WO2011023824A2 - Modulateurs de récepteur oestrogénique pour le traitement de maladies impliquant les cellules dendritiques plasmacytoïdes - Google Patents

Modulateurs de récepteur oestrogénique pour le traitement de maladies impliquant les cellules dendritiques plasmacytoïdes Download PDF

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WO2011023824A2
WO2011023824A2 PCT/EP2010/062698 EP2010062698W WO2011023824A2 WO 2011023824 A2 WO2011023824 A2 WO 2011023824A2 EP 2010062698 W EP2010062698 W EP 2010062698W WO 2011023824 A2 WO2011023824 A2 WO 2011023824A2
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treatment
pdcs
compound according
estrogen receptor
dendritic cells
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WO2011023824A3 (fr
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Jean-Charles Guery
Victorine Douin-Echinard
Cyril Seillet
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INSERM (Institut National de la Santé et de la Recherche Médicale)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients

Definitions

  • the present invention relates to the general field of therapy of diseases involving plasmacytoid dendritic cells, namely autoimmune, infectious and cancerous diseases.
  • DCs Dendritic cells
  • cDC myeloid DCs
  • pDC plasmacytoid DC
  • pDCs are the main cell population in the blood producing type-1 interferon following viral infection, and can exert potent paracrine effect on cDC or other cell types via the secretion of IFN-CC.
  • pDCs are precursors of DCs (pre-DCs) which can aquire cDC morphology and functional properties upon activation in vitro [Wu, L. et AL, 2007 and Shortman, K. et Al., 2007].
  • IFN- ⁇ could activate immature myeloid DCs to become fully mature DCs able to activate autoreactive T cells that would help, together with pDCs, to expand autoreactive B cells in SLE.
  • preautoimmune (NZB x NZW) Fl mice accelerated disease development leading to early lethality due to severe immune complex glomerulonephritis.
  • the inventors evaluated the effect of an E2 -treatment of post-menopausal women on the capacity of pDCs to produce IFN- ⁇ and TNF- ⁇ in response to TLR-9 and TLR-7 stimulation. Their data showed that in vivo E2 -treatment enhanced TLR9-mediated IFN- ⁇ production by pDCs present in circulating peripheral blood mononuclear cells. This effect of E2 was not attributable to changes in pDCs numbers. Indeed, by intracellular cytokine staining by flow cytometry the inventors found that the frequency of pDCs producing IFN- ⁇ or TNF- ⁇ after TLR7- stimulation was significantly higher after estrogen-supplementation. In conclusion, their data point to a direct causal link between estrogens and type I IFN-production by human pDCs upon TLR- activation.
  • the invention relates to the use of a compound which is a modulator of the estrogen receptor of plasmacytoid dendritic cells (pDCs) for the treatment of diseases involving plasmacytoid dendritic cells.
  • pDCs plasmacytoid dendritic cells
  • the invention relates to a compound which is an antagonist of the estrogen receptor of plasmacytoid dendritic cells (pDC) for the treatment of an autoimmune disease.
  • pDC plasmacytoid dendritic cells
  • the invention relates to a compound which is an agonist of the estrogen receptor of plasmacytoid dendritic cells (pDC) for the treatment of a cancer, for the stimulation of the immune system in a vaccine therapy, to improve natural immune response against viral infections or to improve natural immunity in patients chronically infected with viruses such as hepatitis C virus.
  • pDC plasmacytoid dendritic cells
  • estrone As used herein, the term "estrogens” denotes a group of steroidal compounds, known for their role in the oestrous cycle, and functioning as the primary female sex hormone.
  • the three major naturally occurring estrogens in women are estrone (El), estradiol (E2), and estriol (E3).
  • estrogen receptor denotes to a group of receptors which are activated by the hormone 17 ⁇ -estradiol[l] (estrogen).
  • Estrogen There are two different estrogen receptors (ER), usually referred to as ERa and ER ⁇ , each encoded by a separate gene (ESRl and ESR2 respectively).
