe and competing PD-L1-A and PD-L1-B probes. Nuclear extracts caused shifts, with supershifting by FoxA1 -specific antibody, but not IgG control. Shift dose- dependently competed using 10-, 100- or 1000-fold molar excess of unlabeled PD-L1- B, but not PD-L1-A. First lane (probe only) as negative control, (n) FoxA1 directly binds labeled PD-L1-B, but not PD-L1-A. EMSA on nuclear extracts from 3T3-FoxA1 cells with labeled PD-L1-A and PD-L1-B probes. Nuclear extract nonspecifically shifted PD- L1-A probe, different from the positive control, which was not supershifted with a FoxA1 -specific antibody. PD-L1-B shifted similar to the positive control, and
supershifted with FoxA1 antibody. First three lanes, probe-only negative controls; followed by supershifted positive control mTTR. Data are representative of two independent experiments, (o) FoxA1 positively regulates PD-L1 . 3T3-L1 cells were transfected with empty vector or PD-L1 -promoter reporter plasmids (pGL3basic, pGL3 PD-L1 prom). Co-transfection was conducted with empty mammalian expression vector (pCDNA3) or FoxA1 -containing expression vector (pCDNA3foxA1 ). Reporter assay shows FoxA1 regulated PD-L1 promoter. Data are one of two independent
experiments. Bar, mean relative luciferase activity (RLU/relative to renilla luciferase) with SD. * P < 0.05 by one-way ANOVA with Tukey's multiple testing correction.
Figure 6. IFN-β generates suppressive FoxA1 expressing TCRa +CD4+PD-L1hl RFoxAis i RRMS-R but not RRMS-NR patients, (a) Representative FACS
micrographs of TRFoxAis in PBMCs showing gating strategy. FACS of TRFoxAis on gated live cells, excluding duplet cells, then gated on surface expression of
TCRap+CD47+CD4hiPD-L1 hi T-cells. Percent CD4+CD47+PD-L1 hi TRFoxAis by FACS (b) at baseline, (c) after 24 months IFN-β. Dots are Percent (with mean) of TRFoxA1s. N=10, 9 and 16 per group, ψ P < 0.05, non-parametric Mann-Whitney test, (d) Percent RFoxAis before and after IFN-β treatment in RRMS-NR and RRMS-R patients, sampling blood from non-treated healthy controls at indicated time points served as control. N=9 and 16 per group respectively, ψ P < 0.05, non-parametric Mann-Whitney test, ** P < 0.01 Student's paired i-test to compare IFN-β treatment effect in groups at baseline versus 24 months of treatment.
Figure imgf000014_0001
responsiveness in RRMS-R patients, (e) Representative FACS micrograph of FoxA1 in gated TRFoxAis (Τ^αβ+ΰ04+ΡΟ-Ι_1 ί). (f) Percent FoxA1 + TRFoxAis before and after IFN-β in RRMS-NR and RRMS-R patients. N=7 and 15 per group respectively. ** P < 0.01 by Student's paired i-test comparing effect of IFN-β treatment in groups at baseline versus 24 months of treatment. Purified TggnxAi cells from RRMS-R inhibit proliferation and kill activated T-cells. (g-k) Co-culture of enriched CD4+ T-cells from peripheral blood of RRMS-R patients with purified TRFoxAis (gated on Τ^αβ+ΰϋ4+ΡΟ-Ι_1 hi). (g)
Representative FACS micrographs show gating strategy for TRFoxAi purification, (h) Representative FACS micrograph and (i) percent of proliferative responder T-cells (CFSE-labeled activated CD4+ T-cells from the same patient served as responder T- cells) after co-culture with purified TRFoxAis or non-TRFoxA1s from a RRMS-R patient, (j) Representative FACS micrograph of 7AAD+ responder T-cells after co-culture with purified TRFOXAIS- (k) Cell death as percent of 7AAD+ responder T-cells alone, after co- culture with non-TRFoxAis or TRFoxAis purified from an RRMS-R patient. Graphs are mean ± SD, representative of 3 individual patients. ** P < 0.01 , *** P < 0.001 by Student's unpaired i-test.
Figure 7. Ifnb-/- mice develop severe relapsing-remitting form of EAE. (a, b, c)
Disease development in five representative EAE-affected mice with different disease courses in Ifnb''', heterozygous (HT), and WT groups: mice developed different rate number of relapses, with different duration of remission in between. The mean score per group from these five selected individuals with EAE is also shown for the respective groups to demonstrate that due to variability in disease development, the mean score at each time point becomes relatively low, although all five clearly suffer from severe EAE. As no statistical differences were found between WT and HT mice, we will refer to them as WT in the rest of figures. Data are representative of two independent experiments (N = 5).
Figure 8. Tregs (CD4+CD25+Foxp3+) are not defective in Ifnb-/- mice capable of suppressing EAE. (a) Percent of CD4+Foxp3+ Treg cells in the CNS of C57BL/6 WT and Ifnb''' mice after active MOG35-55-induced EAE. N=3-5 mice per group, (b) Percent of CD4+Foxp3+ Treg cells in the CNS of C57BL/B10.RIII and C57BL/6 mice during peak of diseases after induction of EAE with MBP89-ioi and MOG35-55 respectively, (c) FACS dot plots show gating strategy for CD4+CD25+ T cells purification, which are also mainly Foxp3+ T cells, (d) CFSE-labeled purified murine CD4+ T cells were activated with plate-coated anti-CD3 antibody for 24 hours that served as responder T cells, then co-cultured with purified CD4+CD25+ T cells from WT and Ifnb''' mice for an additional 24 hours. Bars show the percent of proliferating responder T cells after co-cultures. Data are meansiSD, N=3, ***P < 0.001 , using one-way ANOVA with Newman-Keuls post hoc test for multiple comparison correction, (e) Adoptive EAE was established in irradiated Ifnb''' C57BL/6 mice by transferring 10x106 splenocytes from MOG35-55 immunized mice, day 0 and 13. Purified natural CD4+CD25+ Tregs, or TGF-3-induced Tregs were used for treatment. No functional differences were seen between nTregs and iTregs hence the results are combined. Groups received either equal number of Ctrl activated T-cells, Tregs from WT or Ifnb''' mice. Data are the mean of 12-14 mice per group. No differences were found in EAE-suppressive capacity of WT vs. Ifnb''' Tregs, but both groups equally suppressed EAE, *P < 0.05 using one-way ANOVA Kruskal-Wallis test with Dunn's multiple comparisons test for multiple comparison correction, (f) FACS dot plots show neuron-induced Tregs R1 -gated (CD4+TGF-31 + T cells) which are also Foxp3+ Treg cells (upper panels), isotype controls (lower panels). Figure 9. TRFoxAI has distinct gene profile than known Tregs. (a) nTRFoxAi
Transcription Factor profile. Plot of transcription factor genes differentially regulated in nTRFoxAi vs. their progenitor EncT-cells. Y-axis is log2 of fold change. The only relevant transcription factors known to regulate Treg signature (Fu et al. 2012 Nat Imm) were found to be downregulated in ηΤΚΡοΧΑΐ indicated in red. (b) Heatmap of ηΤΚΡοΧΑΐ gene profile. Genes on the y-axis correspond to nTRFoXAi gene profile, which is based on differentially regulated genes between nTRFoXAi cells and EncT-cells. (c) Heatmap of nTRFoxAi gene profile in comparison to the published gene expression sets of n/iTregS. All included gene sets are normalized together. Genes on the y-axis correspond to nTRFoxAi profile, (d) Heatmap of Treg signature genes (the published gene expression sets) in comparison to nTRFoxAi data sets. All included gene sets are normalized together. Genes on the y-axis correspond to Treg signature (Hill et al. 2007 Imm). (e) Heatmap of nTreg signature genes in comparison to iTregs (the published gene expression sets). All included gene sets are normalized together. Genes on the y-axis correspond to Treg signature (Hill et al. 2007 Imm).
Figure 10. TRFoxAls have distinct gene signature and surface markers compared to Exhausted T cells, (a) Work flow used to analyze gene expression profiles of the available data-set for exhausted T cells (ExhT-cells) (Wherry et. al, Immunity 2007, 27, 670-84) to compare with TRFoxAis- (b) Venn diagram of genes differentially regulated in TRFoxAi and ExhT cells. TRFoxAis "signature" consists of 1498 genes (21 12 probe sets, non-assigned probes and probes matching to multiple genes included), and ExhCD8+ T cell "signature" consists of 455 genes (504 probes). These two sets share only 43 genes, 31 of which are regulated in the same direction (i.e. up or downregulated in both exhausted and TRFoxAis)- Additional analyses were performed using analogous approach to compare TRFoxAi cells with exhausted CD8/CD4 T cells from (Doering et al. Immunity 2012, 37, 1 130-1 144) and (Quigley et al. Nature medicine 2010, 16, 1 147-51 ) datasets. Overlapping genes were only 6 (data not shown). Figure 11. Addition of IL-2 has partial effect on inhibitory activity of TRFoxA 1 cells. TRFOXAI cells were purified on the basis of their surface expression of CD4 PD- L1 hi by FACSAria from OVA-activated OT-II cells co-cultured with CGNs. Purified CD4+ T cells were labeled with CFSE and activated with anti-CD3/anti-CD28 for 24 hours (responder T cells), before co-culturing with purified ΤΚΡΟΧΑΙ cells for an additional 24 hours. Where indicated recombinant IL-2 (2.4 pg/ml) was added to the co-cultures. FACS histograms show proliferative (CFSE) or dead (7AAD) responder T cells alone and after co-culture with TRFOXAI cells (with or without IL-2). IL-2 addition rescues TRFoxAi-inhibition of cell proliferation, while there is no effect on their capacity to induce cell death of activated responder T cells. Representative FACS data are from two independent experiments.
Figure 12. IFN- -treatment of lfnb~'~ mice with EAE leads to generation of TRFoxAis in vivo. MOG35-55-EAE was induced in WT and \1nb~'~ mice in C57BL/6 background. RFOXAI cells were generated upon treatment of lfnb~;~ mice with recombinant mlFN-β (5000 U/ml x 3 times). Data indicate successful generation of TRFOXAI (CD4+FOXA1 + T cells) in the spleens of lfnb~;~ mice upon mIFN-p-treatment. Bars are the mean±SD of 3-5 mice per group, ***P < 0.001 using one-way ANOVA with Newman-Keuls post hoc test for multiple comparison correction. Figure 13. hlFN-β treatment do not lead to the generation of Treg cells expressing FoxP3 or IL-35* cells. Purified CD4+ T cells from healthy blood donors were treated in vitro with or without recombinant hlFN-β (1000U/ml) for 3 days, (a) FACS dot plots show no differences in the FoxP3 expression with or without hlFN-β treatment, (b) FoxP3 expression do not differ in purified iTRFoxAis (R1 -gated) compared to non-TRFoxAis (R2-gated cells), (c) Histograms of IL-35 expression are shown in gated iTRFoxAis in comparison with non-TRFoxAis. (d) FACS histogram shows PD-1 expression after different treatments. Representative FACS data are showing from three independent experiments. Figure 14. iTRFoxAi cells suppress effector function ofAPCs by inhibiting their pro-inflammatory cytokines production. MACS purified APCs from healthy blood donors were treated with LPS for 2 days prior to co-culture with iTRFoxAi cells. After 24 hours of co-culture of APCs with iTRFoxAi cells, intracellular^ stained cytokines were gated on APCs (HLA-DR+TCR"), supernatant were analyzed for different cytokines using ELISA. (a) One representative of FACS dot plots show IL-10, (b) IL-12, and (c) IL-17 productions by ELISA. Bars represent mean ± SD. *P≤ 0.05. Student's unpaired i-tests were used for analysis, N = 3.
Figure 15. Rescue of TRFoxAI phenotype by ectopic expression of FoxA1 confirms specificity of siRNAs targeting, (a) Scheme for gene targeting and rescue strategy depicts pcDNA3.1 foxal , foxal siRNAs and amplified regions by indicated PCR primers, (b) Murine purified CD4+ T cells were transfected with siRNAs. Efficiency of FoxA 1 KD was confirmed by qPCR after 72 h of transfection, using 4 different siRNAs and a smartpool foxal siRNA (4 siRNA pooled), (c) Purified CD4+ T cells were transfected by pcD N A3.1 foxal . pcDNA3.1 vector was used as a control. qPCR was performed for the indicated regions, (d-e) Ectopic FoxA1 is not affected by siRNAs targeting 3'UTRs. Purified CD4+ T cells were transfected with a smartpool foxal siRNAs for 24 h, followed by transfection with pcDNA3.1foxa1 for additional 48h. (d) Expression levels of FoxA 1 gene were measured by qPCR which indicates that foxal siRNAs are specifically targeting and deleting endogenous FoxA1 , (e) while
pcDNA3.1foxa1 is rescuing FoxA1 determined by positive ORF primer readout but not 3'UTR primers. Data are meansiSD from duplicates.
Figure 16. iTRFoxAis suppress responder T cells via PD-L1-PD-1 mediated inhibition ofpAKT, pP38 and upregulation of cleaved caspase3. Purified CD4+ T cells from healthy blood donors were treated in vitro with hlFN-β 1000U/ml for 2 days and ITRFOXAI cells were purified. CFSE-labeled purified CD4+ T cells from the same healthy blood donors were transfected either with control siRNA (UNC) or PD-1 siRNA (PD-1 KD) for 3 days, then activated with plate-coated anti-CD3 antibody for 24 h (responder T cells). Control siRNA and PD-1 siRNA silenced responder T cells were co-cultured with purified iTRFoxAi for additional 24 h. A representative CSFE FACS histogram shows that iTRFoxAi inhibits proliferation of (a) phosphorylated AKT and (b) cleaved caspase 3. (c) Phosphorylated p38 in responder T cells in a PD-1 dependent manner. Data represent from three independent experiments.
Figure 17. Changes on EDSS scores over time in RRMS patients treated with IFN- β. (a) EDSS scores in RRMS-R patients before (baseline) and after 24 months of treatment with IFN-β show no progression on neurological disability during the follow- up period, (b) EDSS scores in RRMS-NR patients indicate progression on neurological disability over time. N=9-15 per group. One-way ANOVA with repeated measures (€€P < 0.01 ,€€€P < 0.001 ), post-test Dunnett's Multiple Comparison (*P≤ 0.05, **P≤ 0.01 ) and linear trend tests (###P≤ 0.001 ) were used. The linear trend test revealed a P≤ 0.001 with positive slope (increasing EDSS) in the RRMS-NR and P≤ 0.001 with a negative slope (decreasing EDSS) in the RRMS-R group.
Figure 18. IFN-β induced FoxA1+Treg cells kill glioma tumor cells. Purified CD4+ T cells from C57BL/6 mice spleens were treated with murine IFN-β (100U/ml) for 48 hours. IFN-β treated CD4+ T cells were sorted by CD4+PD-L1 high gated cells, served as iFoxAI +T cells. GL261 (mouse Glioma tumor cell line) were labeled with CFSE and then seeded at 4000/well in 96-welll plate for 24 hours prior to co-culture with FoxA1 + T cells. Next FoxA1 + T cells were co-cultured with GL261 cells at 10:1 (iFoxAUreg : GL261 ) ratio. After 24 h, GL261 cells (CFSE+) cell death (7AAD+) was analysed by FACS. Results indicate that FoxA1 + T cells are capable of killing cancer cells i.e. 37.2% dead tumor cells compared to original 7.9%.
Figure 19. FoxA1+Treg cells are significantly lacking in the gut environment (i.e. colon and small intestine) of ifnb~'~ mice associated with gut inflammation.
Lymphocytes were purified from thymus, spleens, colons, mesenteric lymph nodes (MLN) and small intestines (BR10III wild type/WT mice and ifnb'1' mice). Single cells were prepared and stained with antibodies against CD4-APC and PD-L1 -PE. Next stained lymphocytes driven from different tissues were analyzed using FACS.
Percentage of FoxA1 + T cells was defined as CD4+PD-L1 high T cells. Number of mice per group=5-8, ***p<0.001 shows significant difference between normal gut environment in wild type mice versus inflammatory gut in ifnb''' mice. Conclusion: results determined that ifnb''' mice are defective in FoxA1 + T cells specifically in small intestine suggesting inflammatory condition modeling inflammatory bowel disease.
Definitions
A "treatment effect" or "therapeutic effect" is manifested if there is a change in the condition being treated, as measured by the criteria constituting the definition of the terms "treating" and "treatment." There is a "change" in the condition being treated if there is at least 5% improvement, preferably 10% improvement, more preferably at least 25%, even more preferably at least 50%, such as at least 75%, and most preferably at least 100% improvement. The change can be based on improvements in the severity of the treated condition in an individual, or on a difference in the frequency of improved conditions in populations of individuals with and without treatment with the bioactive agent, or with the bioactive agent in combination with a pharmaceutical composition of the present invention. "Pharmacologically effective amount", "pharmaceutically effective amount" or
"physiologically effective amount" of a bioactive agent is the amount of an active agent present in a pharmaceutical composition as described herein that is needed to provide a desired level of active agent in the bloodstream or at the site of action in an individual (e.g. the lungs, the gastric system, the colorectal system, prostate, etc.) to be treated to give an anticipated physiological response when such composition is administered. The precise amount will depend upon numerous factors, e.g., the active agent, the activity of the composition, the delivery device employed, the physical characteristics of the composition, intended patient use (i.e. the number of doses administered per day), patient considerations, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein. An "effective amount" of a bioactive agent can be administered in one administration, or through multiple administrations of an amount that total an effective amount. It can be determined using standard clinical procedures for determining appropriate amounts and timing of administration. It is understood that the "effective amount" can be the result of empirical and/or
individualized (case-by-case) determination on the part of the treating health care professional and/or individual.
A "polypeptide", "peptide" or "protein" is a polymer of amino acid residues preferably joined exclusively by peptide bonds, whether produced naturally or synthetically. The term "polypeptide" as used herein covers proteins, peptides and polypeptides, wherein said proteins, peptides or polypeptides may or may not have been post-translationally modified. A peptide is usually shorter in length than a protein.
An "isolated polypeptide" is a polypeptide separated and/or recovered from a component of their natural, typically cellular, environment, that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature. Typically, a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. One way to show that a particular protein preparation contains an isolated polypeptide is by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining of the gel. However, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers, tetramers or alternatively glycosylated or derived forms. An "amino acid residue" can be a natural or non-natural amino acid residue linked peptide bonds or bonds different from peptide bonds. The amino acid residues can be in D-configuration or L-configuration. An amino acid residue comprises an amino terminal part (NH2) and a carboxy terminal part (COOH) separated by a central part comprising a carbon atom, or a chain of carbon atoms, at least one of which comprises at least one side chain or functional group. NH2 refers to the amino group present at the amino terminal end of an amino acid or peptide, and COOH refers to the carboxy group present at the carboxy terminal end of an amino acid or peptide. The generic term amino acid comprises both natural and non-natural amino acids. Natural amino acids are Y, G, F, M, A, S, I, L, T, V, P, K, H, Q, E, W, R, D, N and C. Non-natural amino acids are those not listed here. Also, non-natural amino acid residues include, but are not limited to, modified amino acid residues, L-amino acid residues, and stereoisomers of D-amino acid residues. Where the L or D form (optical isomers) has not been specified it is to be understood that the amino acid in question has the natural L form.
An "equivalent amino acid residue" refers to an amino acid residue capable of replacing another amino acid residue in a polypeptide without substantially altering the structure and/or functionality of the polypeptide. Equivalent amino acids thus have similar properties such as bulkiness of the side-chain, side chain polarity (polar or non-polar), hydrophobicity (hydrophobic or hydrophilic), pH (acidic, neutral or basic) and side chain organization of carbon molecules (aromatic/aliphatic). As such, "equivalent amino acid residues" can be regarded as "conservative amino acid substitutions".
The classification of equivalent amino acids refers in one embodiment to the following classes: 1 ) HRK, 2) DENQ, 3) C, 4) STPAG, 5) MILV and 6) FYW.
Within the meaning of the term "equivalent amino acid substitution" as applied herein, one amino acid may be substituted for another, in one embodiment, within the groups of amino acids indicated herein below: i) Amino acids having polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys,) ii) Amino acids having non-polar side chains (Gly, Ala, Val, Leu, lie, Phe, Trp, Pro, and Met)
iii) Amino acids having aliphatic side chains (Gly, Ala Val, Leu, lie)
iv) Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro)
v) Amino acids having aromatic side chains (Phe, Tyr, Trp)
vi) Amino acids having acidic side chains (Asp, Glu)
vii) Amino acids having basic side chains (Lys, Arg, His)
viii) Amino acids having amide side chains (Asn, Gin)
ix) Amino acids having hydroxy side chains (Ser, Thr)
x) Amino acids having sulphur-containing side chains (Cys, Met),
xi) Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser, Thr)
xii) Hydrophilic, acidic amino acids (Gin, Asn, Glu, Asp), and
xiii) Hydrophobic amino acids (Leu, lie, Val)
A "Bioactive agent" (i. e., biologically active substance/agent) is any agent, drug, compound, composition of matter or mixture which provides some pharmacologic, often beneficial, effect that can be demonstrated in-vivo or in vitro. It refers herein to a FoxA1 peptide according to the present invention, compounds or compositions comprising these and nucleic acid constructs encoding said peptides, as well as the immunosuppressive T cells expressing FoxA1 (FoxA1 + T cells). As used herein, this term further includes any physiologically or pharmacologically active substance that produces a localized or systemic effect in an individual. Further examples of bioactive agents include, but are not limited to, agents comprising or consisting of an
oligosaccharide, agents comprising or consisting of a polysaccharide, agents comprising or consisting of an optionally glycosylated peptide, agents comprising or consisting of an optionally glycosylated polypeptide, agents comprising or consisting of a nucleic acid, agents comprising or consisting of an oligonucleotide, agents comprising or consisting of a polynucleotide, agents comprising or consisting of a lipid, agents comprising or consisting of a fatty acid, agents comprising or consisting of a fatty acid ester and agents comprising or consisting of secondary metabolites. It may be used either prophylactically, therapeutically, in connection with treatment of an individual, such as a human or any other animal.
Due to the imprecision of standard analytical methods, molecular weights and lengths of polymers are understood to be approximate values. When such a value is expressed as "about" X or "approximately" X, the stated value of X will be understood to be accurate to +/- 20%, such as +/- 10%, for example +/- 5%.
Detailed description of the invention
Immune function that preserves tolerance to self, while retaining antimicrobial function, is imperative for preventing chronic inflammation and autoimmunity. Regulation of inflammation is critical for the disease management in tissue-specific chronic inflammatory diseases, including multiple sclerosis (MS). This is naturally achieved when inflammatory counteracting, functionally intact immune cells are generated.
Inflammation is a self-destructive process that can lead to irreversible chronic tissue destruction, and regulatory T-cell (Treg) mediated suppression is vital in negatively regulating inflammation. Conversely, defects in Treg generation or function are risk factors for inflammation and autoimmune diseases. For instance, Treg (Neuron-induced, FoxP3-expressing Tregs) defects are reported in experimental autoimmune
encephalomyelitis (EAE), a model for multiple sclerosis (MS), a tissue-specific inflammatory disease affecting the central nervous system (CNS). Mice lacking Ifnb and Ifna/b-receptor genes develop chronic inflammatory and demyelinating EAE (Ifnb''' mice). The present inventors have found no defects associated with Foxp3+Tregs in the inflamed CNS of Ifnb''' mice; however, the inventors did identify a novel population of T-cells in wildtype mice that was absent in Ifnb''' mice. These newly identified immunosuppressive T cells, denoted herein FoxA1 +T-cells (or TRFOXAI in the figure legends and the examples), are largely defined by expression of the transcription factor FoxA1.
As shown herein, FoxA1 +T-cells suppress T cell activation, inhibit proliferation and induce cell death in activated T cells in vitro, which effect translates in vivo into an inhibition of CNS inflammation and RR-EAE in Ifnb''' mice.