  • ESRl and ESR2 Hormone activated estrogen receptors form dimers, and since the two forms are coexpressed in many cell types, the receptors may form ERa (aa) or ER ⁇ ( ⁇ ) homodimers or ERa ⁇ (a ⁇ ) heterodimers.
  • Estrogen receptor alpha and beta show significant overall sequence homology, and both are composed of seven domains.
  • ER iso forms Due to alternative RNA splicing, several ER iso forms are known to exist. At least three ERalpha and five ERbeta isoforms have been identified. The ERbeta isoforms receptor subtypes can only transactivate transcription when a heterodimer with the functional ERJ31 receptor of 59 kDa is formed. The ERJ33 receptor was detected at high levels in the testis. The two other ERalpha isoforms are 66 and 46kDa. Only in fish, but not in humans, an ERgamma receptor has been described. As used herein, the term "autoimmune disease" denotes an overactive immune response of the body against a substances or tissue normally present in the body.
  • the immune response may be restricted to certain organs (e.g. in thyroiditis) or involve a particular tissue in different places (e.g. Goodpasture's disease which may affect the basement membrane in both the lung and the kidney).
  • the treatment of autoimmune diseases is typically through immunosuppressive drugs that decrease the immune response.
  • pDC plasmacytoid dendritic cells
  • pDC plasmacytoid dendritic cells
  • these cells express the surface markers CD123, BDCA-2(CD303) and BDCA-4(CD304), but do not express CDl Ic or CD 14, which distinguishes them over conventional dendritic cells or monocytes, respectively.
  • these cells express intracellular Toll-like receptors 7 and 9, which enable the detection of viral and bacterial nucleic acids, such as ssRNA or CpG DNA motifs, respectively.
  • type I interferon mainly IFN- ⁇ (alpha) and IFN- ⁇ (beta)
  • IFN- ⁇ critical pleiotropic anti-viral compounds mediating a wide range of effects.
  • a modulator of the estrogen receptor of plasmacytoid dendritic cells denotes a compound that is capable of binding ERs and that is either an agonist or an antagonist.
  • the term "disease involving plasmacytoid dendritic cells” denotes diseases where plasmacytoid dendritic cells are involved such as autoimmune diseases, infectious diseases and cancer. Indeed, Type I IFNs produced by pDCs can act as adjuvant for a variety of vaccine strategies not only directed against infectious agents such as influenza virus [Bracci, L., et Al., 2006] but also against tumors such as melanoma [Sikora, A. G., et Al., 2009].
  • SERM selective estrogen receptor modulator
  • SERMs are scientifically accepted SERMs from a botanical source.
  • SERMs include but are not limited to af ⁇ moxifene (4-hydroxytamoxifen), arzoxifene, clomifene, femarelle (DT56a), lasofoxifene, ormeloxifene, raloxifene, tamoxifen, toremifene [Kuiper et Al., 2009].
  • the term "pure antagonist of the estrogen receptor (or pure anti- estrogen)” denotes a molecule capable to bind the estrogen receptor without any estrogenic activity, either in vitro or in vivo, in any species or tissue studied, including all estrogen targeted tissues, e.g. uterus, mammary gland, ovaries or bone [Hermenegildo, C, 2000].
  • progestin denotes a synthetic progestagen that has progestinic effects similar to progesterone.
  • the two most frequent uses of progestins are for hormonal contraception (either alone or with an estrogen), and to prevent endometrial hyperplasia from unopposed estrogen in hormone replacement therapy.
  • Progestins are also used to treat secondary amenorrhea, dysfunctional uterine bleeding and endometriosis, and as palliative treatment of endometrial cancer, renal cell carcinoma, breast cancer, and prostate cancer.
  • Progestin include but are not limited to norethynodrel (Enovid), norethindrone, norgestimate (Ortho Tricyclen, Ortho-Cyclen), norgestrel, levonorgestrel, medroxyprogesterone, desogestrel, and drospirenone.
  • Treating denotes reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such a disorder or condition.
  • a first aspect of the invention relates to a compound which is a modulator of the estrogen receptor of plasmacytoid dendritic cells (pDCs) for the treatment of a disease involving plasmacytoid dendritic cells.