The immunosuppressive properties of FoxA1 in T cells makes FoxA1 and the immunosuppressive FoxA1 +T-cells potentially useful in the treatment of a range of disorders where immunosuppression is desired, such as diseases having an inflammatory component including inflammatory disorders. The immune system
The immune system is a system of biological structures and processes within an organism that protects against disease. To function properly, an immune system must detect a wide variety of agents, from bacteria and viruses to parasitic worms, and distinguish them from the organism's own healthy tissue.
The immune system protects organisms from infection with layered defenses of increasing specificity. In simple terms, physical barriers prevent pathogens such as bacteria and viruses from entering the organism. If a pathogen breaches these barriers, the innate immune system provides an immediate, but non-specific response. If pathogens successfully evade the innate response, vertebrates possess a second layer of protection, the adaptive immune system, which is activated by the innate response. Here, the immune system adapts its response during an infection to improve its recognition of the pathogen. This improved response is then retained after the pathogen has been eliminated, in the form of an immunological memory, and allows the adaptive immune system to mount faster and stronger attacks each time this pathogen is encountered.
The immune system is thus tightly controlled system, with many inhibitory and stimulatory effectors coordinating the final outcome.
The innate immune system
Microorganisms or toxins that successfully enter an organism encounter the cells and mechanisms of the innate immune system. Innate immune defenses are non-specific, meaning these systems respond to pathogens in a generic way. This system does not confer long-lasting immunity against a pathogen. The innate immune system comprises inflammation, the complement system, cellular barriers and natural killer cells (or NK cells). Inflammation is one of the first responses of the immune system to infection. The symptoms of inflammation are redness, swelling, heat, and pain, which are caused by increased blood flow into tissue. Inflammation is produced by eicosanoids and cytokines, which are released by injured or infected cells. Eicosanoids include prostaglandins that produce fever and the dilation of blood vessels associated with inflammation, and leukotrienes that attract certain white blood cells (leukocytes). Common cytokines include interleukins that are responsible for communication between white blood cells; chemokines that promote chemotaxis; and interferons that have anti-viral effects, such as shutting down protein synthesis in the host cell. Growth factors and cytotoxic factors may also be released. These cytokines and other chemicals recruit immune cells to the site of infection and promote healing of any damaged tissue following the removal of pathogens.
The complement system is a biochemical cascade that attacks the surfaces of foreign cells. It contains over 20 different proteins and is named for its ability to "complement" the killing of pathogens by antibodies. Complement is the major humoral component of the innate immune response. In humans, this response is activated by complement binding to antibodies that have attached to these microbes or the binding of complement proteins to carbohydrates on the surfaces of microbes. This recognition signal triggers a rapid killing response and signal amplification.
Cellular barriers. Leukocytes (white blood cells) act like independent, single-celled organisms and are the second arm of the innate immune system. The innate leukocytes include the phagocytes (macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. These cells identify and eliminate pathogens, either by attacking larger pathogens through contact or by engulfing and then killing microorganisms. Innate cells are also important mediators in the activation of the adaptive immune system.
Adaptive immune system
The adaptive immune system evolved in early vertebrates and allows for a stronger immune response as well as immunological memory, where each pathogen is
"remembered" by a signature antigen. The adaptive immune response is antigen- specific and requires the recognition of specific "non-self antigens during a process called antigen presentation. Antigen specificity allows for the generation of responses that are tailored to specific pathogens or pathogen-infected cells. The ability to mount these tailored responses is maintained in the body by "memory cells". Should a pathogen infect the body more than once, these specific memory cells are used to quickly eliminate it. The cells of the adaptive immune system are special types of leukocytes, called lymphocytes. B cells and T cells are the major types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow. B cells are involved in the humoral immune response, whereas T cells are involved in cell-mediated immune response. Both B cells and T cells carry receptor molecules that recognize specific targets. T cells recognize a "non-self target, such as a pathogen, only after antigens (small fragments of the pathogen) have been processed and presented in combination with a major histocompatibility complex (MHC) molecule. T-cells can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T- cell receptor (TCR) on the cell surface. There are two major subtypes of T cells: the killer T cell and the helper T cell. Killer T cells only recognize antigens coupled to Class I MHC molecules, while helper T cells only recognize antigens coupled to Class II MHC molecules. These two mechanisms of antigen presentation reflect the different roles of the two types of T cell. A third, minor subtype are the γδ T cells that recognize intact antigens that are not bound to MHC receptors. In contrast, the B cell antigen-specific receptor is an antibody molecule on the B cell surface, and recognizes whole pathogens without any need for antigen processing. Each lineage of B cell expresses a different antibody, so the complete set of B cell antigen receptors represent all the antibodies that the body can manufacture.
Killer T cells (CD8+) (or cytotoxic T cells, Tc cells, CTL) are activated when their T cell receptor (TCR) binds to this specific antigen in a complex with the MHC Class I receptor of another cell. Recognition of this MHC:antigen complex is aided by a co- receptor on the T cell, called CD8. The T cell then travels throughout the body in search of cells where the MHC I receptors bear this antigen. When an activated T cell contacts such cells, it releases cytotoxins. T cell killing of host cells is particularly important in preventing the replication of viruses. T cell activation is tightly controlled and generally requires a very strong MHC/antigen activation signal, or additional activation signals provided by helper T cells
Helper T cells, Th cells, (CD4+) regulate both the innate and adaptive immune responses and help determine which immune responses the body makes to a particular pathogen. These cells have no cytotoxic activity and do not kill infected cells or clear pathogens directly. They instead control the immune response by directing other cells to perform these tasks. Helper T cells express T cell receptors (TCR) that recognize antigen bound to Class II MHC molecules. The MHC:antigen complex is also recognized by the helper cell's CD4 co-receptor, which recruits molecules inside the T cell that are responsible for the T cell's activation. The activation of a resting helper T cell causes it to release cytokines that influence the activity of many cell types.
Cytokine signals produced by helper T cells enhance the microbicidal function of macrophages and the activity of killer T cells
Regulatory T cells (Tregs) are a subpopulation of T cells (CD4+) which modulate the immune system, maintain tolerance to self-antigens, and abrogate autoimmune disease. Mouse models have suggested that modulation of Tregs can treat
autoimmune disease and cancer, and facilitate organ transplantation.
T regulatory cells are a component of the immune system that suppresses immune responses of other cells in order to prevent excessive reactions. Regulatory T cells come in many forms with the most well-understood being those that express CD4, CD25, and Foxp3. CD4+ Foxp3+ regulatory T cells have been called "naturally- occurring" regulatory T cells (nTreg). An additional regulatory T cell subset, denoted induced regulatory T cells (iTreg) (also CD4+ CD25+ Foxp3+), are needed for tolerance and suppression. Regulatory T cells are defined by expression of the forkhead family transcription factor FOXP3 (forkhead box p3). Thus, FOXP3 can be used as a good marker for CD4+CD25+ T cells, although also expressed in CD4+CD25" T cells and conventional T-cells.
Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with "memory" against past infections. Memory T cells comprise three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells may be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.
Natural killer T cells (NKT cells - not to be confused with natural killer cells of the innate immune system) bridge the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CD1 d. Once activated, these cells can perform functions ascribed to both Th and Tc cells (i.e., cytokine production and release of cytolytic/cell killing molecules). They are also able to recognize and eliminate some tumor cells and cells infected with herpes viruses.
FoxA1
FoxA1 or forkhead box A1 (gene FOXA1 ) is a transcription factor of 472 amino acids (aa) in length (UniProt accession number P55317 (FOXA1_HUMAN); NCBI
NM_004496). An often used synonym is hepatocyte nuclear factor 3, alpha (HNF-3- alpha).
The protein sequence of FoxA1 is (SEQ ID NO:1 ):
MLGTVKMEGHETSDWNSYYADTQEAYSSVPVSNMNSGLGSMNSMNTYMTMNTMTTSGNMTPASFNMSYAN PGLGAGLSPGAVAGMPGGSAGAMNSMTAAGVTAMGTALSPSGMGAMGAQQAASMNGLGPYAAAMNPCMSP MAYAPSNLGRSRAGGGGDAKTFKRSYPHAKPPYSYISLITMAIQQAPSKMLTLSEIYQWIMDLFPYYRQN QQRWQNSIRHSLSFNDCFVKVARSPDKPGKGSYWTLHPDSGNMFENGCYLRRQKRFKCEKQPGAGGGGGS GSGGSGAKGGPESRKDPSGASNPSADSPLHRGVHGKTGQLEGAPAPGPAASPQTLDHSGATATGGASELK TPASSTAPPISSGPGALASVPASHPAHGLAPHESQLHLKGDPHYSFNHPFSINNLMSSSEQQHKLDFKAY EQALQYSPYGSTLPASLPLGSASVTTRSPIEPSALEPAYYQGVYSRPVLNTS
The DNA binding region (Fork-head) has been identified as aa 169-206, namely the
Sequence AKPPYSYISLITMAIQQAPSKMLTLSEIYQWIMDLFPY (SEQ ID NO:2). Natural variations in the amino acid sequence of FoxA1 occur, and comprises
G^A at position 72 (VAR_015183),
A^T at position 83 (VAR_013457),
G^E at position 87 (VAR_055835)
MET-> INS at position 124 (VAR_015184)
Q^R at position 185 (VAR_015185)
S^N at position 448 (VAR_013458)
The FOXA1 gene comprises a DNA sequence of 3396 bp (SEQ ID NO:3):
1 gggcttcctc ttcgcccggg tggcgttggg cccgcgcggg cgctcgggtg actgcagctg
61 ctcagctccc ctcccccgcc ccgcgccgcg cggccgcccg tcgcttcgca cagggctgga
121 tggttgtatt gggcagggtg gctccaggat gttaggaact gtgaagatgg aagggcatga
181 aaccagcgac tggaacagct actacgcaga cacgcaggag gcctactcct ccgtcccggt
241 cagcaacatg aactcaggcc tgggctccat gaactccatg aacacctaca tgaccatgaa
301 caccatgact acgagcggca acatgacccc ggcgtccttc aacatgtcct atgccaaccc
361 gggcctaggg gccggcctga gtcccggcgc agtagccggc atgccggggg gctcggcggg
421 cgccatgaac agcatgactg cggccggcgt gacggccatg ggtacggcgc tgagcccgag
481 cggcatgggc gccatgggtg cgcagcaggc ggcctccatg aatggcctgg gcccctacgc 541 ggccgccatg aacccgtgca tgagccccat ggcgtacgcg ccgtccaacc tgggccgcag
601 ccgcgcgggc ggcggcggcg acgccaagac gttcaagcgc agctacccgc acgccaagcc
661 gccctactcg tacatctcgc tcatcaccat ggccatccag caggcgccca gcaagatgct
721 cacgctgagc gagatctacc agtggatcat ggacctcttc ccctattacc ggcagaacca
781 gcagcgctgg cagaactcca tccgccactc gctgtccttc aatgactgct tcgtcaaggt
841 ggcacgctcc ccggacaagc cgggcaaggg ctcctactgg acgctgcacc cggactccgg
901 caacatgttc gagaacggct gctacttgcg ccgccagaag cgcttcaagt gcgagaagca
961 gccgggggcc ggcggcgggg gcgggagcgg aagcgggggc agcggcgcca agggcggccc
1021 tgagagccgc aaggacccct ctggcgcctc taaccccagc gccgactcgc ccctccatcg
1081 gggtgtgcac gggaagaccg gccagctaga gggcgcgccg gcccccgggc ccgccgccag
1141 cccccagact ctggaccaca gtggggcgac ggcgacaggg ggcgcctcgg agttgaagac
1201 tccagcctcc tcaactgcgc cccccataag ctccgggccc ggggcgctgg cctctgtgcc
1261 cgcctctcac ccggcacacg gcttggcacc ccacgagtcc cagctgcacc tgaaagggga
1321 cccccactac tccttcaacc acccgttctc catcaacaac ctcatgtcct cctcggagca
1381 gcagcataag ctggacttca aggcatacga acaggcactg caatactcgc cttacggctc
1441 tacgttgccc gccagcctgc ctctaggcag cgcctcggtg accaccagga gccccatcga
1501 gccctcagcc ctggagccgg cgtactacca aggtgtgtat tccagacccg tcctaaacac
1561 ttcctagctc ccgggactgg ggggtttgtc tggcatagcc atgctggtag caagagagaa
1621 aaaatcaaca gcaaacaaaa ccacacaaac caaaccgtca acagcataat aaaatcccaa
1681 caactatttt tatttcattt ttcatgcaca acctttcccc cagtgcaaaa gactgttact
1741 ttattattgt attcaaaatt cattgtgtat attactacaa agacaacccc aaaccaattt
1801 ttttcctgcg aagtttaatg atccacaagt gtatatatga aattctcctc cttccttgcc
1861 cccctctctt tcttccctct ttcccctcca gacattctag tttgtggagg gttatttaaa
1921 aaaacaaaaa aggaagatgg tcaagtttgt aaaatatttg tttgtgcttt ttccccctcc
1981 ttacctgacc ccctacgagt ttacaggtct gtggcaatac tcttaaccat aagaattgaa
2041 atggtgaaga aacaagtata cactagaggc tcttaaaagt attgaaagac aatactgctg
2101 ttatatagca agacataaac agattataaa catcagagcc atttgcttct cagtttacat
2161 ttctgataca tgcagatagc agatgtcttt aaatgaaata catgtatatt gtgtatggac
2221 ttaattatgc acatgctcag atgtgtagac atcctccgta tatttacata acatatagag
2281 gtaatagata ggtgatatac atgatacatt ctcaagagtt gcttgaccga aagttacaag
2341 gaccccaacc cctttgtcct ctctacccac agatggccct gggaatcaat tcctcaggaa
2401 ttgccctcaa gaactctgct tcttgctttg cagagtgcca tggtcatgtc attctgaggt
2461 cacataacac ataaaattag tttctatgag tgtataccat ttaaagaatt tttttttcag
2521 taaaagggaa tattacaatg ttggaggaga gataagttat agggagctgg atttcaaaac
2581 gtggtccaag attcaaaaat cctattgata gtggccattt taatcattgc catcgtgtgc
2641 ttgtttcatc cagtgttatg cactttccac agttggacat ggtgttagta tagccagacg
2701 ggtttcatta ttatttctct ttgctttctc aatgttaatt tattgcatgg tttattcttt
2761 ttctttacag ctgaaattgc tttaaatgat ggttaaaatt acaaattaaa ttgttaattt
2821 ttatcaatgt gattgtaatt aaaaatattt tgatttaaat aacaaaaata ataccagatt
2881 ttaagccgtg gaaaatgttc ttgatcattt gcagttaagg actttaaata aatcaaatgt
2941 taacaaaaga gcatttctgt tatttttttt cacttaacta aatccgaagt gaatatttct
3001 gaatacgata tttttcaaat tctagaactg aatataaatg acaaaaatga aaataaaatt
3061 gttttgtctg ttgttataat gaatgtgtag ctagtaaaaa ggagtgaaag aaattcaagt
3121 aaagtgtata agttgattta atattccaag agttgagatt tttaagattc tttattccca
3181 gtgatgttta cttcattttt tttttttttt ttgacaccgg cttaagcctt ctgtgtttcc
3241 tttgagcctt ttcactacaa aatcaaatat taatttaact acctttcctc cttccccaat
3301 gtatcacttt tctttatctg agaattcttc caatgaaaat aaaatatcag ctgtggctga
3361 tagaattaag ttgtgtccaa aaaaaaaaaa aaaaaa
FoxA1 is a transcription factor involved in embryonic development, establishment of tissue-specific gene expression and regulation of gene expression in differentiated tissues. Originally described as a transcription activator for a number of liver genes such as AFP, albumin, tyrosine aminotransferase, PEPCK, etc. Is thought to act as a 'pioneer' factor opening the compacted chromatin for other proteins through interactions with nucleosomal core histones and thereby replacing linker histones at target enhancer and/or promoter sites. Also proposed to play a role in translating the epigenetic signatures into cell type-specific enhancer-driven transcriptional programs. FoxA1 is involved in the development of multiple endoderm-derived organ systems such as liver, pancreas, lung and prostate.
Furthermore, FoxA1 modulates the transcriptional activity of nuclear hormone receptors (oestrogen receptor-a (ER) and androgen receptor (AR)) and is involved in ESR1 -mediated transcription in breast cancer cells. It is also involved in regulation of apoptosis by inhibiting the expression of BCL2, and in cell cycle regulation by activating expression of CDKN1 B, alone or in conjunction with BRCA1.
FoxA1 expression has been positively associated with tumorigenesis / tumour progression at least in prostate cancer, esophageal and lung adenocarcinomas, invasive bladder cancer and breast cancer.
Variants of FoxA 1
It is an aspect of the present invention to provide FoxA1 , or a functional variant or functional fragment of FoxA1 ; either as comprised or expressed in T cells, as a protein or as DNA. When in the form of DNA it is preferably comprised in a nucleic acid construct, as defined herein below.
According to the present invention, FoxA1 as defined herein above may be a functional variant or functional fragment of said FoxA1. Variants and fragment according to the present invention are meant to be the functional equivalents of FoxA1 , i.e. retaining the same biological activity or capabilities as the sequence from which it is derived.
In one embodiment, a functional variant or functional fragment of FoxA1 is able to render T cells immunosuppressive, such as being able to suppress activated T cells. In one embodiment said FoxA1 is in the form of a protein (SEQ ID NO:1 ), or a functional variant or functional fragment thereof (of SEQ ID NO:1 ).
In another embodiment said FoxA1 is in the form of DNA (SEQ ID NO:3), or a functional variant or functional fragment thereof (of SEQ ID NO:3). It is understood that a functional variant or functional fragment of FoxA1 is meant to include also a functional variant of a fragment of FoxA1 (i.e. a fragment also being a variant). In one embodiment, a functional variant or functional fragment of FoxA1 according to the present invention i) is able to suppress production of one or more pro-inflammatory cytokines, such as pro-inflammatory cytokines selected from the group consisting of IL- 12 and IL-17 in APCs; ii) do not induce production of IL-10 in APCs, iii) induce expression in T cells of one or more of CD4, PD-L1 , PD-1 , CD47, CD69, CD25, CD45Rb and TCRa3+; iv) is able to suppress activated T cells, such as inhibit proliferation and/or induce cell death of activated T cells; v) is able to down-regulate c- Fos expression in T cells; and/or vi) is able to reduce nuclear pc-Fos in T cells.
Lack of expression of IL-10 is a feature separating the present immunosuppressive FoxA1 + cells from native Tregs.
In one embodiment, a functional variant or functional fragment of FoxA1 according to the present invention has between 60-99.9% sequence identity, such as between 60- 70%, for example 70-75%, such as 75-80%, for example 80-85%, such as 85-90%, for example 90-95%, such as 95-96%, for example 96-97%, such as 97-98%, for example 98-99%, such as 99-99.9% sequence identity to SEQ ID NO:1 (protein) or SEQ ID NO:3 (DNA).
In one embodiment, a functional variant or functional fragment of FoxA1 according to the present invention has at least 60% sequence identity, such as at least 65% sequence identity, for example at least 70% sequence identity, such as at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity to SEQ ID NO:1
(protein) or SEQ ID NO:3 (DNA).
In one embodiment, a functional variant or functional fragment of FoxA1 according to the present invention comprises from 10 to 471 consecutive amino acids of SEQ ID NO: 1 , such as from 10-25, 25-50, 50-75, 75-100, 100-125, 125-150, 150-175, 175-200, 200-250, 250-300, 300-350, 350-400, 400-450 or from 450-471 consecutive amino acids of SEQ ID NO:1.
In yet an embodiment, a functional variant or functional fragment of FoxA1 according to the present invention comprises the FoxA1 Fork-head DNA-binding region (aa 169- 260) (SEQ ID NO:2).
In a further embodiment, a functional variant or functional fragment of FoxA1 according to the present invention comprises the naturally occurring variants of FoxA1 , including but not limited to VAR_015183, VAR_013457, VAR_055835, VAR_015184,
VAR_015185 and VAR_013458.
In one embodiment, a functional variant or functional fragment of FoxA1 is a sequence wherein any amino acid specified in SEQ ID NO:1 is changed to a different amino acid, provided that no more than 20 of the amino acid residues in the sequence are so changed, such as provided than no more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 of the amino acid residues in the sequence are so changed. In one embodiment, the 1 -20 amino acids so changed are changed to equivalent amino acids.
A Clustal W alignment of the FoxA1 protein sequence between different species can be used to predict which amino acid residues can be substituted without substantially affecting the biological activity of the protein; and which are deemed conservative or semi-conservative. Based on the fully conserved residues, a consensus sequence for mature FoxA1 protein can be derived, which consensus sequences may be regarded as conserved domains. In particular, it is contemplated that the conserved and/or semi- conserved amino acid residues must be located at corresponding positions in a variant.
The FoxA1 protein and its use according to the present invention may be further modified or optimized in order to obtain favorable properties with respect to e.g.
delivery and solubility. Such optimizations are known to the skilled person.
In one embodiment, the FoxA1 protein as defined herein may be linked covalently or non-covalently with a carrier molecule, with a protective molecule, with a vehicle or delivery molecule or with a localization molecule known to the skilled person. Protein transduction domains (PTDs, sometimes termed cell permeable proteins (CPP) or membrane translocating sequences (MTS)) are small peptides that are able to ferry much larger molecules into cells independent of classical endocytosis. This property makes PTDs ideal tools to transfer proteins and other molecules into living cells.
PEGylation defines the modification of a protein by the linking of one or more polyethylene glycol (PEG) chains. This polymer is non-toxic, non-immunogenic, non- antigenic, highly soluble in water and FDA approved. The PEG-drug conjugates have several advantages: a prolonged residence in body, a decreased degradation by metabolic enzymes and a reduction or elimination of protein immunogenicity.
In one embodiment of the invention, the FoxA1 protein of the present invention, having sequence SEQ ID NO:1 , or a functional variant or functional fragment thereof, further comprises one or more protein transduction domains (PTDs).
In one embodiment of the invention, the FoxA1 protein of the present invention, having sequence SEQ ID NO:1 , or a functional variant or functional fragment thereof, further comprises one or more polyethylene glycol (PEG) chains
Immunosuppressive FoxA1 + T cells
It is an aspect of the present invention to provide immunosuppressive T cells comprising FoxA1 or a functional variant or functional fragment of FoxA1
(immunosuppressive FoxA1 + T cells, or simply FoxA1 + T cells). These cells are referred to as "TRFOXAI" in the figure legends and examples.
In one embodiment there is provided an isolated population of immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment of FoxA1 . In another embodiment there is provided an in vitro cell culture comprising the immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment of FoxA1 . In one embodiment, said in vitro cell culture further comprises a cell culture medium. As the present inventors have shown, the immunosuppressive properties of the FoxA1 + T cells are mediated via FoxA1 , making FoxA1 a lineage-specification factor that defines the FoxA1 + T cells, directing their lineage and immunosuppressive properties. In one embodiment, said immunosuppressive FoxA1 + T cells are derived from a population of T cells selected from the group consisting of Regulatory T cells (Tregs), Helper T cells (Th cells), Cytotoxic T cells (Tc cells, TCL), Natural killer T cells (NKT) and Memory T cells. In one embodiment, said immunosuppressive FoxA1 + T cells further comprise or express, or are positive for, one or more of the group consisting of CD4, PD-L1 , PD-1 , CD47, CD69, CD25, CD45Rb and TCRa3.
In one embodiment, said immunosuppressive T cells are FoxA1 +, CD4+, PD-L1 +, PD- 1 +, CD47+, CD69+, CD25+, CD45Rb+ and/or TCRa3+.
In this respect, it is understood that for a given marker, the attributed suffix '+' or 'hi' has clear meaning to the skilled person. For instance, '-' (e.g. FoxP3-) means that the marker is undetectable, absent or low, at least lower than an intermediate expressing population. Likewise, '+' (e.g. CD4+ and FoxA1 +) means that expression is detectable, and/or have higher intensity of staining than '-' cells and possibly higher than intermediate staining populations. Furthermore, 'hi' cells (e.g. PD-L1 hl) have higher expression and/or intensity of staining than the '+' cells (e.g. PD-L1 +) cells. In one embodiment determination of expression or intensity of staining is detected by the FACS technique, wherein the intensity of staining correlates to degree expression. In another embodiment determination of expression is determined by gene expression analysis (gene array, western blot, ELISA, and any technique known to the skilled person).