  • pDCs plasmacytoid dendritic cells
  • the estrogen receptor according to the invention is the estrogen receptor alpha.
  • the modulator is an antagonist of the estrogen receptor of plasmacytoid dendritic cells (pDC) useful for the treatment of an autoimmune disease.
  • pDC plasmacytoid dendritic cells
  • the antagonist may be useful for the treatment of autoimmune diseases including , but not limited to systemic lupus erythematosus, Sjogren's syndrome, psoriasis, dermatitis herpetiformis, vitiligo, mycosis fungoides, allergic contact dermatitis, atopic dermatitis, lichen planus, Pityriasis lichenoides and varioliforms acuta (PLEVA).
  • autoimmune diseases including , but not limited to systemic lupus erythematosus, Sjogren's syndrome, psoriasis, dermatitis herpetiformis, vitiligo, mycosis fungoides, allergic contact dermatitis, atopic dermatitis, lichen planus, Pityriasis lichenoides and varioliforms acuta (PLEVA).
  • the antagonist has a dissociation constant between 10 "11 M and 10 " ' M, preferably between l(T -10 ⁇ M and 10 ⁇ y M.
  • the antagonist according to the invention is preferably a "SERM” and /or a "pure antagonist".
  • the antagonist according to the invention is a pure antagonist and is selected from ICI 164384, ICI 182780, RU 58668, EM-139 and EM-800 [Hermenegildo, C, 2000].
  • the pure antagonist has a dissociation constant between 10 "
  • the modulator is an agonist of the estrogen receptor of plasmacytoid dendritic cells (pDC) useful for the treatment of cancer, infectious diseases and for the stimulation of the immune system in a vaccine therapy, to improve natural immune response against viral infections or to improve natural immunity in patients chronically infected with viruses such as hepatitis C virus.
  • pDC plasmacytoid dendritic cells
  • the agonist of the estrogen receptor of plasmacytoid dendritic cells may be useful for the treatment of a disease or a condition for which stimulation of the immune system is required.
  • the disease may be an infectious disease, whereby the agonist of the estrogen receptor acts to prevent an infection to occur or treats an existing infection.
  • the infectious disease is a viral disease like HCV, herpes, influenza.
  • the disease may be a cancer.
  • the cancer includes but is not limited to tissue specific cancers like colon cancer, hairy-cell leukemia, Kaposi's sarcoma, chronic myeloid leukemia and melanoma [Dunn et Al., 2006].
  • the agonist may be used in a vaccine therapy.
  • the agonist has a dissociation constant between 10 "10 M and 10 "8 M, preferably the dissociation constant is 10 "9 M.
  • antagonist or agonist of the invention may be a small chemical entity, e. g. a small organic molecule (natural or not).
  • small organic molecule refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
  • antagonist or agonist of the invention may consist in an antibody which inhibits or activates the estrogen receptor of plasmacytoid dendritic cells or an antibody fragment which inhibits or activates the estrogen receptor of plasmacytoid dendritic cells.
  • Antibodies directed against the estrogen receptor of plasmacytoid dendritic cells can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • Various adjuvants known in the art can be used to enhance antibody production.
  • antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.
  • Monoclonal antibodies against the estrogen receptor of plasmacytoid dendritic cells can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture.
  • Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985).
  • techniques described for the production of single chain antibodies can be adapted to produce anti-estrogen receptor single chain antibodies.
  • Estrogen receptor antagonists and agonists useful in practicing the present invention also include anti-estrogen receptor antibody fragments including but not limited to F(ab') 2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to the estrogen receptor of plasmacytoid dendritic cells.
  • Humanized anti-estrogen receptor antibodies and antibody fragments thereof may also be prepared according to known techniques.
  • “Humanized antibodies” are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non- human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the estrogen receptor antagonists or agonists may be selected from aptamers.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al, 1996).
  • Another object of the invention relates to a therapeutic composition
  • a therapeutic composition comprising a compound according to the invention for the treatment of diseases according to the invention.
  • Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • compositions for example, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
  • compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
  • compositions include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.
  • the therapeutic composition comprising an agonist according to the invention comprises in addition a progestin substance.
  • compounds of the invention which inhibit or activate the estrogen receptor signalling pathway can be further identified by screening methods as hereinafter described. Screening methods:
  • Another object of the invention relates to a method for screening a compound which inhibits or activates the estrogen receptor signalling pathway of plasmacytoid dendritic cells.
  • the invention provides a method for screening an estrogen receptor of plasmacytoid dendritic cells antagonist or agonist for the treatment of different disorder.
  • the screening method may measure the binding of a candidate compound to the estrogen receptor, or to cells or membranes bearing the estrogen receptor, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound.
  • a screening method may involve measuring or, qualitatively or quantitatively, detecting the competition of binding of a candidate compound to the receptor with a labelled competitor (e.g., antagonist).
  • screening methods may test whether the candidate compound results in a signal generated by an agonist of the estrogen receptor, using detection systems appropriate to cells bearing the receptor.
  • the screening method of the invention comprises the step consisting of:
  • the inventors have designed in vitro assays for the selection of ER-ligands that behave as agonist or antagonist regarding their capacity to modulate type I IFNs production by pDCs upon stimulation with TLR7 and TLR9 synthetic ligands.
  • Two type of pDCs are used: i) ex vivo purified pDCs from PBMCs and ii) pDCs differentiated in vitro from CD34+ progenitors cultured in the presence of appropriate cytokines.
  • Agonist ER-ligands are added to the culture medium and selected for their capacity to enhance type I IFNs production by pDCs stimulation with various doses of TLR7 and TLR9 synthetic ligands.
  • the compounds to be tested are added to E2- supplemented medium (E2 10 ⁇ 9 M) at the initiation of the culture. We then measure their capacity to inhibit type I IFNs production by TLR7 and TLR9-stimulated pDCs.
  • cells are selected from the group consisting of pDC isolated from PBMCs or pDC generated from cord blood CD34+ cells cultured with Flt3-L and IL-7 on OP9-Deltal stroma cells (Olivier et al., Blood 2006 107:2694).
  • the screening method of the invention may be employed for determining an agonist or an antagonist by contacting such cells with compounds to be screened and determining whether such compound activates or not the receptor through its capacity to modulate IFNa production by TLR-stimulated pDCs.
  • the candidate compound may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo or natural compounds.
  • the candidate compound may be selected from the group of (a) proteins or peptides,
  • libraries of pre-selected candidate nucleic acids may be obtained by performing the SELEX method as described in documents US 5,475,096 and US 5,270,163.
  • the candidate compound may be selected from the group of antibodies directed against the estrogen receptor.
  • Such the method may be used to screen estrogen receptor antagonists or agonists according to the invention.
  • FIGURES
  • FIG. 1 Enumeration of conventional and plasmacytoid dendritic cells in postmenopausal women after E2 treatment.
  • A Protocol of the clinical trial, blood samples were collected at two time points before initiation of the treatment (Sl , S2). E2-treatment started at S2 and lasted for one month, sample S3 was collected at the end of this period.
  • B PBMCs were gated on the side scatter (SSC) and CD45 expression.
  • SSC side scatter
  • CD45 + cells DCs are identified as negative for lineage markers, CD3, CD 14, CD 16, CD 19 and CD56 (Lin) and positive for HLA-DR.
  • Conventional DCs are distinguished by the expression of CDl Ic from plasmacytoid DCs whose expressed CD123.
  • FIG. 2 IFN- ⁇ production by pDCs in PBMCs after stimulation with the TLR-9 ligand CpG.
  • PBMCs purified from whole blood were cultured in X- Vivo synthetic medium at 5 x 10 6 cells/well in 96-well plates in the presence of the indicated concentrations of CpG 2216. Supernatants were collected at 24 hours and frozen. IFN- ⁇ concentration in culture supernatants was measured by ELISA. Data from the ELISA are normalized on the basis of the respective percentage of plasmacytoid DCs present in the PBMC fraction calculated from FACS analysis described in Figure IB.