In one embodiment, said immunosuppressive T cells do not comprise or express, or are negative for, one or more of FoxP3, TGF-β, IFN-gamma, TNF-alpha, CTLA-4, FAS, FASL, CD8, IL-17, IL-4, IL-10 and IL-13. In one embodiment, said immunosuppressive T cells do not comprise or express, or are negative for, FoxP3 (are FoxP3").
By the term 'do not comprise or express', is meant to comprise no or essentially no expression or content, or undetectable expression or content, such as identified by (essentially) no staining by FACS. In this respect they may be classified as 'negative' (i.e. FoxP3 negative). '+' and/or 'hi' means the cells may be classified as 'positive' (e.g. CD4+ or CD4hl). In one embodiment 'do not comprise or express' or being classified as 'negative' for the marker, means that the cells express less than those that are classified as 'positive' (+ or hl).
In one embodiment, said immunosuppressive T cells further comprise or express CD4, or are CD4 positive. In one embodiment, said immunosuppressive T cells are CD4+ and/or CD4hi (CD4+/hi).
In one embodiment, said immunosuppressive T cells further comprise or express PD- L1 , or are PD-L1 positive. In one embodiment, said immunosuppressive T cells are PD- L1 + and/or PD-L1 hi. In one embodiment, said immunosuppressive T cells further comprise or express both CD4 and PD-L1 , or are CD4 and PD-L1 positive (i.e. CD4+ and PD-L1 +; CD4+ and PD- L 1 hi. CD4hi and P D.L1 +. or CD4hi and PD-L1 hi).
In one embodiment, said immunosuppressive T cells further comprise or express one or more of CD4, PD-L1 , PD-1 , CD47 and CD69; or are positive for (+ or hi) one or more of CD4, PD-L1 , PD-1 , CD47 and CD69.
CD4+/hi
In one embodiment, the immunosuppressive T cells according to the present invention are FoxA1 +CD4+PD-L1 + T cells, FoxA1 +CD4hiPD-L1 hi T cells or FoxA1 +CD4+PD-L1 hi T cells.
In one embodiment, the immunosuppressive T cells according to the present invention are FoxA1 +CD4+PD-L1 +FoxP3" T cells.
In one embodiment, the immunosuppressive T cells according to the present invention are FoxA1 +CD4hiPD-L1 hiFoxP3" T cells. In one embodiment, the immunosuppressive T cells according to the present invention are FoxA1 +CD4+PD-L1 hiFoxP3" T cells.
In one embodiment, the immunosuppressive T cells according to the present invention are FoxA1 + CD4+/hiPD-L1 hi TCRa3+ T cells.
In one embodiment, the immunosuppressive T cells according to the present invention are FoxA1 + CD4+/hiPD-L1 hi TCRa3+FoxP3" T cells.
In one embodiment, the immunosuppressive T cells according to the present invention are FoxA1 + CD4+/hiPD-L1 hi CD47+ T-cells.
In one embodiment, the immunosuppressive T cells according to the present invention are FoxA1 + CD4+/hiPD-L1 hi CD47+FoxP3" T-cells.
In one embodiment, the immunosuppressive T cells according to the present invention are FoxA1 + CD4+/hiPD-L1 hi CD47+CD69+T-cells.
In one embodiment, the immunosuppressive T cells according to the present invention are FoxA1 + CD4+/hiPD-L1 hi CD47+CD69+FoxP3" T-cells.
In one embodiment, the immunosuppressive T cells according to the present invention are FoxA1 + CD4+/hiPD-L1 hi CD47+CD69+TCRa3+ T-cells.
In one embodiment, the immunosuppressive T cells according to the present invention are FoxA1 + CD4+/hiPD-L1 hi CD47+CD69+TCRa3+FoxP3" T-cells. In one embodiment said immunosuppressive T cells have reduced expression of one or more of the group consisting of TNF-alpha, IL-1 a (interleukin l alpha), I L-1 b, IL-4, IL- 5, IL-7r (IL-7 receptor), IL-10, IL-13, IL-16, IL-17rd (receptor D), IL-17, IL-18 (receptor 1 ), Cerebral endothelial cell adhesion molecule 1 (Cercaml ), Procollagen-lysine, 2- oxoglutarate 5-dioxygenase 3 (Plod3), Procollagen-proline, 2-oxoglutarate 4- dioxygenase (proline 4-hydroxylase), alpha 1 polypeptide (P4ha1 ), at least as compared to encephalitogenic (EncT cells/MBP89-ioi-specific) progenitor CD4+T cells.
In one embodiment said immunosuppressive T cells have reduced expression of one or more of the group consisting of CD47 and CD60, at least as compared to
encephalitogenic (EncT cells/MBP89-ioi-specific) progenitor CD4+T cells.
In one embodiment said immunosuppressive T cells further comprise or express one or more transcription factors selected from the group consisting of Tcf7l2, Spic, Pou3f1 , Nfib, Mafb, Mef2c, Zbtb16, Tcf7l2, Bach2, Esr1 , Mef2c, Klf2, Aff3, Spib, Tcf7, Rorc, BarhH , Hoxb13, Zic3, Six3, Trp73, Hoxa13, Rora, Foxb2, PrrxH , Neurog3 and Zfp369. In one embodiment said immunosuppressive T cells are non-proliferative. In another embodiment, c-Fos is down-regulated in said immunosuppressive T cells. In yet another embodiment, nuclear pc-Fos (phosphorylated c-Fos) is reduced in said immunosuppressive T cells.
In one embodiment, the immunosuppressive T cells according to the present invention i) are able to suppress production of one or more pro-inflammatory cytokines, such as pro-inflammatory cytokines selected from the group consisting of IL-12 and IL-17 in APCs; ii) do not induce production of IL-10 in APCs, and/or iii) suppress activated T cells, such as inhibit proliferation and/or induce cell death of activated T cells.
A method of making FoxA 1+ T cells
It is also an aspect of the present invention to provide a method of making the immunosuppressive T cells comprising FoxA1 or a functional variant or functional fragment of FoxA1 according to the present invention, said method comprising one or more steps of
i. Introducing into T cells FoxA1 protein, or a functional variant or functional fragment thereof,
ii. Introducing into T cells a nucleic acid construct encoding FoxA1 or a functional variant or functional fragment thereof, and/or
iii. treating T cells with an effective amount of IFN-β.
In one embodiment the method of making the immunosuppressive T cells comprises one or more steps of introducing into T cells FoxA1 protein, or a functional variant or functional fragment thereof, and treating said T cells with an effective amount of IFN-β.
In one embodiment the method of making the immunosuppressive T cells comprises one or more steps of introducing into T cells a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof, and treating said T cells with an effective amount of IFN-β. In one embodiment the method of making the immunosuppressive T cells comprises introducing into T cells FoxA1 protein, or a functional variant or functional fragment thereof.
In one embodiment the method of making the immunosuppressive T cells comprises introducing into T cells a nucleic acid construct encoding FoxA1 or a functional variant or functional fragment thereof. In one embodiment the method of making the immunosuppressive T cells comprises treating T cells with an effective amount of IFN-β.
In one embodiment, an effective amount of IFN-β is an amount sufficient to induce expression of FoxA1 in said T cells.
A nucleic acid construct encoding FoxA1 or a functional variant or functional fragment thereof will in one embodiment comprise all or part of the FOXA1 gene (SEQ ID NO:3).
It is to be understood that introducing FoxA1 into T cells, whether in the form of protein, gene/DNA or a nucleic acid construct, may be by any technical means known to the skilled person.
In one embodiment, said T cells are cultured animal cells. In one embodiment, said T cells are extracted from an individual, such as an individual having an inflammatory disease or disorder as specified herein. In another embodiment, said T cells are selected from the group consisting of Regulatory T cells (Tregs), Helper T cells (Th cells), Cytotoxic T cells (Tc cells, TCL), Natural killer T cells (NKT) and Memory T cells.
In one embodiment, said method of making the immunosuppressive T cells of the present invention is performed in vitro or ex vivo. In another embodiment, said method of making the immunosuppressive T cells of the present invention is performed in vivo.
In one embodiment, introducing into T cells FoxA1 protein, or a functional variant or functional fragment thereof, comprise one or more steps of simply adding protein in solution (such as media) to a T cell culture; microinjection of protein into T cells; one or more membrane-vesicle methods or one or more physical methods.
The term microinjection is usually associated with direct pressure injection of proteins or other molecules into cells through glass microcapillaries ('needle microinjection'). This is one of the simplest microinjection procedures, and the one most frequently used, but the term is commonly applied to other methods of introducing
macromolecules into cells. Most of the other methods fall into two categories: (1 ) membrane-vesicle methods in which pre-loaded membrane vesicles (erythrocyte ghosts, liposomes, protoplasts) are caused to fuse with cultured cells and release their contents into the cytoplasm; and (2) physical methods, which rely on macromolecules entering cells by diffusion through holes transiently introduced in their plasma membranes by mechanical means (scraping from substratum, agitating with glass beads).
In one embodiment, introducing into T cells the FoxA1 gene or a functional variant or functional fragment thereof; or introducing a nucleic acid construct encoding FoxA1 or a functional variant or functional fragment thereof, comprise one or more steps of transfection. Transfection is the process of deliberately introducing nucleic acids into cells. The term is often used for non-viral methods in eukaryotic cells.
Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane, to allow the uptake of material. Transfection can be chemical-based (carried out using e.g. calcium phosphate, dendrimers or cationic polymers); by non- chemical methods such as electroporation, sonoporation or optical transfection; by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell membrane and deposit their cargo inside. Liposome transfection is termed lipofection. Also particle-based methods and viral methods may be employed. Medical treatment
It is an aspect of the present invention to provide methods for treatment, prevention or alleviation of an inflammatory condition (or inflammatory disease or disorder) in the tissue of one or more organs. Such methods according to the present invention in one embodiment comprise one or more steps of administration or release of an effective amount of a bioactive agent (FoxA1 protein, nucleic acid construct comprising FoxA1 or FoxA1 + T cells, as defined herein) according to the present invention, or a pharmaceutical composition comprising one or more such bioactive agents, to an individual in need thereof. In one embodiment, such steps of administration or release according to the present invention are simultaneous, sequential or separate.
An individual in need as referred to herein, is in one embodiment an individual that benefits from the administration of a bioactive agent according to the present invention. Such an individual in one embodiment suffers from an inflammatory condition in the tissue of one or more organs, or is at risk of suffering therefrom. The term "Individual" refers preferably to vertebrates, particular members of the mammalian species, preferably primates including humans. The individual is in one embodiment any human being, male or female, infant, middle-aged or old. The disorder to be treated or prevented in the individual in one embodiment relates to the age of the individual, the general health of the individual, the medications used for treating the individual and whether or not the individual has a prior history of suffering from diseases or disorders that may have or have induced ischemic and/or inflammatory conditions in the individual.
The terms "treatment" and "treating" as used herein refer to the management and care of a patient for the purpose of combating a condition, disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of the bioactive agent for the purpose of: alleviating or relieving symptoms or complications; delaying the progression of the condition, partially arresting the clinical manifestations, disease or disorder; curing or eliminating the condition, disease or disorder; and/or preventing or reducing the risk of acquiring the condition, disease or disorder, wherein "preventing" or "prevention" is to be understood to refer to the management and care of a patient for the purpose of hindering the development of the condition, disease or disorder, and includes the administration of the active compounds to prevent or reduce the risk of the onset of symptoms or complications. The term "palliation", and variations thereof, as used herein, means that the extent and/or undesirable manifestations of a physiological condition or symptom are lessened and/or time course of the progression is slowed or lengthened, as compared to not administering compositions of the present invention.
The patient to be treated is preferably a mammal, in particular a human being.
Treatment of animals, such as mice, rats, dogs, cats, cows, horses, sheep and pigs, is, however, also within the scope of the present invention. The patients to be treated according to the present invention can be of various ages, for example, adults, children, children under 16, children age 6-16, children age 2-16, children age 2 months to 6 years or children age 2 months to 5 years.
The invention is thus, in one embodiment, directed to a bioactive agent selected from the group consisting of FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined herein above; for use as a medicament.
In another embodiment the invention relates to a bioactive agent selected from the group consisting of FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined herein above, for use in the treatment of an inflammatory disease or disorder. In yet an embodiment the present invention relates to use of a bioactive agent selected from the group consisting of FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined herein above, for the manufacture of a medicament for the treatment of an inflammatory disease or disorder.
Also provided is a method of treating an inflammatory disease or disorder, said method comprising administering to an individual in need thereof an effective amount of one or more bioactive agents selected from FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined herein. In one embodiment said treatment is prophylactic, ameliorative and/or curative. In one embodiment, said mammal is a human (homo sapiens). Said treatment may be initiated prior to symptom onset or after symptom onset. In one embodiment, the present invention relates to FoxA1 protein, or a functional fragment or functional variant thereof; for use as a medicament or for use in the treatment of an inflammatory disease or disorder.
In one embodiment, the present invention relates to use of FoxA1 protein, or a functional fragment or functional variant thereof, for the manufacture of a medicament for the treatment of an inflammatory disease or disorder.
In one embodiment, the present invention relates to a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; for use as a medicament or for use in the treatment of an inflammatory disease or disorder.
In one embodiment, the present invention relates to use of a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; for the manufacture of a medicament for the treatment of an inflammatory disease or disorder.
In one embodiment the inflammatory disease or disorder is an inflammatory condition in the tissue of one or more organs, such as one or more organs selected from the group consisting of kidney, liver, brain, heart, muscles, bone marrow, skin, skeleton, lungs, the respiratory tract, spleen, exocrine glands, bladder, endocrine glands, reproduction organs including the phallopian tubes, eye, ear, vascular system, the gastroinstestinal tract including small intestines, colon, rectum, canalis analis and the prostate gland.
In one embodiment the inflammatory disease to be treated according to the present invention is an autoimmune disorder. Autoimmune disorders are characterized by an overactive immune response. Here, the immune system fails to properly distinguish between self and non-self, and attacks part of the body.
In one embodiment, the present invention relates to immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells); for use as a medicament or for use in the treatment of an inflammatory disease or disorder.
In one embodiment, the present invention relates to use of immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells); for the manufacture of a medicament for the treatment of an inflammatory disease or disorder.
It is also an aspect of the present invention to provide a method of treating an inflammatory disease or disorder, said method comprising the steps of
a. collecting T cells from an individual having an inflammatory disease or
disorder, and
b. transforming said collected T cells into immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells) i. by introducing into said collected T cells FoxA1 protein, or a functional fragment or functional variant thereof; or a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof, and/or ii. by treating said collected T cells with an effective amount of IFN-β, and c. re-introducing said immunosuppressive T cells comprising FoxA1 , or a
functional variant or functional fragment thereof (FoxA1 + T cells), to said individual having an inflammatory disease or disorder.
In one embodiment said collected T cells are transformed into immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), by introducing into said collected T cells i) FoxA1 protein, or a functional fragment or functional variant thereof; or ii) a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof.
In one embodiment said collected T cells are transformed into immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), by treating said collected T cells with an effective amount of IFN-β. It is understood that an effective amount of IFN-β corresponds to a dosage sufficient to transform the T cells into immunosuppressive T cells expressing FoxA1 (FoxA1 + T cells).
In one embodiment said collected T cells are transformed into immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), by introducing into said collected T cells i) FoxA1 protein, or a functional fragment or functional variant thereof; or ii) a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof, and also treating said collected T cells with an effective amount of IFN-β.
In one embodiment said T cells are collected from the blood of an individual, from the cerebrospinal fluid (CSF) of an individual and/or from the lymph of an individual.
In one embodiment said T cells are re-introduced into the blood of an individual, into the cerebrospinal fluid (CSF) of an individual, and/or into the lymph of an individual.
In a preferred embodiment, said T cells are collected from the same individual that receives them after transformation into FoxA1 + T cells.
In one embodiment one or more subpopulations of T cells are specifically collected and re-introduced.
In one embodiment one or more subpopulations of T cells selected from the group consisting of Regulatory T cells (Tregs), Helper T cells (Th cells), Cytotoxic T cells (Tc cells, TCL), Natural killer T cells (NKT) and Memory T cells, are specifically collected and re-introduced.
Inflammatory diseases
Inflammatory abnormalities are a large group of disorders which underlie a vast variety of human diseases. The immune system is often involved with inflammatory disorders, demonstrated in both allergic reactions and some myopathies, with many immune system disorders resulting in abnormal inflammation. Non-immune diseases with etiological origins in inflammatory processes include cancer, atherosclerosis, and ischaemic heart disease.
An inflammatory disease or disorder may be defined as a disorder having an inflammatory component, an inflammatory abnormality and/or an etiological origin in inflammatory processes.
Inflammation is a localized defensive response of the body against pathogens and injury. Immune cells and soluble factors take part in this process to neutralize the injurious agent and initiate tissue repair to restore homeostasis. Loss of regulation of these mechanisms can prevent the final resolution of the inflammatory process, leading to chronic inflammation. Chronic inflammation is extremely relevant in today's modern medicine, as it contributes to the pathogenesis of the most important diseases of the industrialized societies including atherosclerosis, acute and chronic heart failure, cancer, diabetes, and obesity-associated diseases.
Arthritis is a form of joint disorder that involves inflammation of one or more joints. There are over 100 different forms of arthritis. The most common form, osteoarthritis (degenerative joint disease), is a result of trauma to the joint, infection of the joint, or age. Other arthritis forms are rheumatoid arthritis, psoriatic arthritis, and related autoimmune diseases. Septic arthritis is caused by joint infection
Joint diseases such as rheumatoid arthritis (RA) and gout (a type of arthritis) are characterized by episodes with acute exacerbations, in RA the exacerbations (often described as flairs) typically develop on top of chronic symptoms and develop despite intense pharmacological treatment. A similar pattern can be seen in gout, with the major difference that most gout patients are without symptom between the
exacerbations. In both conditions significant neutrophil infiltration into the synovial membrane and joint fluid are the primary pathological hallmark of the exacerbations. In one embodiment the inflammatory disease to be treated according to the present invention is an inflammatory disease selected from the group consisting of arthritis, an arthropathy (a disease of a joint, Arthritis (including diseases associated with arthritis), osteoartritis, rheumatoid arthritis; spondylarthropathies (e.g. ankylosing spondilitis), reactive arthritis (including arthritis following rheumatic fever), Henoch-Schonlein purpura, Reiter's disease, Juvenile Chronic arthritis including Still 's disease, juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, psoriasis, osteoarthritis, osteoarthritis secondary to hypermobilty, congenital dysplasias, slipped femoral epiphysis, Perthes' disease, intra-articular fractures, meniscectomy, obesity, recurrent dislocation, repetitive actions, crystal depositions and diseases and metabolic abnormalities of cartilage including pyrophosphate arthropathy, ochronosis, haemochromatosis, avascular necrosis including Sickle Cell disease, therapy with corticoids or other drugs, Caisson disease, septic or infectious arthitis (including tuberculous arthritis, meningococcal arthritis, gonococcal arthritis, salmonella arthritis), infective endocarditis, viral arthritis, recurrent haemarthrosis, and all kinds of deposition diseases including crystal deposition diseases such as Gout, pyrophosphate arthopathy and acute calcific periarthritis. In a particular embodiment of the present invention the inflammatory disease to be treated is arthritis, such as rheumatoid arthritis.
In another embodiment, said inflammatory disease is a connective tissue disorder; in one embodiment selected from the group consisting of systemic lupus erythematosus, polymyositis/dermatomyositis, systemic sclerosis, mixed connective tissue disease, sarcoidosis and primary Sjogrens syndrome including keratoconjunctivitis sicca, polymyalgia rheumatica, and other types of vasculitis.
In one embodiment, said inflammatory disease is a soft-tissue rheumatism including bursitis, tenosynovitis or peritendonitis, enthesitis, nerve compression, periarthritis or capsulitis, muscle tension and muscle dysfunction.
In one embodiment, said inflammatory disease is selected from the group consisting of vasculitis including vasculitis secondary to rheumatoid arthritis, infective vasculitis due to infections with bacterial species including spirochaetal diseses as Lyme disease, syphilis, rickettsial and mycobacterial infections, fungal, viral or protozoal infections, non-infective vasculitis secondary to hypersensibility and leucocytoplastic vasculitis including Serum Sickness and Henoch-Schonlein purpura, Drug induced vasculitis, essential mixed cryoglobulinaemia, hypocomplentaemia, Vasculitis associated with other kinds of malignancy, non-infective vascultitis including Takayasu's
arteritis/disease, Giant Cell Arteritis (Temporal arteritis and polymyalgia rheumatica), Buerger's disease, polyarteritis nodosa, microscopic polyarteritis, Wegener's granulomatose, Churg-Strauss syndrome, and vasculitis secondary to connective tissue diseases including Systemic Lupus Erythematosus, Polymyositis/
Dermatomyositis, Systemic Sclerosis, Mixed Connetive Tissue Disease, sarcoidosis and Primary Sjogrens syndrome.
In one embodiment, said inflammatory disease is an inflammatory disease of the gastrointestinal system. Said inflammatory disease of the gastrointestinal system is in one embodiment selected from the group consisting of inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behget's disease, indeterminate colitis,
coeliac disease, gluten sensitive enteropathy, eosinophilic gastroenteritis, intestinal lympangiectasia, diverticular disease of the colon, radiation enteritis, irritable bowel syndrome, Whipple 's diease, stomatitis of all kinds, salivary gland diseases (such as sarcoidosis, salivary duct obstruction and Sjogrens syndrome), inflammaton of the oesophagus (e.g. due to gastro- oesophagel reflux or infections with Candida species, herpes simplex and cytomegalus virus), inflammatory diseases of the stomach (including acute and chronic gastritis, helicobacter pylori infection and Mentriers disease), and inflammation of the small intestine. In a particular embodiment of the present invention the inflammatory disease to be treated is an inflammatory bowel disease, such as an inflammatory bowel disease selected from the group consisting of Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behget's disease and indeterminate colitis.
In one embodiment, said inflammatory disease is selected from the group consisting of dermatitis, pemfigus, bulloid pemphigoid, benign mucous membrane pemphigoid, dermatitis herpitiformis, tropical sprue, systemic amyloidosis, primary biliary cirrhosis, Goodpasture syndrome, all kinds of deposition diseases as Gout, pyrophosphate arthopathy and acute calcific periarthritis, pancreatitis, septic discitis, tuberculosis, malignancies (such as metastases, myeloma and others), spinal tumours, ancylosing spondylitis, acute disc prolapse, chronic disc disease/osteoarthritis, osteoporosis, and osteomalacia, Pagets disease, hyperparathyroidism, renal osteodystrophy,
spondylolisthesis, spinal senosis congenital abnormalities and fibromyalgia.
In one embodiment, said inflammatory disease is selected from the group consisting of upper and lower airway diseases such as chronic obstructive pulmonary diseases (COPD), allergic and non-allergic asthma, allergic rhinitis, allergic and non-allergic conjunctivitis, allergic and non-allergic dermatitis and lung inflammation.
CNS disorders
Post mitotic neurons are vastly incapable of regeneration. Therefore, it is essential that these indispensable cell types with highly sophisticated functions be protected from inflammatory damage. From an evolutionary standpoint, this is a rather logical explanation for CNS being relatively an immune privileged organ. However, immune cells are infiltrating the CNS, and it is becoming increasingly known that inflammatory cells and mediators are involved in progression of several of neurodegenerative diseases. Neurons have been neglected as cells with major immune regulatory function. Recently, two signaling pathways by which neurons are controlling function of inflammatory T cells that are causing CNS inflammation were identified. These two pathways include cytokines and their receptor interactions as well as co-stimulatory signaling (via receptor-ligand interaction). These important signaling pathways, in addition to many other signaling pathways, are the communicative language of the cells. Any defect in genes and proteins regulating this communication could disturb the tissue homeostasis and result in pathological conditions.