  • FIG. 3 Effect of E2 administration on the frequency of pDC expressing IFN- ⁇ and TNF- ⁇ in TLR7-stimulated PBMCs.
  • A A representative experiment from one post-menopausal woman is shown.
  • CDl Ic + is indicated for the different culture condition.
  • B-D pDC differentiated in presence of E2 (black line) or not (filled grey) were stained for MHC II (B), CD40 (C) and CD86 (D).
  • Column bar indicate GMFI of CD40 and CD86 expression calculated from the gated population indicated in (A). The value represent mean ⁇ SEM from 4 independent cultures and P values were determined by use of Mann- Whitney U test.
  • FIG. 5 Intrinsic expression of ERa is required to promote pDC maturation in the presence of E2.
  • CD45.2 bone marrow cells from WT or ERa 7" were mixed with CD45.1 bone marrow cells at a 1 :1 ratio in steroid- free medium with Flt3L during 9 days in presence or absence of E2 (10 "9 M) as indicated.
  • A The percentage Of MHCII + pDC (B220 + CDl Ic + ) developed from CD45.1 (R2) or CD45.2 (Rl) bone marrow is indicated for the different culture conditions.
  • B Column bar indicates the ratio between CD45.1 and CD45.2 MHC H + pDC. The value represent mean ⁇ SEM from 4 independent cultures and P values were determined by use of Mann- Whitney U test (*, p ⁇ 0.05).
  • EXAMPLE Example 1: Effect of E2 administration on TLR-mediated responses of pDCs in post-menopausal women
  • FITC-conjugated lineage cocktail (Lin) containing anti-CD3, -CD14, -CD16, CD56, -CD19, and -CD20, anti-HLA- DR-PerCp, anti-CD 11 c-allophycocyanin (APC), anti-CD 123 -phycoerythrin (PE) and anti- CD45-PE-Cyanin7 from BD Biosciences.
  • Lin flurorochrome conjugated monoconal antibodies
  • APC anti-CD 11 c-allophycocyanin
  • PE anti-CD 123 -phycoerythrin
  • PE- CD45-PE-Cyanin7 from BD Biosciences.
  • the absolute number of circulating cDCs and pDCs was calculated by comparing cellular events to bead events following manufacturer instructions (BD Biosciences) as determined by the analysis of flow cytometry. DC subsets were also expressed as a pourcentage of peripheral blood CD45 + monuclear cells.
  • CpG-2216 efficiently stimulate IFN- ⁇ production in pDCs.
  • Data from the ELISA were normalized on the basis of the respective percentage of pDCs present in the PBMC fraction to show IFN- ⁇ production on a per-pDC basis as described [Longman, R. S., 2005]. These results were then used to establish at each time points an IFN- ⁇ /CpG score by summing the data obtained for 3 different doses of CpG- 2216 tested (from l ⁇ g/ml to 0.1 ⁇ g/ml) excepted for the first patients (up to P#5) for which the titration started at 3 ⁇ g/ml.
  • the data obtained for the two time points at pretreatment served at evaluating the intra-individual variability and the basal level of TLR9 responsiveness in post-menopausal women.
  • the mean basal IFN- ⁇ /CpG score ( ⁇ SD) was 0.78 ⁇ 0.73 pg/ml per pDC.
  • ⁇ SD mean basal IFN- ⁇ /CpG score
  • Intracellular cytokine staining of pDCs The functional activity of circulating pDC subset was assessed ex vivo by intracellular cytokine staining in response to short term TLR7- stimulation of freshly isolated PBMCs. Cytokine production was analyzed on the Alexa700 or PE-channel on pDC subset defined as BDCA-2 + lineage negative cells. PBMCs (2.5 x 10 6 cells/ml) were stimulated in X- Vivo 15 medium for 5 hrs in vitro in the presence of R-848 (3 or 1 ⁇ g/ml) a potent ligand of TLR- 7 expressed on pDCs. Brefeldin A (5 ⁇ g/ml) was added for the last 2 hrs of culture.