Thus, in one embodiment of the present invention the inflammatory disease or disorder to be treated is a CNS disorder, such as a CNS disorder having an inflammatory component.
Neurodegeneration is the umbrella term for the progressive loss of structure or function of neurons, including death of neurons. Many neurodegenerative diseases including Parkinson's, Alzheimer's, and Huntington's occur as a result of neurodegenerative processes. In one embodiment of the present invention the CNS disorder having an inflammatory component is a cancer of the CNS, a neurodegenerative disorder, a stroke, or trauma to the head. A stroke, sometimes referred to as a cerebrovascular accident (CVA), is the rapid loss of brain function due to disturbance in the blood supply to the brain. This can be due to ischemia caused by blockage (thrombosis, arterial embolism), or a hemorrhage.
In one embodiment, said inflammatory disease is a neurodegenerative disease, such as a neurodegenerative disease having an inflammatory component.
Neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS (Amyotrophic lateral sclerosis), Polyglutamine (PolyQ) Diseases (These include Huntington's disease, spinocerebellar ataxias, DRPLA (Dentatorubropallidoluysian atrophy) and SBMA (Spinobulbar muscular atrophy or Kennedy disease)) and Non-Polyglutamine Diseases.
Glioblastoma/glioma
Gliomas are tumours arising from glial cells and may occur in the spinal cord or the brain, the latter being more common. Gliomas are the most common type of brain tumour and can be either supratentorial or infratentorial. There are four main types of glioma:
• Ependymomas (ependymal cells).
• Astrocytomas (astrocytes), of which glioblastoma multiforme (GBM) is the most common.
· Oligodendrogliomas (oligodendrocytes).
• Mixed gliomas, eg oligoastrocytomas.
Glioblastoma multiforme (GBM) is the most common and most aggressive type of primary brain tumour. It involves glial cells and has small areas of necrotising tissue surrounded by anaplastic cells. There are also hyperplastic blood vessels.
In one embodiment of the present invention the inflammatory disease or disorder to be treated is a cancer of the CNS, for example glioma, including ependymomas, astrocytomas, oligodendrogliomas and mixed gliomas. In one embodiment the inflammatory disease or disorder to be treated is glioblastoma multiforme. In one embodiment of the present invention the inflammatory disease or disorder to be treated is cancer. Multiple sclerosis
Multiple sclerosis (MS, also known as disseminated sclerosis or encephalomyelitis disseminata) is an inflammatory disease in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged, leading to demyelination and scarring as well as a broad spectrum of signs and symptoms.
Almost any neurological symptom can appear with the disease, and often progresses to physical and cognitive disability. MS takes several forms, with new symptoms occurring either in discrete attacks (relapsing forms) or slowly accumulating over time (progressive forms). Between attacks, symptoms may go away completely, but permanent neurological problems often occur, especially as the disease advances.
There is no known cure for Multiple sclerosis. Treatments attempt to return function after an attack, prevent new attacks, and prevent disability. MS medications can have adverse effects or be poorly tolerated, and many patients pursue alternative
treatments, despite the lack of supporting scientific study. Life expectancy of patients is 5 to 10 years lower than that of the unaffected population.
In relapsing-remitting MS (RRMS) patients have relapses of MS and periods of stability in between relapses. RRMS affects around 85 per cent of everyone diagnosed with MS.
10-15% of diagnosed MS patients have primary progressive MS (PPMS), which from the first (primary) symptoms is progressive. Symptoms gradually get worse over time, rather than appearing as sudden attacks (relapses).
During symptomatic or acute attacks, administration of high doses of IV corticosteroids, such as methylprednisolone, is the usual therapy. Eight disease-modifying treatments have been approved by regulatory agencies for relapsing-remitting multiple sclerosis (RRMS) including: interferon beta-1 a, interferon beta-1 b, glatiramer acetate, mitoxantrone, natalizumab, fingolimod, teriflunomide and dimethyl fumarate. In RRMS they are modestly effective at decreasing the number of attacks. The interferons and glatiramer acetate are first-line treatmentsand are roughly equivalent, reducing relapses by approximately 30%. Early-initiated long-term therapy is safe and improves outcomes. Natalizumab reduces the relapse rate more than first-line agents; however, due to issues of adverse effects is a second-line agent reserved for those who do not respond to other treatments or with severe disease. Mitoxantrone, whose use is limited by severe adverse effects, is a third-line option for those who do not respond to other medications. The disease-modifying treatments have several adverse effects. Also, an autologous attenuated T-cell vaccine (Tovaxin®) is proposed for treatment of MS.
No treatment has been shown to change the course of primary progressive MS and only mitoxantrone has been approved for secondary progressive MS. In this population tentative evidence supports mitoxantrone moderately slowing the progression of the disease and decreasing rates of relapses over two years.
The clinical benefit of interferon-β in relapsing-remitting multiple sclerosis is attributed to its immunomodulatory effects on inflammatory mediators and T cell reactivity.
Interferon beta 1 a is sold under the trade names Avonex (Biogen Idee) and Rebif (Merck Serono), (Pfizer); CinnoVex (CinnaGen) is biosimilar. Interferon beta 1 b is marketed only by Bayer in the US as Betaseron and outside the US as Betaferon. The present inventors have shown that IFN-β can induce FoxA1 and FoxA1 -mediated PD-L1 , leading to the generation of the FoxA1 + T cells; and furthermore that the immunosuppressive FoxA1 + T cells are generated in IFN-p-responsive relapsing- remitting MS (RRMS-R) patients. FoxA1 + T cells have also been identified in PPMS IFN-p-responders (PPMS-R). Thus, the FoxA1 + T cells as defined herein are associated with favorable clinical outcomes in IFN-p-responder MS patients.
Thus, the use of a bioactive agent as defined herein, whether in the form of FoxA1 protein, DNA or FoxA1 + T cells, can potentially increase the treatment effect of IFN-β administered in RRMS-R and/or PPMS-R patients; and be a co-treatment or an alternative treatment to IFN-β in IFN-p-non-responder RRMS patients (RRMS-NR) and/or IFN-p-non-responder PPMS patients (PPMS-NR).
In one embodiment the invention relates to a bioactive agent selected from the group consisting of FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), for use in the treatment of multiple sclerosis.
In one embodiment the invention relates to a method of treating multiple sclerosis comprising administering to an individual in need thereof an effective amount of a bioactive agent selected from the group consisting of FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells).
In one embodiment, said multiple sclerosis is IFN-p-responding MS (MS-R).
In one embodiment, said multiple sclerosis is IFN-p-non-responding MS (MS-NR).
In one embodiment, said multiple sclerosis is selected from the group consisting of relapse-remittent MS and primary progressive MS. In one embodiment, said multiple sclerosis is relapse-remittent MS (RRMS).
In one embodiment, said multiple sclerosis is IFN-p-responding relapse-remittent MS (RRMS-R).
In one embodiment, said multiple sclerosis is IFN-p-non-responding relapse-remittent MS (RRMS-NR).
In one embodiment, said multiple sclerosis is primary progressive MS (PPMS).
In one embodiment, said multiple sclerosis is IFN-p-responding primary progressive
MS (PPMS-R).
In one embodiment, said multiple sclerosis is IFN-p-non-responding primary progressive MS (PPMS-NR). In one embodiment, the bioactive agent for use in the treatment of multiple sclerosis according to the present invention is administered in combination with IFN-β
(comprising at least IFN-p-1 a and IFN-β-Ι b).
In one embodiment, there is provided a bioactive agent according to the present invention for the treatment of IFN-p-non-responding relapse-remittent MS (RRMS-NR). In one embodiment, said bioactive agent is administered to said RRMS-NR alone or at least without co-administration of IFNB.
In one embodiment, there is provided a bioactive agent according to the present invention for the treatment of IFN-p-non-responding primary progressive MS (PPMS- NR). In one embodiment, said bioactive agent is administered to said PPMS-NR alone or at least without co-administration of IFNB.
In one embodiment, there is provided a bioactive agent according to the present invention for the treatment of IFN-p-responding relapse-remittent MS (RRMS-R), wherein said bioactive agent is administered in combination with IFN-β. In one embodiment, there is provided a bioactive agent according to the present invention for the treatment of IFN-p-responding primary progressive MS (PPMS-R), wherein said bioactive agent is administered in combination with IFN-β.
Also provided is a method of distinguishing an IFN-β non-responding multiple sclerosis (MS-NR) patient from an IFN-β responding multiple sclerosis (MS-R) patient comprising one or more steps of
a. treating a MS patient with an effective amount of IFN-β,
b. collecting a sample from said MS patient, and
c. identifying whether immunosuppressive FoxA1 + T cells are present in said sample,
wherein the positive identification or presence of immunosuppressive FoxA1 + T cells in said sample is indicative of said MS being IFN^-responsive, and negative identification or absence of immunosuppressive FoxA1 + T cells in said sample is indicative of said MS being IFN^-non-responsive. In one embodiment said IFN-β non-responding multiple sclerosis (MS-NR) patient comprises IFN-β non-responding relapse-remittent multiple sclerosis (RRMS-NR) patients and/or IFN-β non-responding primary progressive multiple sclerosis (PPMS- NR) patients.
In one embodiment said IFN-β responding multiple sclerosis (MS-R) patient comprises IFN-β responding relapse-remittent multiple sclerosis (RRMS-R) patients and/or IFN-β responding primary progressive multiple sclerosis (PPMS-R) patients.
A method of distinguishing an IFN-β non-responding multiple sclerosis patient from an IFN-β responding multiple sclerosis patient is equivalent to a method for identifying an IFN-β non-responding multiple sclerosis patient and a method for identifying an IFN-β responding multiple sclerosis patient.
A method of distinguishing is equivalent to a method for separating, discriminating or differentiating MS-R and MS-NR.
In one embodiment, said sample is a blood sample. In one embodiment said blood sample is whole blood, optionally treated with an anticoagulant. In one embodiment said blood sample is blood plasma. In one embodiment said blood sample is a buffy coat. In one embodiment, the lymphocytes from said blood sample are separated or isolated. It is understood that said IFN-β treatment may be according to a conventional IFN-β treatment scheme for MS patients.
Said sample is preferably collected from said MS patient when IFN-β has been administered at a sufficient dosage and for a sufficient time. In one embodiment said sample is collected from said MS patient 2-48 hours after IFN-β treatment, such as 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-18, 18-20, 20-22, 22-24, 24-26, 26-28, 28-30, 30-32, 32-34 or 34-36 hours after IFN-β treatment. In one embodiment the sample is collected 12-48 hours after treatment, such as 24-36 hours, for example 24-48 hours after treatment. In one embodiment, the presence of FoxA1 + T cells in the sample is detected by FACS analysis. In another embodiment the presence of FoxA1 + T cells in the sample is detected by expression analysis.
In one embodiment, IFN-β treatment is discontinued if said MS patient is characterized as an IFN-β non-responding multiple sclerosis (MS-NR) patient. In one embodiment, said MS-NR patient is treated instead with a bioactive agent according to the present invention and/or other known treatment options for MS including but not limited to disease-modifying treatments.
In one embodiment IFN-β treatment is continued if said MS patient is characterized as an IFN-β responding relapse-remittent multiple sclerosis (MS-R) patient. Hypersensitivity
Hypersensitivity is an immune response that damages the body's own tissues. They are divided into four classes (Type I - IV) based on the mechanisms involved and the time course of the hypersensitive reaction. Type I hypersensitivity is an immediate or anaphylactic reaction, often associated with allergy. Symptoms can range from mild discomfort to death. Type I hypersensitivity is mediated by IgE, which triggers degranulation of mast cells and basophils when cross-linked by antigen. Type II hypersensitivity occurs when antibodies bind to antigens on the patient's own cells, marking them for destruction. This is also called antibody-dependent (or cytotoxic) hypersensitivity, and is mediated by IgG and IgM antibodies. Immune complexes (aggregations of antigens, complement proteins, and IgG and IgM antibodies) deposited in various tissues trigger Type III hypersensitivity reactions. Type IV hypersensitivity (also known as cell-mediated or delayed type hypersensitivity) usually takes between two and three days to develop. Type IV reactions are involved in many autoimmune and infectious diseases, but may also involve contact dermatitis (poison ivy). These reactions are mediated by T cells, monocytes, and macrophages.
Delayed type hypersensitivity (DTH)
CD4+ helper T cells recognize antigen in a complex with Class 2 major
histocompatibility complex. The antigen-presenting cells in this case are macrophages that secrete IL-12, which stimulates the proliferation of further CD4+ Th1 cells. CD4+ T cells secrete IL-2 and interferon gamma, further inducing the release of other Th1 cytokines, thus mediating the immune response. Activated CD8+ T cells destroy target cells on contact, whereas activated macrophages produce hydrolytic enzymes and, on presentation with certain intracellular pathogens, transform into multinucleated giant cells.
In one embodiment the invention relates to FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined herein above, for use in the treatment of one or more of hypersensitivity, a disease associated with hypersensitivity, delayed type hypersensitivity or a disease associated with delayed type hypersensitivity. According to the invention a disease associated with delayed type hypersensitivity or diseases with a DTH-component comprise diabetes mellitus type I, multiple sclerosis, rheumatoid arthritis, some peripheral neuropathies, Hashimoto's thyroiditis, Crohn's disease, allergic contact dermatitis, psoriasis, temporal or giant-cell arteritis (GCA), symptoms of leprosy, symptoms of tuberculosis, coeliac disease, graft-versus host disease and chronic transplant rejection.
Diabetes Mellitus Type I
Diabetes mellitus type 1 is a form of diabetes mellitus that results from autoimmune destruction of insulin-producing beta cells of the pancreas. The subsequent lack of insulin leads to increased blood and urine glucose causing classical symptoms of polyuria, polydipsia, polyphagia, and weight loss. Glutamic acid decarboxylase autoantibodies (GADA), islet cell autoantibodies (ICA), insulinoma-associated (IA-2) autoantibodies, and zinc transporter autoantibodies (ZnT8) are present in individuals with type 1 diabetes.
In one embodiment the invention relates to FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined herein above, for use in the treatment of diabetes mellitus type 1 . Psoriasis
Psoriasis is a common, chronic immune-mediated skin disease which may also affect the joints.
In one embodiment the invention relates to FoxA1 protein, or a functional fragment or functional variant thereof; a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; or immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined herein above, for use in the treatment of psoriasis.
FOXA1 Gene therapy
FoxA1 may be administered in the form of a protein or in the form of a gene (DNA; nucleic acid construct), by gene therapy.
Gene therapy is the use of DNA as a pharmaceutical agent to treat disease. A common form of gene therapy involves using DNA that encodes a functional, therapeutic gene to replace a mutated gene. Other forms involve directly correcting a mutation, or using DNA that encodes a therapeutic protein drug (rather than a natural human gene) to provide treatment.
In gene therapy, DNA that encodes a therapeutic protein is packaged within a vector from which, once inside cells, the DNA becomes expressed by the cell machinery, resulting in the production of the therapeutic protein. In 2012, Glybera became the first gene therapy treatment to be approved for clinical use.
It is an aspect of the present invention to provide a nucleic acid construct encoding for and/or being capable of expressing a FoxA1 peptide according to the present invention. In one embodiment said nucleic acid construct encoding FoxA1 comprises all or part of SEQ ID NO:3 (FOXA1 gene).
Preferably said nucleic acid construct will be able to continuously express a peptide according to the present invention for a prolonged period of time, such as at least 1 month, for example at least 2 months, such as at least 3 months, for example at least 4 months, such as at least 5 months, for example at least 6 months, such as at least 7 months, for example at least 8 months, such as at least 9 months, for example at least 12 months.
In one embodiment of the present invention there is provided a nucleic acid construct encoding for and being capable of expressing FoxA1 , or a functional variant or functional fragment thereof.
It is also an aspect of the present invention to provide a nucleic acid construct encoding and/or being capable of expressing FoxA1 according to the present invention for use in the treatment of an inflammatory disease or disorder, such as an inflammatory disease as defined herein.
In one embodiment the encoded FoxA1 of the nucleic acid construct is a functional variant having at least 60% sequence identity, such as at least 65% sequence identity, for example at least 70% sequence identity, such as at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity to SEQ ID NO:1 (protein) or SEQ ID NO:3 (DNA).
In one embodiment the encoded FoxA1 of the nucleic acid construct is a functional variant having from 60 to 65% sequence identity, for example from 65 to 70% sequence identity, such as from 70 to 75% sequence identity, for example from 75 to 80% sequence identity, such as from 80 to 85% sequence identity, for example from 85 to 90% sequence identity, such as from 90 to 95% sequence identity, for example from 95 to 99% sequence identity, such as 99 to 99.9 sequence identity to SEQ ID NO:1 (protein) or SEQ ID NO:3 (DNA).
In one embodiment the encoded FoxA1 of the nucleic acid construct is a functional fragment of SEQ ID NO: 1 comprising from 10 to 471 consecutive amino acids of SEQ ID NO:1 , such as from 10-25, 25-50, 50-75, 75-100, 100-125, 125-150, 150-175, 175- 200, 200-250, 250-300, 300-350, 350-400, 400-450 or from 450-471 consecutive amino acids of SEQ ID NO:1. By nucleic acid construct is understood a genetically engineered nucleic acid. The nucleic acid construct may be a non-replicating and linear nucleic acid, a circular expression vector or an autonomously replicating plasmid. A nucleic acid construct may comprise several elements such as, but not limited to genes or fragments of same, promoters, enhancers, terminators, poly-A tails, linkers, polylinkers, operative linkers, multiple cloning sites (MCS), markers, STOP codons, internal ribosomal entry sites (IRES) and host homologous sequences for integration or other defined elements. It is to be understood that the nucleic acid construct according to the present invention may comprise all or a subset of any combination of the above-mentioned elements.
Methods for engineering nucleic acid constructs are well known in the art (see, e.g., Molecular Cloning: A Laboratory Manual, Sambrook et al., eds., Cold Spring Harbor Laboratory, 2nd Edition, Cold Spring Harbor, N.Y., 1989). Further, nucleic acid constructs according to the present invention may be synthesized without template, and may be obtained from various commercial suppliers (e.g. Genscript Corporation).
In one embodiment, the nucleic acid construct are naked DNA constructs comprising sequences encoding the peptide of the invention. An expression cassette is a part of a vector DNA used for cloning and transformation. An expression cassette is composed of one or more genes and the sequences controlling their expression. Three components comprise an expression cassette: a promoter sequence, an open reading frame, and a 3' untranslated region that, in eukaryotes, usually contains a polyadenylation site. Different expression cassettes can be transformed into different organisms including bacteria, yeast, plants, and mammalian cells as long as the correct regulatory sequences are used.
The present invention also provides the nucleic acid construct as described herein above comprised within a delivery vehicle. A delivery vehicle is an entity whereby a nucleotide sequence or polypeptide or both can be transported from at least one media to another. Delivery vehicles are generally used for expression of the sequences encoded within the nucleic acid construct and/or for the intracellular delivery of the construct or the polypeptide encoded therein. In one embodiment, there is provided a delivery vehicle comprising the nucleic acid construct according to the present invention. A delivery vehicle may be selected from the group consisting of: plasmid vectors, RNA based vehicles, DNA based vehicles/ vectors, lipid based vehicles (such as a liposome), polymer based vehicles (such as a cationic polymer DNA carrier), colloidal gold particles (coating) and virally derived DNA or RNA vehicles or vectors.
Methods of non-viral delivery include physical (carrier-free delivery) and chemical approaches (synthetic vector-based delivery).
Physical approaches, including needle injection, gene gun, jet injection,
electroporation, ultrasound, and hydrodynamic delivery, employ a physical force that permeates the cell membrane and facilitates intracellular gene transfer. Said physical force may be electrical or mechanical.
Examples of chemical delivery vehicles include, but are not limited to: biodegradable polymer microspheres, lipid based formulations such as liposome carriers, cationically charged molecules such as liposomes, calcium salts or dendrimers,
lipopolysaccharides, polypeptides and polysaccharides.
Another embodiment of the present invention comprises a vector which herein is denoted a viral vector (i.e. not a virus) as a delivery vehicle. Viral vectors according to the present invention are made from a modified viral genome, i.e. the actual DNA or RNA forming the viral genome, and introduced in naked form. Thus, any coat structures surrounding the viral genome made from viral or non-viral proteins are not part of the viral vector according to the present invention.
In one embodiment the virus from which the viral vector is derived is selected from the non-exhaustive group consisting of: adenoviruses, retroviruses, lentiviruses, adeno- associated viruses, herpesviruses, vaccinia viruses, foamy viruses, cytomegaloviruses, Semliki forest virus, poxviruses, RNA virus vector and DNA virus vector. Such viral vectors are well known in the art.
In one embodiment, said viral vector is selected from the group consisting of adenoviruses, lentiviruses, adeno-associated viruses (AAV) and recombinant adeno- associated viruses (rAAV). In one embodiment, said viral vector is a therapeutic rAAV vector.
An adenovirus is a group of double-stranded DNA containing viruses. Adenoviruses can be genetically modified making them replication incompetent or conditionally replication incompetent. In this form, as adenoviral constructs or adenovectors, they can be used as gene delivery vehicles for vaccination or gene therapy.
Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell. These features make AAV a very attractive candidate for creating viral vectors for gene therapy. To date, AAV vectors have been used in over 80 clinical trials worldwide.
At least 1 1 serotypes of AAV exist, and all of these are encompassed by the present invention.
In one embodiment the nucleic acid construct and/or the delivery vehicle is designed in order to achieve T-cell specific delivery and/or expression, i.e. the construct is delivered and/or expressed exclusively, predominantly or mainly in T cells. Such T cells may be any given T cell, such as one or more subpopulations selected from the group consisting of Regulatory T cells (Tregs), Helper T cells (Th cells), Cytotoxic T cells (Tc cells, TCL), Natural killer T cells (NKT) and Memory T cells.
In one embodiment there is provided a viral vector comprising the nucleic acid construct according to the present invention, wherein said viral vector specifically targets T cells.
In one embodiment there is provided a nanoparticle comprising the protein or nucleic acid construct according to the present invention, wherein said nanoparticle specifically targets T cells. This may be achieved by providing a nanoparticle with a T cell selective and/or specific surface.
In one embodiment, the nucleic acid construct and/or the delivery vehicle according to the present invention is CD4+ and/or CD8+ specific. In one embodiment, said nucleic acid construct comprises a T-cell specific promoter or T cell specific expression cassette.
An expression cassette is composed of one or more genes and the sequences controlling their expression. Three components comprise an expression cassette: a promoter sequence, an open reading frame, and a 3' untranslated region that, in eukaryotes, usually contains a polyadenylation site.
Recombinant cell
An aspect of the present invention relates to a cell comprising the nucleic acid construct according to the present invention. Such a recombinant cell can be used a tool for in vitro research, as a delivery vehicle for the nucleic acid construct or as part of a gene-therapy regime. The nucleic acid construct according to the invention can be introduced into cells by techniques well known in the art and which include
microinjection of DNA into the nucleus of a cell, transfection, electroporation, lipofection/liposome fusion and particle bombardment. Suitable cells include
autologous and non-autologous cells, and may include xenogenic cells.
In one embodiment there is provided a recombinant cell, such as a recombinant T cell, comprising a nucleic acid construct comprising FoxA1 , or a functional variant or functional fragment thereof.
Administration and dosage
According to the present invention, a FoxA1 peptide or a nucleic acid construct encoding said peptide, or FoxA1 + T cells, or a composition comprising the same, is to be administered to an individual in need of treatment in pharmaceutically effective doses or a therapeutically effective amount. The dosage requirements will vary with the particular drug composition employed, the route of administration and the particular subject being treated, which depend on the severity and the sort of the disorder as well as on the weight and general state of the subject. It will also be recognized by one skilled in the art that the optimal quantity and spacing of individual dosages will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optima can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of a compound given per day for a defined number of days, can be ascertained using conventional course of treatment determination tests.
A therapeutically effective amount according to the present invention is in one embodiment an amount sufficient to cure, prevent, reduce the risk of, alleviate or partially arrest the clinical manifestations of a given disease or disorder and its complications. The amount that is effective for a particular therapeutic purpose will depend on the severity and the sort of the disorder as well as on the weight and general state of the subject. An amount adequate to accomplish this is defined as a "therapeutically effective amount".
A 'bioactive agent' will be used to denote collectively a peptide, a nucleic acid construct encoding said peptide, FoxA1 + T cells, and a composition comprising the same, according to the present invention.
In one embodiment of the present invention, the bioactive agent is administered in doses of from 1 μg day to 100 mg/day; such as from 1 μg/day to 10 Mg/day, such as 10 g/day to 100 Mg/day, such as 100 Mg/day to 250 Mg/day, such as 250 Mg/day to 500 Mg/day, such as 500 Mg/day to 750 Mg/day, such as 750 Mg/day to 1 mg/day, such as 1 mg/day to 2 mg/day, such as 2 mg/day to 5 mg/day, or such as 5 mg/day to 10 mg/day, such as 10 mg/day to 20 mg/day, such as 20 mg/day to 30 mg/day, such as 30 mg/day to 40 mg/day, such as 40 mg/day to 50 mg/day, such as 50 mg/day to 75 mg/day, or such as 75 mg/day to 100 mg/day. In one embodiment of the present invention, one single dose of the bioactive agent is administered and may comprise of from 1 Mg/kg body weight to 100 mg/kg body weight; such as from 1 to 10 Mg/kg body weight, such as 10 to 100 Mg/day, such as 100 to 250 Mg/kg body weight, such as 250 to 500 Mg/kg body weight, such as 500 to 750 Mg/kg body weight, such as 750 Mg/kg body weight to 1 mg/kg body weight, such as 1 mg/kg body weight to 2 mg/kg body weight, such as 2 to 5 mg/kg body weight, such as 5 to 10 mg/kg body weight, such as 10 to 20 mg/kg body weight, such as 20 to 30 mg/kg body weight, such as 30 to 40 mg/kg body weight, such as 40 to 50 mg/kg body weight, such as 50 to 75 mg/kg body weight, or such as 75 to 100 mg/kg body weight. A dose according to the present invention may be administered one or several times per day, such as from 1 to 6 times per day, such as from 1 to 5 times per day, such as from 1 to 4 times per day, such as from 1 to 3 times per day, such as from 1 to 2 times per day, such as from 2 to 4 times per day, such as from 2 to 3 times per day. A dose may also be administered in intermittent intervals, or intervals, whereby a dose is not administered every day. Rather one or more doses may be administered every second day, every third day, every fourth day, every fifth day, every sixth day, every week, every second week, every third week, every fourth week, every fifth week, every sixth week, or intervals within those ranges (such as every 2 to 4 weeks, or 4 to 6 weeks).
Routes of administration
It will be appreciated that the preferred route of administration will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated, the location of the tissue to be treated in the body and the active ingredient chosen.
In one embodiment of the present invention, the route of administration allows for the bioactive agent to cross the blood-brain barrier. Systemic treatment
For systemic treatment according to the present invention the route of administration is capable of introducing the bioactive agent (a peptide, a nucleic acid construct encoding said peptide, FoxA1 + T cells, and a composition comprising the same, according to the present invention) into the blood stream to ultimately target the sites of desired action.
Such routes of administration are any suitable routes, such as an enteral route (including the oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal and intraperitoneal administration), and/or a parenteral route (including subcutaneous, intramuscular, intrathecal, intracerebral, intravenous and intradermal administration).
Appropriate dosage forms for such administration may be prepared by conventional techniques. Parenteral administration
Parenteral administration is any administration route not being the oral/enteral route whereby the medicament avoids first-pass degradation in the liver. Accordingly, parenteral administration includes any injections and infusions, for example bolus injection or continuous infusion, such as intravenous administration, intramuscular administration or subcutaneous administration. Furthermore, parenteral administration includes inhalations and topical administration.
Accordingly, the bioactive agent may be administered topically to cross any mucosal membrane of an animal to which the biologically active substance is to be given, e.g. in the nose, vagina, eye, mouth, genital tract, lungs, gastrointestinal tract, or rectum, preferably the mucosa of the nose, or mouth, and accordingly, parenteral
administration may also include buccal, sublingual, nasal, rectal, vaginal and intraperitoneal administration as well as pulmonal and bronchial administration by inhalation or installation. Also, the agent may be administered topically to cross the skin.
In one embodiment, the intravenous, subcutaneous and intramuscular forms of parenteral administration are generally preferred.
Local treatment
The bioactive agent according to the invention may in one embodiment be used as a local treatment, i.e. be introduced directly to the site(s) of action. Accordingly, the bioactive agent may be applied to the skin or mucosa directly, or the bioactive agent may be injected into the site of action, for example into the diseased tissue or to an end artery leading directly to the diseased tissue.
These administration forms preferably avoid the blood brain barrier, and the blood- retina barrier.
In one particular embodiment, the bioactive agent according to the present invention is applied or injected directly into the brain, such as into a specific region of the brain. Thus, an effect of the bioactive agent may be achieved in the region of the brain where it is mainly required. This may depend on the condition being treated. This may be termed intracerebral administration. In another embodiment, the bioactive agent is administered via intrathecal
administration or injection, i.e. in the space under the arachnoid membrane of the brain or spinal cord. Pharmaceutical formulation
Whilst it is possible for the bioactive agent of the present invention (a peptide, a nucleic acid construct encoding said peptide, or T cells comprising said peptide, and a composition comprising the same) to be administered as the raw chemical, it is sometimes preferred to present them in the form of a pharmaceutical formulation. Such a pharmaceutical formulation may be referred to as a pharmaceutical composition, pharmaceutically acceptable composition or pharmaceutically safe composition.
Accordingly, the present invention further provides a pharmaceutical formulation, which comprises a bioactive agent of the present invention, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier, excipient and/or diluent. The pharmaceutical formulations may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 2005,
Lippincott, Williams & Wilkins. The pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more excipients which may also act as diluents, flavouring agents, solubilizers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrating agents, or an encapsulating material.
Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene, water, saline or a glucose solution. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glycerol monostearate or glycerol distearate, alone or mixed with a wax.
Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations. Such liquid forms include solutions,
suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
The bioactive agent of the present invention may be formulated for parenteral administration and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers, optionally with an added
preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol.
Examples of oily or non-aqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.
The bioactive agent of the invention may also be formulated for topical delivery.
Regions for topical administration include the eye or the cornea, the skin surface and also mucous membrane tissues of the vagina, rectum, nose, mouth, and throat. The topical formulation may include a pharmaceutically acceptable carrier adapted for topical administration. Thus, the composition may take the form of a suspension, solution, ointment, lotion, sexual lubricant, cream, foam, aerosol, spray, suppository, implant, inhalant, tablet, capsule, dry powder, syrup, balm or lozenge, for example.
Formulations for use in nasal, pulmonary and/or bronchial administration are normally administered as aerosols in order to ensure that the aerosolized dose actually reaches the mucous membranes of the nasal passages, bronchial tract or the lung. Typically aerosols are administered by use of a mechanical device designed for pulmonary and/or bronchial delivery, including but not limited to nebulizers, metered dose inhalers, and powder inhalers. With regard to construction of the delivery device, any form of aerosolization known in the art, including but not limited to spray bottles, nebulization, atomization or pump aerosolization of a liquid formulation, and aerosolization of a dry powder formulation, can be used. Liquid aerosol formulations in general contain a compound of the present invention in a pharmaceutically acceptable diluent. Pharmaceutically acceptable diluents include but are not limited to sterile water, saline, buffered saline, dextrose solution, and the like.
Formulations for dispensing from a powder inhaler device will normally comprise a finely divided dry powder containing a pharmaceutical composition of the present invention (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device. Dry powder formulations for inhalation may also be formulated using powder- filled capsules, in particularly capsules the material of which is selected from among the synthetic plastics.
The formulations of the present embodiment may also include other agents useful for pH maintenance, solution stabilization, or for the regulation of osmotic pressure.
Pharmaceutically acceptable salts of the instant peptide compounds, where they can be prepared, are also intended to be covered by this invention. These salts will be ones which are acceptable in their application to a pharmaceutical use. By that it is meant that the salt will retain the biological activity of the parent compound and the salt will not have untoward or deleterious effects in its application and use in treating diseases.
Pharmaceutically acceptable salts are prepared in a standard manner. If the parent compound is a base it is treated with an excess of an organic or inorganic acid in a suitable solvent. If the parent compound is an acid, it is treated with an inorganic or organic base in a suitable solvent.
The compounds of the invention may be administered in the form of an alkali metal or earth alkali metal salt thereof, concurrently, simultaneously, or together with a pharmaceutically acceptable carrier or diluent, especially and preferably in the form of a pharmaceutical composition thereof, whether by oral, rectal, or parenteral (including subcutaneous) route, in an effective amount.
Examples of pharmaceutically acceptable acid addition salts for use in the present inventive pharmaceutical composition include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, for example. Second active ingredients
The bioactive agent of the present invention may be combined with or comprise one or more second active ingredients which are understood as other therapeutical compounds or pharmaceutically acceptable derivatives thereof. "Co-administering" or "co-administration" of bioactive agents of the invention and state- of-the-art medicaments, as used herein, refers to the administration of one or more bioactive agents of the present invention, or administration of one or more bioactive agents of the present invention and a state-of-the-art pharmaceutical composition within a certain time period or administered together. Methods for treatment according to the present invention may thus further comprise one or more steps of administration of one or more second active ingredients, either concomitantly or sequentially, and in any suitable ratios. Such second active
ingredients may, for example, be selected from compounds used to treat or prevent inflammatory disorders or symptoms and complications associated with inflammatory disorders. Exemplary drugs comprise immunosuppressive drugs, anti-inflammatory drugs, and so forth.
Methods of treatment according to the present invention may include a step wherein the bioactive agent as defined herein is administered simultaneously, sequentially or separately in combination with one or more second active ingredients.
It follows, that co-administration should be targeted so that to optimise treatment of the patient, i.e. in a patient with multiple sclerosis, a drug approved for this specific purpose may be complemented with the peptide, compound or composition according to the present invention to optimise and improve treatment outcome for the patient. This is regardless of whether the approved drug for the specific purpose is prophylactic, ameliorating or curative.
In one embodiment, the bioactive agent according to the present invention is administered in combination with IFNB (interferon beta, including IFNB 1 a and 1 b). Examples
FoxA1 directs the lineage and immunosuppressive properties of novel TRFOXAI regulatory cells important in EAE and MS
Inflammation is a self-destructive process that can lead to irreversible chronic tissue destruction. The defective generation or function of Treguiatory cells contributes to chronic autoimmune inflammation. We report the first identification of FoxA1 as a novel transcription factor in T-cells that conveys suppressive properties in a new Treg population, hereby called ΤΚΡΟΧΑΙ (or FoxA1 +Treg or FoxA1 +T-cells). FoxA1 bound to the pdl1 promoter, inducing PD-L1 , which was essential for TRFoxAis to kill activated T-cells. TRFOXAI cells had a distinct transcription profile. In addition to expressing FoxA1 , TRFoxAis were CD4hl, PD-L1 hl and CD47+. \1nb~'~ and Ifnaf'' mice were defective in TRFoxAis- In mice, adoptive transfer of stable TRFoxAi inhibited experimental autoimmune encephalomyelitis mediated by functional FoxA1 and PD-L1. In patients with relapsing-remitting multiple sclerosis, response to IFN-β treatment was associated with expansion of suppressive TRFoxAi cells. Therefore, FoxA1 is a lineage-specification factor with a specialized role in supporting differentiation and the suppressive function of TRFoxAi cells.
Introduction
Immune function that preserves tolerance while retaining antimicrobial function is imperative for preventing chronic inflammation and autoimmunity. Defects in regulatory T-cell (Treg) generation or function are risk factors for autoimmune diseases. Treg-mediated suppression is therefore vital for negatively regulating inflammation1.
T-cell differentiation into effector or Treg cells is determined by the dynamic action of transcription factors. Tbet determines Th1 , GAT A3 decides Th2, and RORyt controls Th17 fate and function. FoxP3 is the cell-lineage commitment factor for natural and induced (n/i)Tregs1 "3. In mice and humans, FoxP3 mutations lead to multiorgan failure and systemic autoimmunity4,5.
Treg defects are reported in experimental autoimmune encephalomyelitis (EAE)6, a model for multiple sclerosis (MS), a tissue-specific inflammatory disease affecting the central nervous system (CNS). Neuron-induced, FoxP3-expressing Tregs control CNS-inflammation in EAE7. However, their role in MS is debated8,9. Although Treg numbers are unchanged in MS, they might be malfunctioning. The effect of interferon (IFN)-p, a leading treatment for MS, on TregS is contentious8"12.
Mice lacking Ifnb and Ifna/b-receptor genes develop chronic inflammatory and demyelinating EAE13,14. However, chronic relapsing-remitting (RR)-EAE in \1nb~'~ mice is prevented upon induction of systemic tolerance resulting in Treg-expansion15. We reported that endogenous IFN-β does not regulate EAE through differential T-cell priming and/or effector cytokines, T-helper-subset shift, B-cell activation, or antibody production, but through limitation of CNS-inflammation13,16. Although \1nb~'~ mice generate Tregs systemically, they might lack the capacity to generate tissue-specific Tregs in the inflamed CNS. We investigated if chronic RR-EAE in lfnb~;~ mice results from a failure to generate tissue-specific TregS.
We found no defects associated with Foxp3+Tregs in the inflamed CNS of \1nb~'~ mice. However, we discovered a novel population of Tregs in wildtype mice that was absent in lfnb' mice. These suppressive cells, hereby called TRFOXAI, were generated in IFN-p-responsive relapsing-remitting MS (RRMS) patients. TRFOXAI cells express FoxA1 , an epigenetic-modifying pioneer transcription factor17 important in embryonic development, stem cell differentiation, hepatocyte development, and cancer epigenetics18"22. FoxA1 modulates postembryonic tissue functions, including the brain. FoxA1 is necessary for epithelial cells, and mediates lineage specification23,24.
Previously, no function was reported for FoxA1 in T-cells. Here, we demonstrate that FoxA1 is a lineage-specification factor that defines TRFOXAI cells and direct function of these novel Treg cells.
Results
Novel murine CD4hiPD-L1hi Treg cells
We hypothesized that defects in generating tissue-specific Tregs were responsible for severe RR-EAE in \1nb~'~ mice. We utilized MBP89-ioi-EAE, a chronic demyelinating RR-EAE13, a model of RRMS. Ifnb~;~ mice exhibited higher symptoms, relapses, chronic-EAE and CNS-inflammation than their littermates (Fig. 1 a, Fig. 7a-c, Table I). In addition to massive spinal cord and cerebellar inflammation, \1nb~'~ mice had cortical inflammation absent in WT (Fig. 1 b), mimicking cortical inflammation in early MS25.
We found no differences in Treg(CD4+CD25+FoxP3+) numbers in the CNS of lfnb~;~ compared to WT mice (Fig. 1 d, Fig. 8a-b). Tregs from lfnb~;~ and WT were equally suppressive in vitro and in vivo ameliorating EAE (Fig. 8c-e). However, we consistently found a population of CD4hiPD-L1 hiFoxp3" T-cells in the WT-CNS that were lacking in \fnb~1' mice (Fig. 1 c-f).
The CD4hiPD-L1 hiT-cells were enriched in the CNS of WT RR-EAE mice compared to spleen or lymph nodes (Fig. 1 g). We hypothesized that these cells affected inflammation control and their absence in Ifnb mice contributed to disease chronicity.
FoxA1 is the unique transcription factor of TRFOXAI cells
We established an ex-vivo primary encephalitogenic-MBP89-ioi-reactive T-cell line (EncT) capable of adoptive EAE-transfer13. PD-L1 -PD-1 signaling is involved in generating PD-1 hlCD8+ T-cells in HIV-infected patients, a result of hyperactivation26. We checked if multiple antigen-activation of EncT-cells led to the CD4hlPD-L1 hl phenotype. Multiple activation rounds did not generate CD4hlPD-L1 hl, but co-culture of EncT-cells with cerebellar granular neurons (CGNs), which also induces TGF- p+FoxP3+Tregs 7 (Fig. 8f) and regulates CNS homeostasis27, led to CD4hiPD-L1 hi T-cell generation (Fig. 2a).
We compared the CD4hlPD-L1 hl gene expression profile to EncT-progenitors and neuron-induced TregS. Compared to EncT-progenitors, the absolute number of genes constrained to either CD4hlPD-L1 hl or Treg cells was equal; thus these profiles indicated two distinct cell types (Fig. 2b-c).
FoxA1 , a gene critical for epigenetic reprogramming and cell-lineage commitment24 was robustly upregulated in CD4hiPD-L1 hiT-cells (Fig. 2b, Table II). FoxA1 was found as the first ranking regulated transcription factor using Gene
Ontology (Fig. 9a) and the top ranking overrepresented canonical pathway by GSEA analysis (Table III), suggesting its involvement in initiation of a specific gene expression program. CD4hiPD-L1 hiT-cells expressed CD47, CD69 and high levels of nuclear FoxA1 (Fig. 2d, Table IV); hence, we named them ΤΚΡΟΧΑΙ cells.
By gene and phenotype profile comparison, neuron-induced (n)TRFoxAi cell profile (Fig. 9b) was distinct from n/iTregs28"31 (Fig. 9c-e, Table lll-V) and exhausted T- cells32 (Fig. 10, Table VI). We therefore examined in vitro and in vivo properties of the novel TRFoxAi-population.
Ectopic overexpression of FoxAI decreased phosphorylated c-Fos
Unlike CGN-induced Tregs7 and their EncT-progenitors, nTRFoxAis were nonproliferative measured by Ki-67+ (Fig. 2e). We investigated signaling associated with FoxA1 , regarding nuclear translocation of phosphorylated (p)c-Fos, as c-Fos is involved in T-cell proliferation and possibly T-lymphocyte development and function33.
Ectopic expression of FoxA1 in purified CD4+T-cells (Fig. 2f-i) led to c-Fos downregulation (Fig. 2f, g). Overexpressed FoxA1 was translocated to the nucleus in association with reduced nuclear pc-Fos (Fig. 2h,i). These findings supported that TRFOXAIS were nonproliferative and FoxA1 influenced c-Fos signaling.
RFOXA suppresses T-cell activation and tissue inflammation
RFOXA-IS inhibited proliferation and increased cell-death of responder T-cells (ResT) (Fig. 2j-k). nTRFoxAi cells generated by co-culturing of OVA-activated CD4+OTII- cells with CGNs also suppressed ResT-cells. IL-2 rescued ResT-cell proliferation but not cell-death (Fig. 1 1 , indicating that TRFoxAis regulated these events independently.
To investigate if TRFOXAIS were suppressive in vivo, purified nTRFoxAis were adoptively transferred to ears, in a murine delayed type hypersensitivity model of tissue inflammation. Ears receiving nTRFoxAis had significantly less inflammation (Fig. 2I). nTRFoXAis also significantly reduced adoptive-EAE incidence, prevented clinical severity and inhibited CNS-inflammation in lfnb~;~ mice (Fig. 2m-n, Table VII). These results supported the in vivo anti-inflammatory and suppressive properties of TRFoxAis- IFN induction of suppressive T requires IFNA-receptor signaling
As lfnb~;~ mice lacked TRFoxAis, we investigated whether IFN-p-treatment restored in vivo TRFoXAi-generation. lfnb' mice develop severe MOG35-55-EAE, and treatment of EAE with IFN-β reduces clinical symptoms15. Treatment of lfnb~;~ mice with murine (m)IFN-p restored generation of TRFoxAis in the CNS (Fig. 3a-c) and spleen (Fig. 12). mIFN-p-treatment of CD4+T-cells induced TCRap+CD4hiPD-L1 hi T-cells in vitro (Fig. 3d), which highly expressed FoxA1 (Fig. 3e-f), hereby referred to as IFN-β- induced (i)TRFoxAis- Immunocytochemistry revealed nuclear FoxA1 expression after mIFN-p-stimulation (Fig. 3g). Compared to PD-L1 l0T-cells, only PD-L1 hiT-cells expressed nuclear FoxA1 (Fig. 3h-i).
CD4+T-cells were purified from healthy donors and cultured with or without human (h)IFN-p. Treatment did not induce FoxP3 and IL-35, markers of classical Tregs, or PD-1 hl, an exhausted T-cell phenotype (Fig. 13a-d). However, hlFN-β significantly induced TCRap+CD4+PD-L1 hiFoxA1 + iTRFoxAis (Fig. 3j-k). Purified iTRFoxAis (R1 -gated) expressed nuclear FoxA1 compared to PD-L1 l0T-cells (R2-gated) (Fig. 3I).
We studied if iTRFoxAi-generation required IFN-p-receptor signaling. In vitro mIFN-p-treatment of CD4+T-cells purified from \fnaf'~ mice did not increase the FoxA1 (Fig. 3m). To determine the requirement for IFN-p-IFNAR signaling for TRFoxAi generation in vivo, and to exclude that IFN-β induces TRFoxAis through other cells, we purified CD4+T-cells from \fnaf'~ and WT mice, and transferred them to NOG mice. mlFN-β induced generation of FoxA1 +TRFoxAis in mice receiving WT but not \fnaf'~ CD4+T-cells (Fig. 3 m-o). Thus, IFN-p-induction of FoxA1 and TRFoxAis in vitro and in vivo required IFN-p-receptor signaling of CD4+ T-cells.
We studied the in vitro suppressive capacity of iTRFoxA1s. hlFN-p-induced TRFOXAIS inhibited proliferation of ResT-cells by inhibiting entry into S-phase, and induced ResT- cell death in the sub-G1 population (Fig. 3p-s). Purified mIFN-p-induced TRFoxAis were similarly suppressive (Fig. 3t-u).
Tregs and Th2-cells modulate antigen-presenting cells (APCs)34 and effective IFN-p-treatment of RRMS is associated with APC modulation35'36. IFN-β induces IL-10 in APCs associated with reduced MS-symptoms37. In contrast to Tregs and Th2-cells, iTRFoxAis did not induce IL-10 in APCs. However, iTRFoxAis significantly suppressed production of pro-inflammatory cytokines (IL-12 and IL-17) by APCs (Fig. 14).
Using chimeric mice, we investigated if in vivo IFN-p-generated TRFoxAis suppressed activated ResT-cells with an IFN-p-IFNAR-signaling requirement.
Suppression of ResT-cells was only observed in mice receiving IFN-p-treated WT- CD4+T-cells but not /fnar/"-CD4+T-cells (Fig. 3v-w). Thus, generation of suppressive TRFoxAis required IFN-p-IFNAR signaling.
Gene profile homology between nTregs and TGF-p-induced (i)Tregs is reported28"31 (Fig.9e). To identify common gene profile of TRFoxAi cells, we compared the gene profile of nTRFoxAis and iTRFoxA1s. As shown by Venn diagram (Fig. 3x), 936 genes were similarly up- or down-regulated in nTRFoxAis and iTRFoxA1s. Common genes of TRFoxAis vs. EncT-cells were compiled to determine TRFoxAiS' heatmap profile (Fig. 3y). Using Gene Ontology analysis, FoxA1 was also found among the top 20 ranking transcription factors differentially expressed in iTRFoxAis (Table VIII). To identify common canonical signaling pathways, the common 936 genes were analyzed by GSEA. Genes in the top 20 pathways were tabulated and compared to find the overlapped in known biological pathways (Table IX-X). A heatmap of the shared pathways (8/20) by i/nTRFoxA1s vs. EncT-cells is shown in Fig. 3z.