  • Estrogen administration in post-menopausal women does not modify the numbers of circulating cDCs and pDCs.
  • E2-treatment enhances type I-IFN production by TLR9-stimulated PBMCs.
  • CpG-2216 triggered the production of IFN- ⁇ in a dose dependent-manner, with low intra-individual variability in estrogen-deprived post-menopausal women for the two time points analyzed (Fig. 2A and B).
  • Fig. 2A and B show that after one month of E2 treatment there was a significant rise in IFN- ⁇ production by CpG-stimulated PBMCs in many patients (Fig. 2A).
  • longitudinal analysis of all post-menopausal women demonstrated a highly significant enhancing effect of E2 -treatment on TLR9-responsiveness (p ⁇ 0.01, Fig. 2C).
  • Estrogens turn on human plasmacytoid dendritic cells in vivo to produce type I IFN and TNF in response to TLR7- activation.
  • TLR9-mediated type I IFN-production by PBMCs from E2-treated patients could result from an increased numbers of IFN- ⁇ -producing cells or an enhanced intrinsic capacity of pDCs to produce IFN- ⁇ on a per cell basis.
  • intracellular cytokine analysis was performed on R-848-stimulated PBMCs.
  • R-848-stimulated PBMCs We analyzed the production of IFN- ⁇ and TNF- ⁇ in BDCA-2 + cells from PBMCs cultured for 5 hrs with the TLR7 ligand R-848.
  • Cytokine-production in pDCs was dependent on ex vivo TLR activation since the frequency of IFN- ⁇ - and TNF- ⁇ -producing pDCs was usually below 0.1 % in unstimulated PBMCs at all time points tested (not shown). Furthermore, the mean fluorescence intensity of IFN-OC + or TNF-OC + pDCs in TLR7-stimulated cultures was not affected by treatment (not shown). Altogether these data demonstrate that administration of E2 in post-menopausal women, modulate the functional state of circulating pDCs by increasing the frequency cells able to produce IFN- ⁇ and TNF- ⁇ upon TLR7 activation.
  • Example 2 Identification of the estrogen receptor alpha (Esrl gene) as the receptor useful for this invention:
  • BM-pDC were generated as previously described (16). Briefly, BM cells were flushed out from femurs and tibias. After lysis of red blood cells in ammonium chloride potassium (ACK), BM cells were cultured in complete medium (CM) or steroid-free medium (SFM) containing 200 ng/ml murine Flt3L (PeproTech, London, UK) at 2 x 105 cells/ml in bacteriological petri dish (Greiner Bio-One, Poitiers, France). On day 3, an equal volume of fresh medium with 200 ng/ml Flt3L was added to the culture and cells were collected on day 9 for further analysis.
  • CM complete medium
  • SFM steroid-free medium
  • CM Complete medium
  • FCS ATGC Biotechnologie, noisy Le Grand, France
  • 1 mM sodium pyruvate 1 mM sodium pyruvate
  • 1 % non-essential amino acids 1 % non-essential amino acids
  • 2 mM L- glutamine 50 ⁇ M 2-mercaptoethanol
  • 50 ⁇ g/ml gentamicin 50 ⁇ g/ml gentamicin
  • steroid- free medium containing phenol red- free RPMI 1640 (Eurobio) with 10 % dextran charcoal-treated FCS (Hyclone, Logan, Utah, USA) supplemented with 1 mM sodium pyruvate, 1 % nonessential amino acids, 2 mM L-glutamine, 50 ⁇ M 2-mercaptoethanol and 50 ⁇ g/ml gentamicin (Sigma) was used during all the culture period.
  • cultures were supplemented with 1 nM 17B-estradiol (Sigma), or with DMSO vehicle.
  • Maturation status was assessed by flow cytometry after staining for CD40, CD86 costimulatory molecules and MHC class II as described below.
  • IL-12p40 intracellular staining was achieved by incubation with 10 mg/ml brefeldin A (Sigma) for the last 4h of culture.