FoxA1 conveys TRFOXAI suppressive functions
We investigated if FoxA1 was essential for generation and suppressive activity of IFN-p-induced TRFoxA1s, and if FoxA1 alone mediated TRFoxA1 function. FoxA1 was knocked down (KD) in purified CD4+T-cells prior to IFN-p-stimulation (Fig. 4a). To prevent off target effects, 4 different siRNAs targeting 3'UTR region of foxal were utilized, all showed specific KD-effects hence they were pooled (Fig. 15a-b). FoxAI KD prevented PD-L1 expression (Fig. 4b), and resulted in in vitro loss of suppressive function (Fig.4c). FoxAI KD and CtrlsiRNA T-cells were transferred to chimeric NOG mice populated with preactivated ResT-cells. In vivo mIFN-p-treated CD4+T-cells expressed FoxA1 (Fig. 4d). FoxAI KD CD4+T-cells did not suppress ResT-cells (Fig. 4e-f), indicating that FoxA1 was required for functional TRFoxAis- TRFoxAis ectopically overexpressing FoxA1 (Fig. 4g, Fig. 15c) profoundly reduced responder T-cell proliferation (Fig. 4h), suggesting that FoxA1 conveyed the suppressive capacity of TRFoxAis-
IFN-p-induced TRFoxAis suppress EAE mediated by FoxA1 and PD-L1
To verify the in vivo suppressive function of TRFoxAis, iTRFoxAis were transferred to lfnb~;~ mice with adoptive-EAE. While iTRFoxAis suppressed EAE- progression and CNS-inflammation, FoxAI KD eliminated their suppressive function. Rescuing this phenotype by ectopic expression of siRNA-insensitive pcDNA3.1 foxal, expressing ORF sequence of foxal (Fig. 15a, d-e), was sufficient to restore the EAE- suppressive function of iTRFoxAis- PD-L1 KD and anti-PD-L1 prevented the EAE- suppressive capacity of iTRFoxAis (Fig. 4i-j, Table XI). Though the antibody was removed, remaining anti-PD-L1 could have affected encephalitogenicity of cells.
iTRFoxAis and all control variants were CFSE-labeled prior to transfer to EAE. Post-EAE analysis of CFSE+T-cells revealed that while iTRFoxAis did not proliferate, the control T-cells and iTRFoxAis with FoxAI KD and PD-L1 KD proliferated in vivo (Fig. 4k-l). iTRFoxAis phenotype was stable in vivo as they maintained FoxA1 and did not gain FoxP3 expression up to 40 days post-transfer (Fig. 4m-n).
These results indicate that FoxA1 conveys anti-inflammatory and EAE- suppressive functions of TRFoxAis mediated by PD-L1 .
RFOXA suppress by inducing PD-L1 -mediated caspase3
PD-L1 is involved in negative signaling to T-cells, and cell-cycle arrest38.
Suppression of iTRFoxAis was PD-L1 -mediated (Fig. 4i-j). We investigated how TRFOXAIS' PD-L1 functioned. We found that blocking PD-L1 restored ResT-cell proliferation and reduced cell-death. PD-L1 binds to PD-1 and B7.139. Blocking PD-L1 on nTRFoxAis and PD-1 on ResT-cells reduced the killing capacity of nTRFoxAis, but blocking B7.1 or B7.2 did not (Fig. 5a-b).
We studied if human iTRFoxAis use this signaling to suppress activated ResT- cells. The anti-proliferative effect of iTRFoxAis was abrogated when PD-1 was KD in ResT-cells (Fig. 5c).
AKT phosphorylation/pAKT regulates T-cell activation. iTRFoxAis inhibited pAKT in ResT-cells. This was dependent on the PD-1 signaling, as PD-1 KD of ResT-cells rescued pAKT (Fig. 5d, Fig. 16a). iTRFoxAis induced PD-1 -dependent caspase3 cleavage in ResT-cells, which triggers an apoptotic caspase chain (Fig. 5e, Fig. 16b). Phosphorylation of P38 (pP38), a mitogen-activated protein kinase (P38MAPK), regulates the cell-cycle and apoptosis. Supporting TRFOXAIS dual functionality in cell- cycle and cell-death regulation, pP38 was regulated in a PD-1 -dependent fashion in ResT-cells upon co-culturing with iTRFoxAis (Fig. 16c).
FoxA1 -overexpressing iTRFoxAis upregulated cleaved caspase3 in ResT-cells (Fig. 5f), which was diminished by PD-L1 and caspase inhibition (Fig. 5g). iTRFoxAi inhibition of ResT-cells was associated with pAKT reduction since blocking PD-L1 and caspases restored pAKT in ResT-cells (Fig. 5h).
FoxA1 was found necessary for the generation and function of TRFoxAis and FoxA1 was sufficient to activate PD-L1 in TRFoxAis- TRFoxAis killing of ResT-cells was mediated by PD-L1 -PD-1 -signaling that inhibited pAKT and triggered caspase- mediated apoptotic pathway.
FoxA1 binds the pd-11 promoter and regulates PD-L1
FoxA1 binds an enhancer sequence in cancer cells17 and controls TTR transcription, suggesting that in addition to enhancer activity, FoxA1 controls promoters40. We studied if FoxA1 regulated PD-L1. FoxA1 -overexpression resulted in profound elevated PD-L1 mRNA and protein in TRFoxAis (Fig. 5i-j).
We investigated if FoxA1 regulated PD-L1 in T-cells via direct binding to a putative pdl1 promoter. Based on an unconfirmed peak in FoxA1 -chromatin
immunoprecipitation (ChIP) using a breast cancer cell line17, we identified two potential binding sites: Pdl1-A upstream of pdl1, and Pdl1-B, a putative FoxA1 -binding sequence in the pdl1 promoter region (Fig. 5k).
We overexpressed FoxA1 to generate TRFoxAis for ChIP of FoxA1 -binding DNA.
FoxA1 bound to the Pdl1-B promoter site compared to a housekeeping gene (Fig. 5I). To exclude indirect binding, we performed FoxA1 -binding electromobility shift assays with a probe containing the FoxA1 -binding site but lacking the c-Fos-binding site of the control TTR promoter40 (mTTR). Labeled mTTR probe was incubated alone or with unlabeled PD-L1-A and PD-L1-B probes. The PD-L1-B but not the PD-L1-A probe competed for FoxA1 -binding to the mTTR probe (Fig. 5m). To confirm that Pdl1-B was the FoxA1 -binding site in the pd 11 promoter, we labeled PD-L1-A and PD-L1-B probes separately and compared their FoxA1 -binding to mTTR. Supershifts showed that FoxA1 bound PD-L1-B and mTTR but not PD-L1-A (Fig. 5n). Expression of a PD-L1 - promoter luciferase-reporter was induced by co-transfection of a FoxA1 -expressing vector, suggesting positive regulation of the PD-L1 promoter (Fig. 5o). Thus, FoxA1 bound to and regulated the pdl1 promoter and hence PD-L1 protein.
IFN-β responsiveness in RRMS patients is associated with TRFOXAIS
We investigated if good-response to IFN-p-treatment in RRMS patients was associated with generation of TRFoxAis- e included 15 IFN-p-responder RRMS patients (RRMS-R) and 9 non-responders (RRMS-NR)41. RRMS-R patients
experienced neither relapse nor progression after two years of IFN-p-treatment.
RRMS-NR patients showed relapses and increased progression (Fig. 17). TRFoxAis were significantly increased in the IFN-p-responsive group, compared to baseline and the RRMS-NRs (Fig. 6a-d). No expansion of TRFoxAis was detected in the RRMS-NRs (Fig. 6b-d). Only gated TCRaP+CD4+PD-L1 hiT-cells from RRMS-Rs were positive for nuclear FoxA1 ; with a significant FoxA1 increase after 24 months of treatment (Fig. 6e-f).
We investigated if in v/Vo-generated TRFOXAIS from RRMS-R patients are suppressive. Purified TRFoxAis from three additional IFN-p-treated RRMS-Rs significantly inhibited T-cell proliferation and induced ResT-cell death (Fig. 6g-k).
These data showed that TRFoxAis from IFN-p-responder RRMS-R patients suppressed activated T-cells by inhibiting proliferation and exerting killing. Significant elevation of TRFoxAis in RRMS-R patients could be associated with clinical benefit of IFN-β therapy.
Discussion
Regulation of inflammation is critical for the disease management in tissue- specific chronic inflammatory diseases including MS. This is naturally achieved when inflammatory counteracting, functionally intact immune cells are generated. While Tregs are crucial in regulating many human inflammatory diseases42, their relevance to favorable IFN-p-responsiveness in MS patients is debated8,9,12. We anticipated that regulatory/suppressive T-cells are functional in CNS-specific autoimmune
inflammation. This led to discovery of a novel population of regulatory TRFoxAi cells determined by FoxA1. This is the first report on the function of FoxA1 in T-cells.
We showed that FoxA1 -overexpression in CD4+T-cells conferred suppressive functions and a TRFoxAi phenotype. Ectopic FoxA 1 expression led to FoxA1 nuclear localization. FoxA1 bound and induced the pdl1 promoter. Stable PD-L1 expression, characteristic of TRFoxAis, mediated TRFoxAis suppression via induction of caspase3- associated apoptosis, and was critical for prevention of CNS-inflammation and EAE. Although this mechanism of action is not reported for Tregs, in myelodysplastic syndrome cells, PD-L1 induces caspase3-dependent apoptosis in T-cells43. PD-L1 - mediated suppression might be a similarity between the TRFOXAIS and TregS. Initially, PdIT A and WT mice were shown to have similar numbers of Tregs(CD4+CD25hiCD45RBl0)44, but PD-L1 's role in generation and function of Tregs is now reported45"47. PD-L1 - mediated induction of Treg development is associated with downregulation of pAkt/mTOR signaling and ERK2 but not P38MAPK47. However, the signaling molecules attenuated in activated T-cells via PD-L1 -PD-1 are unclear. In TRFoxAis, PD-L1 was required for suppression via inhibition of pAKT and pP38 and induction of caspase3- associated T-cell killing. We found no additional similarities between TRFOXAIS and n/iTregs at transcription levels, surface markers, cytokines or gene-expression profiles. TRFOXAIS are negative for FoxP3, CTLA4, TGF-β, IL-10 and IL-3549'50, commonly associated with Tregs- The genetic signature of TRFOXAIS is distinct from their T-cell progenitors, neuron- induced Tregs, n/iTregs28"31 and exhausted T-cells32.
TRFOXA-IS generated by transfection of T-cells with FoxA1 led to downregulated pc-Fos. FoxA1 a 'pioneer' factor binds to chromatinized DNA directly, opens the chromatin and regulate its target genes but can also enhance binding of other co- factors to their target genes48, such as GATA3 and Tbet49. Additional FoxA1 activity as an activator or repressor, its T-cell target genes, and interaction with other factors remain to be determined.
IFN-β was sufficient to induce FoxA1 and FoxA1 -mediated PD-L1 , leading to TRFOXAI generation. nTRFoxAis and iTRFoxAis shared homology in their gene-profile with
FoxA 1-gene and its signaling pathway in the lead. TRFOXAI cell-profile was stable in vivo leading to EAE-prevention. Additional pathways for IFNp-IFNAR-mediated FoxA1 regulation could include activation of STAT molecules. STAT3 binds near the pdl1-B site in tolerogenic APCs50, hence FoxA1 and STAT3 could interact to direct pdl1 transcription in T-cells.
Our data demonstrated that FoxA1 establishes the TRFoxAi cell-lineage through modification of cell-surface and signaling molecules. We propose that this results in adaptation to signals that are required for suppressive function of TRFoxAis- Suppressive TRFoxAis are also generated in association with favorable clinical outcomes in IFN-β- responder RRMS patients.
Understanding novel functions of TRFoxAis in inflammatory diseases could be promising for designing new therapies.
Methods
Mice
lfnb' and WT mice in C57BL/10.RIII or C57BL/6 (more than 20 generation backcrossed) were bred and kept at conventional animal facilities at the University of Copenhagen. Ifnbaf^-mlce were from B&K Universal, UK. NOG
(NOD.CgPr/ dcscd/72rgfmisl79/jicTac) and OT-II (B6.129S6-Rag2fmiFwaTg/TcraTcrb/425 Cbn) mice were from Taconic and The Jackson Laboratroy, respectively. Experiments were performed in accordance with the ethical committees in Copenhagen, Denmark and approved by the respective Institutional Review Boards, approval number
2007/561 -1364.
EAE induction and clinical evaluation
/fnb"A-C57BL/10.RIII, C57BL/10.RII I heterozygote or WT-littermates were used for active or adoptive EAE13. Shortly for adoptive transfer-EAE, mice were irradiated
(500 rad) and injected in the tail vein with a cell suspension of 2x106 MBP89-ioi-specific T cells. Each mouse received co-transfer of either 2x106 in 300 μΙ of PBS of control T cells, or purified TRFOXAIS- At day 0 and 2, each animal was given an i.p. injection of 500 ng of pertussis toxin. Active MOG35.55.EAE in C57BL/6-/fnt)"/" and WT were induced as previously described7. Mice received i.p. injection of mouse recombinatantlFN-β
(5000U) at day 0, 7 and 14 post immunization (p.i.). Mice were observed for clinical signs of EAE every day.
DTH response
Mice aged 8-15 weeks were immunized with 250 μg of MBP89-ioi emulsified in 50 μΙ of PBS and 50 μΙ of CFA. At day 13 p.i., mice were injected with 100 μg of MBP89- 10i (in PBS) + TRFOXA-IS (3X1 O4 cells/ear) in the right ear or 100 μg of MBP89-10i + control T-cells (3x104 cells/ear) in the left ear. Control mice received an injection of 100 μg of MBP89-10i in the left ear and PBS + control T cells (3x104 cells/ear) in the right ear. DTH response was measured as the difference in thickness (mm) of the right and left ears. Data for the control T-cell group are presented as: (ear thickness after injection with MBP89-10i + control T cells) - (ear thickness after injection with MBP89-10i ). Data for the TRFOXA-IS group are presented as: (ear thickness injected with MBP89-10i + TRFOXAIS) - (ear thickness after injection with MBP89-10i ).
Preparation of CNS infiltrating cells
At the indicated times after active EAE induction, brains and spinal cords were dissected and infiltrating cells isolated as described7.
Real time-PCR
Standard procedures and analysis were followed7. Plasmids
FoxA1 was synthesized by Geneart into pMA with 5' Hind III and 3' Not I sites. FoxA1 was transferred to the mammalian expression vector pCDNA3.1 (Invitrogen) by standard cloning techniques.
Amaxa gene transfection
Purified CD4+T-cells were transfected with pcDNA3.1foxa1 or controlpcDNA3.1 using the Amaxa mouse T-cells Nucleofector Kit (DPA-1007) (program X-001 ) according to the manufacturer's instructions. The transfection efficiency was evaluated by FoxA1 staining and FACS analysis.
Western Blot
Proteins were extracted from pcDNA3.1foxa1 transfected TRFoxAis and pcDNA3.1 transfected non-TRFoxAis- Standard procedures were followed7.
Immunohistochemistry
Brains and Spinal cords of mice with EAE were dissected and stained as described13. Slides were visualized under light microscopy.
Affymetrix data analysis
We extracted RNA from FACSAria-sorted populations by Trizol (Sigma, followed by DNAsel (Invitrogen) digestion, with a subsequent Trizol RNA-purification. RNA was subjected to Affymetrix analysis and data sets were quantile normalized and processed by the PLIER (Affymetrix) algorithm at 1.5-fold (PS0.05).
Chromatin ImmunoPrecipitation
Chromatin ImmunoPrecipitation (ChlP) was performed essentially as described51 with the following modifications. Sonications were performed on a Biruptor Next Gen (Diagenode) set for 30s on, 30s off for 12 cycles. Goat IgG (Sigma) was used as a negative control for FoxA1 antibody (Abeam, ab5089). The ChlPed DNA was purified on QIAquick PCR purification kit (Qiagen cat no 28104) and qPCR was performed with primers (table V) with the Lightcycle 480 DNA SYBR Green I Master Mix (Roche). Primers for the selected sequences were designed using Primer3 (v. 0.4.0).
Electro mobility shift assay (EMSA)
FoxA1 -transduced 3T3L1 -cells were used to extract nuclear fraction52. Pdl1-B was identified from ChlP-seq peak data in ZR751 cells17. The precise location of FoxA1 binding sequence was predicted using Clover and ContraV2. Pdl1-A and Pdl1-B EMSA probes sequence were selected from genomic mm9 assembly (UCSC) (Table VI) and are within the amplicon of the Pdl1-A and Pdl1-B primer pairs used in the ChIP assay. EMSA was run as described previously . FoxA1 antibodies were (2F83) (Millipore, 05-
1466) and (ab5089, Abeam).
Patients
Totally 50 individuals were included, 27 patients with RRMS treated with IFN-β and 23 healthy controls were included in the current study.
24 RRMS patients treated with IFN-β were included in the comparative study. Of these, 15 were good IFN-β responder RRMS (7 females/8 males; mean age
[standard deviation]: 34.3 years [7.8]) and 9 RRMS-NR (7 females/2 males; mean age: 37.1 years [8.6]). Patients with RRMS were classified as good responders to IFN-β based on the absence of relapses and no progression on the EDSS score during the first two years of treatment41. Patients were labeled as RRMS-NR when there was presence of relapses during the follow-up period; one or more relapses and an increase of at least 1 point in the EDSS score that persisted for at least two
consecutive scheduled visits separated by a 6-month interval. Classification of MS patients were done according to Lublin and Reingold53. The study was approved by the Hospital Universitari Vail d 'Hebron Ethics Committee [PR(AG)32/2008].
Nine HC (6 females/3 males; mean age: 32.0 years [6.0]) were also included in the study.
An additional 3 RRMS-R patients (females, mean age: 29.3 years [6.1]) were recruited from the Danish MS Center in Copenhagen for fresh isolation of peripheral blood to be utilized in suppression assays. Patients' blood was collected 36h after IFN-β injection, subjected to TRFOXAIS isolation and suppressive studies, approved by the Scientific Ethics Committee for Copenhagen and Frederiksberg (protocol KF01 - 041/95). Additionally buffy coats from 14 healthy individuals were included for functional studies.
Samples
Peripheral blood was collected by standard venipuncture into vacuum tubes with EDTA. PBMC were isolated by Ficoll-lsopaque density gradient centrifugation (Gibco-BRL) and freshly used or stored in liquid nitrogen until used. PBMC were collected at baseline and 24 mo after IFN^-treatment. In HC, longitudinal PBMC taken at two time points separated by 12 mo (n=2) and 24 mo (n=7). The study was approved by the Hospital Universitari Vail d'Hebron Ethics Committee [PR(AG)32/2008].
Human blood lymphocytes preparation
Blood donors' buffy coats or MS patients' blood (10-12 ml) were used for preparation of lymphocytes using Ficoll-Paque PLUS (7.5 ml) (GE Healthcare, Cat. 17- 1440-02). Lymphocyte layer was collected and used for further studies.
PD-1 silencing of primary human T-cells by a siRNA approach
Purified CD4+T cells from healthy donors were transfected with 100 nM of a PD- I siRNA or a control-siRNA using the Amaxa Human T-cells Nucleofector Kit (VPA- 1002) (program U-014) according to the manufacturer's instructions.
Foxal silencing of primary murine T-cells by a siRNA approach
Accell SMART pool small-interfering RNA (siRNA, combines four different siRNAs) (Foxal -poolsiRNA; cat:B-005000-100, nontargeting controlsiRNA; cat:D- 001910-01 -05, Dharmacon, Thermo Scientific) was introduced into purified CD4+T-cells according to manufacturer's protocol. Delivery efficiency and siRNA specificity were examined by intracellular staining of FoxA1.
FACS staining and antibodies
Standard FACS procedures and analysis were previously described7. TRFoxM-cell sorting from RRMS-R and in vitro IFN-p-induced TRFoxAis
PBMC were prepared freshly and then cultured with 1 ,000 U/ml of human recombinant IFN-β (PBL InterferonSource) for 72h. For TRFoxAi-cell sorting from RRMS patients, PBMC or from in vitro IFN-p-induced, lymphocytes were purified with CD4+T Cell Isolation Kit II (Miltenyi Biotec, Cat. 130-091 -155), stained with anti-CD4, anti-TCR and anti-PD-L1 Abs for 20 min at 4°C in the dark. TRFoxAis (TCRap+CD4+PD-L1 high) were purified utilizing a FACSAria sorting program.
Immunoflourescent cytochemistry
TRFOXA-IS and non-TRFoxAis were generated utilizing purified CD4+T-cells treated with mlFN-β (100U/ml) or hlFN-β (1000U/ml) for 48h, then sorted with FACSaria and applied to slides, and subsequently stained with antibodies.
Suppression assays
Murine TRFoxAis obtained either from mlFN-β (100U/ml) treated CD4+T-cells (for 48 h), or purified CD4+T-cells from WT or Ifnaf'' mice, or purified CD4+T-cells were transfected with pcDNA3.1 FoxA1 or its controlpcDNA3.1 , or with foxal siRNA or its controlsiRNA. Human TRFoxAis obtained from hlFN-β (1000U/ml) treated CD4+T-cells (for 48 h) or purified from RRMS-R patients treated with IFN-β. ResponderT-cells
(purified CD4+T-cells from either mouse spleens or corresponding human peripheral blood) were labeled with CFSE and stimulated with plate-bound anti-CD3/anti-CD28 for 24 h. TRFoxAis were purified by FACSAria, labeled with Texas-red tracker (Genovis) for some experiments. Suppressor and responderT-cells were co-cultured in a new culture plate without any antibody at a 1 :1 ratio. After 24h, cells were stained with violet dead cell marker (Invitrogen) or 7AAD and analyzed by FACS.
In vivo, chimeric mice were generated by transferring OTII-responderT-cells to NOG mice. 24h later mice received foxal siRNA, controlsiRNA tranfected, WT or Ifnar' CD4+T-cells, with or without an in vivo injection of mlFN-β. 24h later the suppression was assayed.
Statistical evaluations
Statistical evaluation was performed using GraphPad Prism. For detail see figure legends and supplementary online M&M.
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Tables
Table I: Ifnb ' mice develop more severe relapsing EAE than WT mice.
Group Relapsing Recovered Mean max Max CS Mean cumulative Incidence
frequency frequency CS+SD CS
WT 10/41 = 24% 30/41 = 74% 1.9±1.3 4 17 71%
(15/21)
Ifnb-'- 10/20 = 50%* 9/20 = 45%* 3.6±0.9*** 5 38 95%*
(19/20)
Relapsing frequency= Number of relapsing mice are calculated from total number of mice. Recovery frequency = Number of recovered mice per total number of mice.
*p<0.05, Chi-square test indicating Ifnb "A mice had higher relapsing frequency, higher recovered frequency and higher incidence in comparison with WT mice.
Mean cumulative CS (clinical score): was calculated by summing up each individual score registered during the follow-up period till day 51 divided by the number of mice per group.
Mean max CS, * * *p<0.001 , Mann- Whitney test.
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Table II. Differential expression of an array of selected genes in CD4 'PD-Ll 1 T cells versus encephalitogenic (EncT cells/MBPj loi-specific) progenitor CD4+T cells.
Gene Name Gene Probe Set ID P Value Fold Change
Symbol T A! VS
EncT
Pro-inflammatory cytokines/receptors
Figure imgf000085_0001
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Table III: Top Canonical Pathways overrepresented in nTRFOxAi versus EncT cells.
Figure imgf000086_0002
Top 15 canonical pathways (gene sets) overrepresented in neuron-induced
Figure imgf000086_0001
compared with their progenitor EncT cells using GSEA analysis.
GSEA analysis was quantile normalized and summarized for each comparison using justPlier implementation of Plier algorithm in R. ES=enrichment score, NES= normalized enrichment score, NOM p-val= nominal p value, FDR q-val = False Discovery Rate, FWER p-val = family- wise error rate, RANK AT MAX= position ' the ranked list at which the maximum enrichment score occurred.
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Figure imgf000087_0001
Top 15 canonical pathways (gene sets) overrepresented in Tregs compared with their control Tn cells (analysis based on Samstein et al. 2012 Cell). GSEA analysis, ES=enrichment score, NES= normalized enrichment score, NOM p-val= nominal p value, FDR q-val = False Discovery Rate, FWER p-val = family- wise error rate, RANK AT MAX= position in the ranked list at which the maximum enrichment score occurred.
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Table V. FACS characterization of murine nTRFOxAi cells.
Figure imgf000088_0001
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Figure imgf000089_0001
pihT-cel¾ et¾rtfflrT-cels>.
Figure imgf000089_0002
The confimied protein, expresses! on lepotted ΕΛΓ-cells .are based on fie following reports: Whmry et. el, Immunity 2007, 27, 670-84, Golden-Mason st.al. Journal of virology 2009, S3,
9122-30, Doming etal. Immunity 2012, 37, 1130-1144, f¾¾isy et el. Nature medicine 2010, 16, ".1147-51.
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Table VII. nTRFOxAi cells suppress EAE in Ifnb' ' mice.
Figure imgf000090_0001
Adoptive EAE was established using 2 x 106 encephalitogenic ΜΒΡ89-ιοι -reactive T cells (EncT-cells). N =4- 5/group. Control (CD4+PD-Lll0) or nTRFoXAi (CD4hlPD-Ll¾) T cells were co -transferred with EncT cells.
1 among sick mice.
Mean cumulative CS (clinical score): was calculated by summing up each individual score registered during the follow-up period until day 24 divided by the number of mice per group.
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Table VIII. Top Transcription Factors overrepresented in iTRFOxAi among commonly re ulated enes b different TRFOXAI cells.
Figure imgf000091_0001
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Table IX: Top Canonical Pathways OTerrep resented in HTRFOSAI among commonly regulated genes by different TRF0IAI cells.
Figure imgf000092_0001
vs. EncT cells were analyzed using GSEA analysis. In bold: common pathways found among the top 20 canonical pathways in nTRFoxAl and IFND - induced i pjasAi. GSEA analysis was quanttte normalized and summarized for each comparison using justPlier implementation of Plier algorithm in R. ES=emichment score. NES= normalized enrichment score. OM p-val= nominal p value, FDR q-val = False Discovery Rate, FWER p-val, = family- rate. RANK AT MAX= position in the ranked list at which the maximum enrichment score occurred.
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Table X: Top Canonical Pathways overrep resented In IFNP-lnduced/lTRFoiAi among commonly regulated genes by different TEFOIAI cells.
Figure imgf000093_0002
Top 20 canonical pathways overrepresented in iTjtFwU compared with EncT cells, .among genes commonly regulated by I KFOAI and nTjufcou vs. EncT cells were analyzed using GSEA analysis. In bold: common pathways found among the top 20 canonical pathways in nTEFoxAl and IFND-induced/iTRFeiAi- GSEA analysis was quanole normalized and summarized for each comparison using ju
normalized enrichment score, NOM p-val= nominal p value, SDR q-val = False
Figure imgf000093_0001
position in the ranked list at which the maximum enrichment score occurred.
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Table XL TRFOXAI cells suppress adoptive EAE in Ifnb" mice mediated by FoxAl and PD-L1 signaling.
Figure imgf000094_0001
Adoptive EAE was established by twice transferring 5-10 x 106 MOG35.55-reactive EncT cells. N = 8-22. Control (CD3 activated T cells, with ctrl siRNA+pcDNA3.1), ITRFOXAI (IFN-β treated 48 hours, with ctrl siRNA), iTRpoxA1 (sifoxal KD for 24 hour + IFN-β treated 48 hours), iTRF0xA1 (sipdll KD for 24 hour + IFN-β treated 48 hours) and TRPoxA1 (foxal siRNA+pcDNA3.1 foxal) cells were co-transferred each time with EncT cells. The results are sum of 3 independent experiments.
1 among sick mice, NA = not applicable since only one mice developed disease in these groups which preclude statistic analysis.
*p<0.05, ** p<0.01, Chi-square test indicating significant low incidence in these groups.
Mean cumulative CS (clinical score): was calculated by summing up each individual score registered during the follow-up period until day 40 divided by the number of mice per group.
Statistics for mean clinical score comparison by one-way Anova and multiple comparisons (*** p<0.001).
Tac vs. TRFOXAI ***
Tac vs. TRFOXAI (foxal siRNA) ns
Tac vs. TRp0XAi(pdll siRNA+anti-PD-Ll) ns
Tac vs. TRPOXAI (foxa 1 siRNA+pcDNA3.1 foxal ) ***
ITRFOXAI VS. TRFOXAI (foxal siRNA) ***
ITRFOXAI VS. TRFOXAI (pdl 1 siRNA+anti-PD-L 1 ) * * *
ITRFOXAI VS. TRFoxAiifoxalsiRNA+pcDNAS. l foxal) ns
Josefowicz, S.Z., Lu, L.F. & Rudensky, A.Y. Regulatory T cells: mechanisms of differentiation and function. Annual Review of Immunology 30, 531 -564 (2012). Sundrud, M.S. & Nolan, M.A. Synergistic and combinatorial control of T cell activation and differentiation by transcription factors. Curr Opin Immunol 22, 286-292 (2010).
Yamane, H. & Paul, W.E. Early signaling events that underlie fate decisions of naive CD4(+) T cells toward distinct T-helper cell subsets. Immunol Rev 252, 12-23 (2013).
Bach, J.F. Autoimmune diseases as the loss of active "self-control". Annals of the New York Academy of Sciences 998, 161 -177 (2003).
Himmel, M.E., Yao, Y., Orban, P.C., Steiner, T.S. & Levings, M.K. Regulatory T-cell therapy for inflammatory bowel disease: more questions than answers. Immunology 136, 1 15-122 (2012).
Kumar, V., Stellrecht, K. & Sercarz, E. Inactivation of T cell receptor peptide- specific CD4 regulatory T cells induces chronic experimental autoimmune encephalomyelitis (EAE). The Journal of experimental medicine 184, 1609- 1617 (1996).
Liu, Y., Teige, I., Birnir, B. & Issazadeh-Navikas, S. Neuron-mediated generation of regulatory T cells from encephalitogenic T cells suppresses EAE. Nat Med 12, 518-525 (2006).
Korn, T et al. Myelin-specific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation. Nat Med 13, 423-431 (2007).
Viglietta, V., Baecher-Allan, C, Weiner, H.L. & Hafler, D.A. Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J Exp Med 199, 971 -979 (2004).
Dominguez-Villar, M., Baecher-Allan, CM. & Hafler, D.A. Identification of T helper type 1 -like, Foxp3+ regulatory T cells in human autoimmune disease. Nature Medicine 17, 673-675 (201 1 ).
Astier, A.L., Meiffren, G., Freeman, S. & Hafler, D.A. Alterations in CD46- mediated Tr1 regulatory T cells in patients with multiple sclerosis. J Clin Invest 116, 3252-3257 (2006).
de Andres, C, et al. Interferon beta-1 a therapy enhances CD4+ regulatory T- cell function: an ex vivo and in vitro longitudinal study in relapsing-remitting multiple sclerosis. J Neuroimmunol 182, 204-21 1 (2007).
Teige, I., et al. IFN-beta gene deletion leads to augmented and chronic demyelinating experimental autoimmune encephalomyelitis. J Immunol 170, 4776-4784 (2003).
Prinz, M et al. Distinct and nonredundant in vivo functions of IFNAR on myeloid cells limit autoimmunity in the central nervous system. Immunity 28, 675-686 (2008).
Liu, Y., Teige, I., Ericsson, I., Navikas, V. & Issazadeh-Navikas, S. Suppression of EAE by oral tolerance is independent of endogenous IFN-beta whereas treatment with recombinant IFN-beta ameliorates EAE. Immunol Cell Biol 88, 468-476 (2010).
Teige, I., Liu, Y. & Issazadeh-Navikas, S. IFN-beta inhibits T cell activation capacity of central nervous system APCs. J Immunol 177, 3542-3553 (2006). Hurtado, A., Holmes, K.A., Ross-lnnes, C.S., Schmidt, D. & Carroll, J.S. FOXA1 is a key determinant of estrogen receptor function and endocrine response. Nat Genet 43, 27-33 (201 1 ). Taube, J.H., Allton, K., Duncan, S.A., Shen, L. & Barton, M.C. Foxal functions as a pioneer transcription factor at transposable elements to activate Afp during differentiation of embryonic stem cells. J Biol Chem 285, 16135-16144 (2010).
Laganiere, J., et al. From the Cover: Location analysis of estrogen receptor alpha target promoters reveals that FOXA1 defines a domain of the estrogen response. Proc Natl Acad Sci U S A 102, 1 1651 -1 1656 (2005).
Li, Z., et al. Foxal and Foxa2 regulate bile duct development in mice. The
Journal of Clinical Investigation 119, 1537-1545 (2009).
Motallebipour, M et al. Differential binding and co-binding pattern of FOXA1 and FOXA3 and their relation to H3K4me3 in HepG2 cells revealed by ChlP- seq. Genome Biol 10, R129 (2009).
Kaestner, K.H. The FoxA factors in organogenesis and differentiation. Curr Opin Genet Dev 20, 527-532 (2010).
Bernardo, G.M. & Keri, R.A. FOXA1 : a transcription factor with parallel functions in development and cancer. Bioscience reports 32, 1 13-130 (2012).
Lupien, M et al. FoxA1 translates epigenetic signatures into enhancer-driven lineage-specific transcription. Cell 132, 958-970 (2008).
Lucchinetti, C.F., et al. Inflammatory cortical demyelination in early multiple sclerosis. N Engl J Med 365, 2188-2197 (201 1 ).
Freeman, G.J., Wherry, E.J., Ahmed, R. & Sharpe, A.H. Reinvigorating exhausted HIV-specific T cells via PD-1 -PD-1 ligand blockade. J Exp Med 203,
2223-2227 (2006).
Liu, Y., Carlsson, R., Ambj0rn, M., Badn, W., Hasan, M., Darabi, A., Siesjo, P., Issazadeh-Navikas, S. PD-L1 expression by neurons nearby tumors indicates better prognosis in glioblastoma patients. J Neurosci. 28;33(35):14231 -45. (2013).
Hill, J.A., et al. Foxp3 transcription-factor-dependent and -independent regulation of the regulatory T cell transcriptional signature. Immunity 27, 786- 800 (2007).
Samstein, R.M., et al. Foxp3 exploits a pre-existent enhancer landscape for regulatory T cell lineage specification. Cell 151 , 153-166 (2012).
Fu, W et al. A multiply redundant genetic switch 'locks in' the transcriptional signature of regulatory T cells. Nat Immunol 13, 972-980 (2012).
Haribhai, D et al. A central role for induced regulatory T cells in tolerance induction in experimental colitis. J Immunol 182, 3461 -3468 (2009).
Wherry, E.J., et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity 27, 670-684 (2007).
Ademuyiwa, F.O., Thorat, M.A., Jain, R.K., Nakshatri, H. & Badve, S.
Expression of Forkhead-box protein A1 , a marker of luminal A type breast cancer, parallels low Oncotype DX 21 -gene recurrence scores. Mod Pathol 23, 270-275 (2010).
Andre, S., Tough, D.F., Lacroix-Desmazes, S., Kaveri, S.V. & Bayry, J.
Surveillance of antigen-presenting cells by CD4+ CD25+ regulatory T cells in autoimmunity: immunopathogenesis and therapeutic implications. The
American journal of pathology 174, 1575-1587 (2009).
Porrini, A.M., De Luca, G., Gambi, D. & Reder, AT. Effects of an anti-IL-10 monoclonal antibody on rlFNbeta-1 b-mediated immune modulation. Relevance to multiple sclerosis. Journal of neuroimmunology 81 , 109-1 15 (1998).
Zhang, X., et al. IFN-beta1 a inhibits the secretion of Th17-polarizing cytokines in human dendritic cells via TLR7 up-regulation. Journal of Immunology 182, 3928-3936 (2009).
Hesse, D et al. Disease protection and interleukin-10 induction by endogenous interferon-beta in multiple sclerosis? Eur J Neuron s, 266-272 (201 1 ). Latchman, Y et al. PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol 2, 261 -268 (2001 ).
Butte, M.J., Keir, M.E., Phamduy, T.B., Sharpe, A.H. & Freeman, G.J.
Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses. Immunity 27, 1 1 1 -122 (2007).
Qian, X., Samadani, U., Porcella, A. & Costa, R.H. Decreased expression of hepatocyte nuclear factor 3 alpha during the acute-phase response influences transthyretin gene transcription. Molecular and cellular biology 15, 1364-1376
(1995).
Rio, J., et al. Defining the response to interferon-beta in relapsing-remitting multiple sclerosis patients. Ann Neurol 59, 344-352 (2006).
Izcue, A., Coombes, J.L. & Powrie, F. Regulatory lymphocytes and intestinal inflammation. Annual Review of Immunology 27, 313-338 (2009).
Kondo, A., et al. Interferon-gamma and tumor necrosis factor-alpha induce an immunoinhibitory molecule, B7-H1 , via nuclear factor-kappaB activation in blasts in myelodysplastic syndromes. Blood 116, 1 124-1 131 (2010).
Latchman, Y.E., et al. PD-L1 -deficient mice show that PD-L1 on T cells, antigen-presenting cells, and host tissues negatively regulates T cells. Proc Natl
Acad Sci U S A 101 , 10691 -10696 (2004).
Sandner, S.E., et al. Role of the programmed death-1 pathway in regulation of alloimmune responses in vivo. J Immunol 174, 3408-3415 (2005).
Keir, M.E., et al. Tissue expression of PD-L1 mediates peripheral T cell tolerance. The Journal of experimental medicine 203, 883-895 (2006).
Francisco, L.M., et al. PD-L1 regulates the development, maintenance, and function of induced regulatory T cells. J Exp Med 206, 3015-3029 (2009).
Jozwik, K.M. & Carroll, J.S. Pioneer factors in hormone-dependent cancers. Nat Rev Cancer 12, 381 -385 (2012).
Nakshatri, H. & Badve, S. FOXA1 in breast cancer. Expert Rev Mol Med , e8 (2009).
Wolfle, S.J., et al. PD-L1 expression on tolerogenic APCs is controlled by
STAT-3. European journal of immunology 41 , 413-424 (201 1 ).
Frank, S.R., Schroeder, M., Fernandez, P., Taubert, S. & Amati, B. Binding of c-
Myc to chromatin mediates mitogen-induced acetylation of histone H4 and gene activation. Genes & development 15, 2069-2082 (2001 ).
Carlsson, R., Thorell, K., Liberg, D. & Leanderson, T. SPI-C and STAT6 can cooperate to stimulate IgE germline transcription. Biochemical and biophysical research communications 344, 1 155-1 160 (2006).
Lublin, F.D. & Reingold, S.C. Defining the clinical course of multiple sclerosis: results of an international survey. National Multiple Sclerosis Society (USA) Advisory Committee on Clinical Trials of New Agents in Multiple Sclerosis. Neurology 46, 907-91 1 (1996).

Claims

Claims
Immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment of FoxA1 (FoxA1 + T cells).
The immunosuppressive T cells according to claim 1 , wherein said T cells are an isolated population and/or are comprised in an in vitro cell culture further comprising a cell culture medium.
The immunosuppressive T cells according to any of the preceding claims, wherein said T cells are derived from a population of T cells selected from the group consisting of Regulatory T cells (Tregs), Helper T cells (Th cells), Cytotoxic T cells (Tc cells, TCL), Natural killer T cells (NKT) and Memory T cells
The immunosuppressive T cells according to any of the preceding claims, wherein said T cells further comprise one or more of CD4, PD-L1 , PD-1 , CD47, CD69, CD25, CD45Rb and TCRa3; and/or are CD4, PD-L1 , PD-1 , CD47, CD69, CD25, CD45Rb and/or TCRa3 positive (+ or hi).
The immunosuppressive T cells according to any of the preceding claims, wherein said T cells further comprise or express PD-L1 ; and/or are PD-L1 positive, such as PD-L1 + or PD-L1 hi.
The immunosuppressive T cells according to any of the preceding claims, wherein said T cells further comprise or express CD4; and/or are CD4 positive, such as CD4+ or CD4hi.
The immunosuppressive T cells according to any of the preceding claims, wherein said T cells are FoxP3, TGF-β, IFN-gamma, TNF-alpha, CTLA-4, FAS, FASL, CD8, IL-17, IL-4, IL-10 and/or IL-13 negative.
The immunosuppressive T cells according to any of the preceding claims, wherein said T cells are FoxP3 negative (FoxP3").
The immunosuppressive T cells according to any of the preceding claims, wherein said T cells further comprise or express one or more transcription factors selected from the group consisting of Tcf7l2, Spic, Pou3f1 , Nfib, Mafb, Mef2c, Zbtb16, Tcf7l2, Bach2, Esr1 , Mef2c, Klf2, Aff3, Spib, Tcf7, Rorc, BarhH , Hoxb13, Zic3, Six3, Trp73, Hoxa13, Rora, Foxb2, PrrxH , Neurog3 and Zfp369.
10. The immunosuppressive T cells according to any of the preceding claims, wherein said T cells are selected from the group consisting of
a. FoxA1 +CD4+PD-L1 + T cells,
b. FoxA1 +CD4+PD-L1 hi T cells,
c. FoxA1 +CD4hiPD-L1 hi T cells,
d. FoxA1 +CD4+PD-L1 hiFoxP3" T cells,
e. FoxA1 +CD4hiPD-L1 hiFoxP3" T cells,
f. FoxA1 +CD4+/hiPD-L1 hi TCRa3+ T cells,
g. FoxA1 +CD4+/hiPD-L1 hi TCRa3+FoxP3" T cells,
h. FoxA1 +CD4+/hiPD-L1 hi CD47+ T-cells,
i. FoxA1 +CD4+/hiPD-L1 hi CD47+FoxP3" T-cells,
j. FoxA1 +CD4+/hiPD-L1 hi CD47+CD69+T-cells,
k. FoxA1 +CD4+/hiPD-L1 hi CD47+CD69+FoxP3" T-cells,
I. FoxA1 +CD4+/hiPD-L1 hi CD47+CD69+TCRa3+ T-cells, and
m. FoxA1 +CD4+/hiPD-L1 hi CD47+CD69+TCRa3+FoxP3" T-cells.
1 1 . The immunosuppressive T cells according to any of the preceding claims, wherein said T cells are non-proliferative.
12. The immunosuppressive T cells according to any of the preceding claims, wherein c-Fos is down-regulated and/or nuclear pc-Fos (phosphorylated c-Fos) is reduced in said immunosuppressive T cells.
13. The immunosuppressive T cells according to any of the preceding claims, wherein said T cells i) i) are able to suppress production of one or more proinflammatory cytokines, such as pro-inflammatory cytokines selected from the group consisting of IL-12 and IL-17 in APCs; ii) do not induce production of IL- 10 in APCs, and/or iii) suppress activated T cells, such as inhibit proliferation and/or induce cell death of activated T cells.
14. A method of making the immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment of FoxA1 , according to any of the preceding claims, said method comprising one or more steps of
i. Introducing into T cells FoxA1 protein, or a functional variant or functional fragment thereof,
ii. Introducing into T cells a nucleic acid construct encoding FoxA1 or a functional variant or functional fragment thereof, and/or iii. treating T cells with an effective amount of IFN-β.
15. The method according to claim 14, wherein said T cells are selected from the group consisting of Regulatory T cells (Tregs), Helper T cells (Th cells),
Cytotoxic T cells (Tc cells, TCL), Natural killer T cells (NKT) and Memory T cells.
16. The method according to any of claims 14-15, wherein said T cells are
extracted from an individual, such as an individual having an inflammatory disease or disorder.
17. A method of treating an inflammatory disease or disorder, said method
comprising administering to an individual in need thereof an effective amount of a bioactive agent selected from the group consisting of:
FoxA1 protein, or a functional fragment or functional variant thereof;
a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; and
immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined in any of claims 1 to 13.
18. A bioactive agent selected from the group consisting of FoxA1 protein, or a functional fragment or functional variant thereof;
a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; and
immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined in any of claims 1 to 13, for use as a medicament.
19. A bioactive agent selected from the group consisting of FoxA1 protein, or a functional fragment or functional variant thereof;
a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof; and
immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), as defined in any of claims 1 to 13, for use in the treatment of an inflammatory disease or disorder.
20. The bioactive agent for use according to any of claims 18-19, or the method according to any of claims 14-17, or the T cells according to any of claims 1 -13, wherein said functional variant or functional fragment of FoxA1 has between 60- 99.9% sequence identity, such as between 60-70%, for example 70-75%, such as 75-80%, for example 80-85%, such as 85-90%, for example 90-95%, such as 95-96%, for example 96-97%, such as 97-98%, for example 98-99%, such as 99-99.9% sequence identity to SEQ ID NO:1 or SEQ ID NO:3.
21 . The bioactive agent for use according to any of claims 18-20, or the method according to any of claims 14-17, or the T cells according to any of claims 1 -13, wherein said functional variant or functional fragment of FoxA1 comprises from 10 to 471 consecutive amino acids of SEQ ID NO:1 , such as from 10-25, 25-50, 50-75, 75-100, 100-125, 125-150, 150-175, 175-200, 200-250, 250-300, 300- 350, 350-400, 400-450 or from 450-471 consecutive amino acids of SEQ ID NO:1 .
22. The bioactive agent for use according to any of claims 18-21 , or the method according to any of claims 14-17, or the T cells according to any of claims 1 -13, wherein said functional variant or functional fragment of FoxA1 comprises the FoxA1 Fork-head DNA-binding region (aa 169-260) (SEQ ID NO:2).
23. The bioactive agent for use according to any of claims 18-22, or the method according to any of claims 14-17, or the T cells according to any of claims 1 -13, wherein said functional variant or functional fragment of FoxA1 comprises the naturally occurring variants of FoxA1 , including but not limited to VAR_015183, VAR_013457, VAR_055835, VAR_015184, VAR_015185 and VAR_013458.
24. The bioactive agent for use according to any of claims 18-23, or the method according to any of claims 14-17, wherein said functional variant or functional fragment of FoxA1 i) is able to suppress production of one or more proinflammatory cytokines, such as pro-inflammatory cytokines selected from the group consisting of IL-12 and IL-17 in APCs; ii) do not induce production of IL-
10 in APCs, iii) induce expression in T cells of one or more of CD4, PD-L1 , PD- 1 , CD47, CD69, CD25, CD45Rb and TCRa3+; iv) is able to suppress activated T cells, such as inhibit proliferation and/or induce cell death of activated T cells; v) is able to down-regulate c-Fos expression in T cells; and/or vi) is able to reduce nuclear pc-Fos in T cells.
25. The bioactive agent for use according to any of claims 18-24, or the method according to any of claims 14-17, wherein said FoxA1 , or a functional variant or functional fragment thereof, further comprises one or more protein transduction domains (PTDs).
26. The bioactive agent for use according to any of claims 18-25, or the method according to any of claims 14-17, wherein said nucleic acid construct is comprised in a delivery vehicle.
27. The bioactive agent for use or the method according to claim 26, wherein said delivery vehicle is selected from the group consisting of: plasmid vectors, RNA based vehicles, DNA based vehicles, lipid based vehicles, polymer based vehicles, colloidal gold particles and virally derived DNA or RNA vehicles.
28. The bioactive agent for use or the method according to claim 26, wherein said delivery vehicle is a viral vector selected from the group consisting of adenoviruses, retroviruses, lentiviruses, adeno-associated viruses (AAV), recombinant adeno-associated viruses (rAAV), herpesviruses, vaccinia viruses, foamy viruses, cytomegaloviruses, Semliki forest viruses, poxviruses, RNA virus vectors and DNA virus vectors.
29. The bioactive agent for use according to any of claims 18-28, or the method according to any of claims 14-17, wherein said nucleic acid construct comprises a T-cell specific promoter or expression cassette.
30. The bioactive agent for use or the method according to claim 29, wherein said T-cell specific promoter or expression cassette is CD4+ and/or CD8+ specific.
31 . The bioactive agent for use according to any of claims 18-30, or the method according to any of claims 14-17, further comprising administering an effective amount of IFN-β.
32. A method of treating an inflammatory disease or disorder, said method
comprising
a. collecting T cells from an individual having an inflammatory disease or disorder, and
b. transforming said collected T cells into immunosuppressive T cells
comprising FoxA1 , or a functional variant or functional fragment thereof
(FoxA1 + T cells)
i. by introducing into said collected T cells FoxA1 protein, or a functional fragment or functional variant thereof; or a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof, and/or ii. by treating said collected T cells with an effective amount of IFN- β, and
c. re-introducing said immunosuppressive T cells comprising FoxA1 , or a functional variant or functional fragment thereof (FoxA1 + T cells), to said individual having an inflammatory disease or disorder.
33. The method according to claim 32, wherein said immunosuppressive T cells are collected from the blood, from the cerebrospinal fluid or from the lymph of an individual; and wherein said immunosuppressive T cells are re-introduced into the blood, into the cerebrospinal fluid or into the lymph of an individual.
34. The method according to any of claims 32-33, wherein one or more
subpopulations of T cells selected from the group consisting of Regulatory T cells (Tregs), Helper T cells (Th cells), Cytotoxic T cells (Tc cells, TCL), Natural killer T cells (NKT) and Memory T cells, are specifically collected.
35. The method according to any of claims 32-34, wherein said IFN-β is
administered in an amount sufficient to transform the T cells into
immunosuppressive T cells expressing FoxA1 (FoxA1 + T cells).
36. The bioactive agent for use according to any of claims 18-35, or the method according to any of claims 14-17, wherein said inflammatory disease or disorder is an autoimmune disease.
37. The bioactive agent for use according to any of claims 18-35, or the method according to any of claims 14-17, wherein said inflammatory disease or disorder is multiple sclerosis.
38. The bioactive agent for use or the method according to claim 37, wherein said multiple sclerosis is relapse-remittent multiple sclerosis (RRMS).
39. The bioactive agent for use or the method according to claim 37, wherein said multiple sclerosis is primary progressive multiple sclerosis (PPMS).
40. The bioactive agent for use or the method according to any of claims 37-39, wherein said multiple sclerosis is IFN-β non-responding multiple sclerosis, such as IFN-β non-responding relapse-remittent multiple sclerosis (RRMS-NR) and/or IFN-β non-responding primary progressive multiple sclerosis (PPMS- NR).
41 . The bioactive agent for use or the method according to any of claims 37-39, wherein said multiple sclerosis is IFN-β responding multiple sclerosis, such as IFN-β responding relapse-remittent multiple sclerosis (RRMS-R) and/or IFN-β responding primary progressive multiple sclerosis (PPMS-R).
42. The bioactive agent for use or the method according to any of claims 37-41 , wherein said treatment further comprises administration of an effective amount of IFN-β.
43. The bioactive agent for use according to any of claims 18-35, or the method according to any of claims 14-17, wherein said inflammatory disease or disorder is a CNS disorder with an inflammatory component.
44. The bioactive agent for use or the method according to claim 43, wherein said CNS disorder with an inflammatory component is a cancer of the CNS.
45. The bioactive agent for use or the method according to claim 44, wherein said cancer of the CNS is glioma, including ependymomas, astrocytomas, oligodendrogliomas and mixed gliomas, such as glioblastoma multiforme.
46. The bioactive agent for use according to any of claims 18-35, or the method according to any of claims 14-17, wherein said inflammatory disease or disorder is an inflammatory disease of the gastrointestinal system.
47. The bioactive agent for use or the method according to claim 46 wherein said inflammatory disease of the gastrointestinal system is selected from the group consisting of inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behget's disease, indeterminate colitis, coeliac disease, gluten sensitive enteropathy, eosinophilic gastroenteritis, intestinal lympangiectasia, diverticular disease of the colon, radiation enteritis, irritable bowel syndrome, Whipple 's diease, stomatitis of all kinds, salivary gland diseases (such as sarcoidosis, salivary duct obstruction and Sjogrens syndrome), inflammaton of the oesophagus (e.g. due to gastro- oesophagel reflux or infections with Candida species, herpes simplex and cytomegalus virus), inflammatory diseases of the stomach (including acute and chronic gastritis, helicobacter pylori infection and Mentriers disease), and inflammation of the small intestine.
48. The bioactive agent for use according to any of claims 18-35, or the method according to any of claims 14-17, wherein said inflammatory disease or disorder is a delayed type hypersensitivity (DTH)-related disorder selected from the group consisting of diabetes mellitus type I, multiple sclerosis, rheumatoid arthritis, peripheral neuropathies, Hashimoto's thyroiditis, Crohn's disease, allergic contact dermatitis, psoriasis, temporal or giant-cell arteritis (GCA), symptoms of leprosy, symptoms of tuberculosis, coeliac disease, graft-versus host disease and chronic transplant rejection.
49. The bioactive agent for use according to any of claims 18-35, or the method according to any of claims 14-17, wherein said inflammatory disease or disorder is selected from the group consisting of arthritis, rheumatoid arthritis, arthropathies, osteoarthritis,, spondylarthropathies (e.g. ankylosing spondylitis), reactive arthritis, Henoch-Schonlein purpura, Reiter's disease, Juvenile Chronic arthritis including Still 's disease, juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, psoriatic arthritis, Caisson disease, septic or infectious arthitis (including tuberculous arthritis, meningococcal arthritis, gonococcal arthritis, salmonella arthritis), viral arthritis, recurrent haemarthrosis, Gout, pyrophosphate arthopathy and acute calcific periarthritis.
50. The bioactive agent for use according to any of claims 18-35, or the method according to any of claims 14-17, wherein said inflammatory disease or disorder is cancer.
51 . The bioactive agent for use according to any of claims 18-35, or the method according to any of claims 14-17, wherein said peptide is to be administered in combination with one or more second active ingredients.
52. A recombinant cell, such as a recombinant T cell, comprising a nucleic acid construct encoding FoxA1 , or a functional variant or functional fragment thereof.
53. A method of distinguishing an IFN-β non-responding multiple sclerosis patient (MS-NR) from an IFN-β responding multiple sclerosis patient (MS-R) comprising one or more steps of
a. treating a MS patient with an effective amount of IFN-β,
b. collecting a sample from said MS patient, and
c. identifying if immunosuppressive FoxA1 + T cells are present in said
sample,
wherein the positive identification or presence of immunosuppressive FoxA1 + T cells in said sample is indicative of said MS being IFN^-responsive, and negative identification or absence of immunosuppressive FoxA1 + T cells in said sample is indicative of said MS being IFN-p-non-responsive.
54. The method according to claim 53, wherein said IFN-β non-responding multiple sclerosis patient (MS-NR) is selected from the group consisting of IFN-β non- responding relapse-remittent multiple sclerosis patients (RRMS-NR) and IFN-β non-responding primary progressive multiple sclerosis patient (PPMS-NR).
55. The method according to claim 51 , wherein said IFN-β responding multiple sclerosis patient (MS-R) is selected from the group consisting of IFN-β responding relapse-remittent multiple sclerosis patients (RRMS-R) and IFN-β responding primary progressive multiple sclerosis patients (PPMS-R).
56. The method according to any of claims 53-55, wherein said sample is a blood sample, such as a blood sample selected from the group consisting of whole blood (optionally treated with an anticoagulant), blood plasma and a buffy coat.
57. The method according to claims 56, wherein the lymphocytes from said blood sample are separated or isolated.
58. The method according to any of claims 53-57, wherein presence of
immunosuppressive FoxA1 + T cells is detected by FACS analysis.
59. The method according to any of claims 53-58, wherein IFN-β treatment is
discontinued if said MS patient is characterized as an IFN-β non-responding multiple sclerosis (MS-NR) patient.
60. The method according to any of claims 53-58, wherein IFN-β treatment is
continued if said MS patient is characterized as an IFN-β responding multiple sclerosis (MS-R) patient.
61 . The method according to any of claims 53-60, wherein said MS-R and/or MS- NR patient is treated with a bioactive agent according to any of claims 18-35, or the method according to any of claims 14-17.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107164378A (en) * 2017-06-20 2017-09-15 浙江大学 Specificity suppresses siRNA and its recombinant vector and the application of SPIB gene expressions
EP4107278A1 (en) * 2020-02-18 2022-12-28 Mayo Foundation for Medical Education and Research Methods and materials for treating cancer

Non-Patent Citations (68)

* Cited by examiner, † Cited by third party
Title
"Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY
"Remington: The Science and Practice of Pharmacy", 2005, LIPPINCOTT, WILLIAMS & WILKINS
ADEMUYIWA, F.O.; THORAT, M.A.; JAIN, R.K.; NAKSHATRI, H.; BADVE, S.: "Expression of Forkhead-box protein A1, a marker of luminal A type breast cancer, parallels low Oncotype DX 21-gene recurrence scores.", MOD PATHOL, vol. 23, 2010, pages 270 - 275
ANDRE, S.; TOUGH, D.F.; LACROIX-DESMAZES, S.; KAVERI, S.V.; BAYRY, J.: "Surveillance of antigen-presenting cells by CD4+ CD25+ regulatory T cells in autoimmunity: immunopathogenesis and therapeutic implications.", THE AMERICAN JOURNAL OF PATHOLOGY, vol. 174, 2009, pages 1575 - 1587
ASTIER, A.L.; MEIFFREN, G.; FREEMAN, S.; HAFLER, D.A.: "Alterations in CD46-mediated Tr1 regulatory T cells in patients with multiple sclerosis.", J CLIN INVEST, vol. 116, 2006, pages 3252 - 3257
BACH, J.F.: "Annals of the New York Academy of Sciences", vol. 998, 2003, article "Autoimmune diseases as the loss of active ''self-control''.", pages: 161 - 177
BERNARDO, G.M.; KERI, R.A.: "FOXA1: a transcription factor with parallel functions in development and cancer.", BIOSCIENCE REPORTS, vol. 32, 2012, pages 113 - 130
BUTTE, M.J.; KEIR, M.E.; PHAMDUY, T.B.; SHARPE, A.H.; FREEMAN, G.J.: "Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses.", IMMUNITY, vol. 27, 2007, pages 111 - 122
CARLSSON, R.; THORELL, K.; LIBERG, D.; LEANDERSON, T.: "SPI-C and STAT6 can cooperate to stimulate IgE germline transcription.", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 344, 2006, pages 1155 - 1160
D. HESSE ET AL: "Absence of MxA induction by interferon in patients with MS reflects complete loss of bioactivity", NEUROLOGY, vol. 73, no. 5, 4 August 2009 (2009-08-04), pages 372 - 377, XP055192052, ISSN: 0028-3878, DOI: 10.1212/WNL.0b013e3181b04c98 *
DE ANDRES, C. ET AL.: "Interferon beta-1a therapy enhances CD4+ regulatory T-cell function: an ex vivo and in vitro longitudinal study in relapsing-remitting multiple sclerosis.", J NEUROIMMUNOL, vol. 182, 2007, pages 204 - 211
DOERING ET AL., IMMUNITY, vol. 37, 2012, pages 1130 - 1144
DOMINGUEZ-VILLAR, M.; BAECHER-ALLAN, C.M.; HAFLER, D.A.: "Identification of T helper type 1-like, Foxp3+ regulatory T cells in human autoimmune disease", NATURE MEDICINE, vol. 17, 2011, pages 673 - 675
FRANCISCO, L.M. ET AL.: "PD-L1 regulates the development, maintenance, and function of induced regulatory T cells.", J EXP MED, vol. 206, 2009, pages 3015 - 3029
FRANK, S.R.; SCHROEDER, M.; FERNANDEZ, P.; TAUBERT, S.; AMATI, B.: "Binding of c-Myc to chromatin mediates mitogen-induced acetylation of histone H4 and gene activation.", GENES & DEVELOPMENT, vol. 15, 2001, pages 2069 - 2082
FREEMAN, G.J.; WHERRY, E.J.; AHMED, R.; SHARPE, A.H.: "Reinvigorating exhausted HIV-specific T cells via PD-1-PD-1 ligand blockade.", J EXP MED, vol. 203, 2006, pages 2223 - 2227
FU ET AL., NAT IMM, 2012
FU, W. ET AL.: "A multiply redundant genetic switch 'locks in' the transcriptional signature of regulatory T cells.", NAT IMMUNOL, vol. 13, 2012, pages 972 - 980
H.-J. JIN ET AL: "Androgen Receptor-Independent Function of FoxA1 in Prostate Cancer Metastasis", CANCER RESEARCH, vol. 73, no. 12, 15 June 2013 (2013-06-15), pages 3725 - 3736, XP055191905, ISSN: 0008-5472, DOI: 10.1158/0008-5472.CAN-12-3468 *
HARIBHAI, D. ET AL.: "A central role for induced regulatory T cells in tolerance induction in experimental colitis.", J IMMUNOL, vol. 182, 2009, pages 3461 - 3468
HESSE, D. ET AL.: "Disease protection and interleukin-10 induction by endogenous interferon-beta in multiple sclerosis?", FUR.I NEUROL, vol. 18, 2011, pages 266 - 272
HILL ET AL., IMM, 2007
HILL, J.A. ET AL.: "Foxp3 transcription-factor-dependent and -independent regulation of the regulatory T cell transcriptional signature.", IMMUNITY, vol. 27, 2007, pages 786 - 800
HIMMEL, M.E.; YAO, Y.; ORBAN, P.C.; STEINER, T.S.; LEVINGS, M.K.: "Regulatory T-cell therapy for inflammatory bowel disease: more questions than answers", IMMUNOLOGY, vol. 136, 2012, pages 115 - 122
HONG ET AL., PNAS, vol. 103, no. 13, 2006, pages 5024 - 5029
HURTADO, A.; HOLMES, K.A.; ROSS-INNES, C.S.; SCHMIDT, D.; CARROLL, J.S.: "FOXA1 is a key determinant of estrogen receptor function and endocrine response", NAT GENET, vol. 43, 2011, pages 27 - 33
HYUN JOO LEE ET AL: "Overexpression of hepatocyte nuclear factor-3[alpha] induces apoptosis through the upregulation and accumulation of cytoplasmic p53 in prostate cancer cells", THE PROSTATE, 1 January 2009 (2009-01-01), pages n/a - n/a, XP055192310, ISSN: 0270-4137, DOI: 10.1002/pros.21069 *
JOSEFOWICZ, S.Z.; LU, L.F.; RUDENSKY, A.Y.: "Regulatory T cells: mechanisms of differentiation and function.", ANNUAL REVIEW OF IMMUNOLOGY, vol. 30, 2012, pages 531 - 564
JOZWIK, K.M.; CARROLL, J.S.: "Pioneer factors in hormone-dependent cancers.", NAT REV CANCER, vol. 12, 2012, pages 381 - 385
KAESTNER, K.H.: "The FoxA factors in organogenesis and differentiation.", CURR OPIN GENET DEV, vol. 20, 2010, pages 527 - 532
KEIR, M.E. ET AL.: "Tissue expression of PD-L1 mediates peripheral T cell tolerance", THE JOURNAL OF EXPERIMENTAL MEDICINE, vol. 203, 2006, pages 883 - 895
KONDO, A. ET AL.: "Interferon-gamma and tumor necrosis factor-alpha induce an immunoinhibitory molecule, B7-H1, via nuclear factor-kappaB activation in blasts in myelodysplastic syndromes.", BLOOD, vol. 116, 2010, pages 1124 - 1131
KORN, T. ET AL.: "Myelin-specific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation.", NAT MED, vol. 13, 2007, pages 423 - 431
KUMAR, V.; STELLRECHT, K.; SERCARZ, E.: "Inactivation of T cell receptor peptide-specific CD4 regulatory T cells induces chronic experimental autoimmune encephalomyelitis (EAE).", THE JOURNAL OF EXPERIMENTAL MEDICINE, vol. 184, 1996, pages 1609 - 1617
LAGANIERE, J. ET AL.: "From the Cover: Location analysis of estrogen receptor alpha target promoters reveals that FOXA1 defines a domain of the estrogen response.", PROC NATL ACAD SCI U S A, vol. 102, 2005, pages 11651 - 11656
LATCHMAN, Y. ET AL.: "PD-L2 is a second ligand for PD-1 and inhibits T cell activation", NAT IMMUNOL, vol. 2, 2001, pages 261 - 268
LATCHMAN, Y.E. ET AL.: "PD-L 1-deficient mice show that PD-LL on T cells, antigen-presenting cells, and host tissues negatively regulates T cells.", PROC NATL ACAD SCI U S A, vol. 101, 2004, pages 10691 - 10696
LI, Z. ET AL.: "Foxa1 and Foxa2 regulate bile duct development in mice", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 119, 2009, pages 1537 - 1545
LIU, Y.; CARLSSON, R.; AMBJORN, M.; BADN, W.; HASAN, M.; DARABI, A.; SIESJO, P.; ISSAZADEH-NAVIKAS, S.: "PD-L1 expression by neurons nearby tumors indicates better prognosis in glioblastoma patients.", J NEUROSCI., vol. 33, no. 35, 28 December 2012 (2012-12-28), pages 14231 - 45
LIU, Y.; TEIGE, I.; BIRNIR, B.; ISSAZADEH-NAVIKAS, S.: "Neuron-mediated generation of regulatory T cells from encephalitogenic T cells suppresses EAE", NAT MED, vol. 12, 2006, pages 518 - 525
LIU, Y.; TEIGE, I.; ERICSSON, I.; NAVIKAS, V.; ISSAZADEH-NAVIKAS, S.: "Suppression of EAE by oral tolerance is independent of endogenous I FN-beta whereas treatment with recombinant IFN-beta ameliorates EAE.", IMMUNOL CELL BIOL, vol. 88, 2010, pages 468 - 476
LUBLIN, F.D.; REINGOLD, S.C.: "Defining the clinical course of multiple sclerosis: results of an international survey. National Multiple Sclerosis Society (USA) Advisory Committee on Clinical Trials of New Agents in Multiple Sclerosis", NEUROLOGY, vol. 46, 1996, pages 907 - 911
LUCCHINETTI, C.F. ET AL.: "Inflammatory cortical demyelination in early multiple sclerosis.", N ENGL J MED, vol. 365, 2011, pages 2188 - 2197
LUPIEN, M. ET AL.: "FoxA1 translates epigenetic signatures into enhancer-driven lineage-specific transcription", CELL, vol. 132, 2008, pages 958 - 970
LZCUE, A.; COOMBES, J.L.; POWRIE, F.: "Regulatory lymphocytes and intestinal inflammation.", ANNUAL REVIEW OF IMMUNOLOGY, vol. 27, 2009, pages 313 - 338
MOTALLEBIPOUR, M. ET AL.: "Differential binding and co-binding pattern of FOXA1 and FOXA3 and their relation to H3K4me3 in HepG2 cells revealed by ChIP-seq.", GENOME BIOL, vol. 10, 2009, pages R129
NAKSHATRI, H.; BADVE, S.: "FOXA1 in breast cancer.", EXPERT REV MOL MED, vol. 11, 2009, pages E8
PORRINI, A.M.; DE LUCA, G.; GAMBI, D.; REDER, A.T.: "Effects of an anti-IL-10 monoclonal antibody on rlFNbeta-1 b-mediated immune modulation. Relevance to multiple sclerosis.", JOURNAL OF NEUROIMMUNOLOGY, vol. 81, 1998, pages 109 - 115
PRINZ, M. ET AL.: "Distinct and nonredundant in vivo functions of IFNAR on myeloid cells limit autoimmunity in the central nervous system.", IMMUNITY, vol. 28, 2008, pages 675 - 686
QIAN, X.; SAMADANI, U.; PORCELLA, A.; COSTA, R.H.: "Decreased expression of hepatocyte nuclear factor 3 alpha during the acute-phase response influences transthyretin gene transcription.", MOLECULAR AND CELLULAR BIOLOGY, vol. 15, 1995, pages 1364 - 1376
QUIGLEY ET AL., NATURE MEDICINE, vol. 16, 2010, pages 1147 - 51
RIO, J. ET AL.: "Defining the response to interferon-beta in relapsing-remitting multiple sclerosis patients.", ANN NEURO/, vol. 59, 2006, pages 344 - 352
SAMSTEIN, R.M. ET AL.: "Foxp3 exploits a pre-existent enhancer landscape for regulatory T cell lineage specification.", CEL1, vol. 151, 2012, pages 153 - 166
SANDNER, S.E. ET AL.: "Role of the programmed death-1 pathway in regulation of alloimmune responses in vivo", J IMMUNOL, vol. 174, 2005, pages 3408 - 3415
SEVERSON; HAFLER, RESULTS PROBL CELL DIFFER., vol. 51, 2010, pages 75 - 98
STEFAN NAGEL ET AL: "Transcriptional Activation of Prostate Specific Homeobox Gene NKX3-1 in Subsets of T-Cell Lymphoblastic Leukemia (T-ALL)", PLOS ONE, vol. 7, no. 7, 27 July 2012 (2012-07-27), pages e40747, XP055192314, DOI: 10.1371/journal.pone.0040747 *
SUNDRUD, M.S.; NOLAN, M.A.: "Synergistic and combinatorial control of T cell activation and differentiation by transcription factors.", CURR OPIN IMMUNOL, vol. 22, 2010, pages 286 - 292
TAUBE, J.H.; ALLTON, K.; DUNCAN, S.A.; SHEN, L.; BARTON, M.C.: "Foxa1 functions as a pioneer transcription factor at transposable elements to activate Afp during differentiation of embryonic stem cells.", J BIOL CHEM, vol. 285, 2010, pages 16135 - 16144
TEIGE, I. ET AL.: "IFN-beta gene deletion leads to augmented and chronic demyelinating experimental autoimmune encephalomyelitis", J IMMUNOL, vol. 170, 2003, pages 4776 - 4784
TEIGE, I.; LIU, Y.; ISSAZADEH-NAVIKAS, S.: "IFN-beta inhibits T cell activation capacity of central nervous system APCs.", J IMMUNOL, vol. 177, 2006, pages 3542 - 3553
VIGLIETTA, V.; BAECHER-ALLAN, C.; WEINER, H.L.; HAFLER, D.A.: "Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis", J EXP MED, vol. 199, 2004, pages 971 - 979
WHERRY, E.J. ET AL.: "Molecular signature of CD8+ T cell exhaustion during chronic viral infection.", IMMUNITY, vol. 27, 2007, pages 670 - 684
WHERRY, IMMUNITY, vol. 27, 2007, pages 670 - 84
WOLFLE, S.J. ET AL.: "PD-L1 expression on tolerogenic APCs is controlled by STAT-3.", EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 41, 2011, pages 413 - 424
YAMANE, H.; PAUL, W.E.: "Early signaling events that underlie fate decisions of naive CD4(+) T cells toward distinct T-helper cell subsets.", IMMUNOL REV, vol. 252, 2013, pages 12 - 23
YAWEI LIU ET AL: "FoxA1 directs the lineage and immunosuppressive properties of a novel regulatory T cell population in EAE and MS", NATURE MEDICINE, vol. 20, no. 3, 16 February 2014 (2014-02-16), pages 272 - 282, XP055191482, ISSN: 1078-8956, DOI: 10.1038/nm.3485 *
YAWEI LIU ET AL: "Neuron-mediated generation of regulatory T cells from encephalitogenic T cells suppresses EAE", NATURE MEDICINE, vol. 12, no. 5, 1 May 2006 (2006-05-01), pages 518 - 525, XP055191958, ISSN: 1078-8956, DOI: 10.1038/nm1402 *
ZHANG, X. ET AL.: "IFN-beta1 a inhibits the secretion of Th17-polarizing cytokines in human dendritic cells via TLR7 up-regulation.", JOURNAL OF IMMUNOLOGY, vol. 182, 2009, pages 3928 - 3936

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107164378A (en) * 2017-06-20 2017-09-15 浙江大学 Specificity suppresses siRNA and its recombinant vector and the application of SPIB gene expressions
CN107164378B (en) * 2017-06-20 2019-12-31 浙江大学 siRNA for specifically inhibiting SPIB gene expression and recombinant vector and application thereof
EP4107278A1 (en) * 2020-02-18 2022-12-28 Mayo Foundation for Medical Education and Research Methods and materials for treating cancer

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