  • cells were permeabilized with 0.5 % saponin and incubated with PE conjugated anti-IL-12p40 mAb or isotype control mAb (BD Pharmingen).
  • cells (5-10 x 105) were incubated 15 min at room temperature with blocking buffer (PBS with 1 % SVF, 3 % normal mouse serum, 3 % normal rat serum, 5 mM EDTA, 1 %o NaN3 and 5 ⁇ g/ml anti CD16/CD32 (2.4G2, ATCC).
  • blocking buffer PBS with 1 % SVF, 3 % normal mouse serum, 3 % normal rat serum, 5 mM EDTA, 1 %o NaN3 and 5 ⁇ g/ml anti CD16/CD32 (2.4G2, ATCC).
  • FACS buffer PBS 1 % SVF, 5 mM EDTA, 1 %o NaN3
  • E2 enhances the expression of maturation markers on pDCs through ERa- signaling.
  • Flt3L allows the development of CDl Ic + cDCs and pDCs from BM progenitors that can be distinguished by the expression of B220 marker. Only pDCs expressed B220 and represent between 27% to 33% of the cells present in the culture after 9 days as shown in Fig. 4A. pDCs were generated from either WT or ERaKO BM cells in steroid-free medium supplemented or not with E2. After 9 days of culture the expression of maturation markers MHC class II and CD40 was analyzed by flow cytometry on CDl Ic + B220 + pDCs. As shown in Fig.
  • MHC class II expression was down-regulated on pDCs generated from WT CD45.1 or CD45.2 pDCs in steroid- free medium as compared to E2-suplemented cultures (Fig. 5A, left lower panels).
  • E2 in cultures of ERa " " pDCs (CD45.2) generated in the presence of WT CD45.1 progenitors was able to selectively increase the frequency of MHC class II hl pDCs on WT CD45.1 cells but had no effect on ERa "7" CD45.2 pDCs.
  • MHC II expression profile of ERa 7" pDCs (CD45.2) showed a 25-30 % reduction in the frequency of pDCs expressing high levels of MHC class II as compared to ER ⁇ -suff ⁇ cient CD45.1 cells (Fig. 5B).
  • This reduced frequency of MHC class II hl pDCs was not influenced by the presence of WT progenitors since a similar phenotype was observed in ERa 7" pDCs generated alone (see Fig. 4).
  • IL-12 is an important polarizing cytokines that drive expansion of naive CD4 + T cells to the ThI and is readily produced by mouse pDCs.
  • pDCs were generated from either WT or ERaKO BM cells in steroid free medium supplemented or not with E2.
  • these data provide the first genetic evidence that activation of ERa by E2 can act in a cell autonomous manner during Flt3L-mediated pDC differentiation to modulate the expression of maturation markers on their cell surface as well as their capacity to produce pro -inflammatory cytokines upon engagement of their TLRs.
  • This potent regulatory effect of estrogens on TLR-mediated IFN- ⁇ production by human pDCs could account for the substantial gender bias observed not only in autoimmune diseases, such as SLE, but also in HIV-I- or HCV-associated pathogenesis or other infectious diseases for which sex-based differences have been observed.
  • HRT hormonal replacement therapy
  • pDCs are known to infiltrate the liver during HCV infection and it has been recently shown that they can sense HCV-infected hepatocytes through TLR7 resulting in the secretion of IFN- ⁇ which in turn inhibit infection [Takahashi KS et al., 2010]. Therefore, we propose that agonists of ERa may be used to stimulate the TLR-mediated production of type I-IFNs by pDCs for the treatment of infectious diseases like HCV.
  • the model system described in the present report may therefore be useful to screen SERMs (selective estrogen receptor modulators) with agonist properties that could mimick the enhancing effect of E2 on TLR-responsiveness of pDCs.
  • Type I IFN as a vaccine adjuvant for both systemic and mucosal vaccination against influenza virus. Vaccine 24 Suppl 2:S2-56-57.
  • BDCA-2, BDCA-3, and BDCA-4 three markers for distinct subsets of dendritic cells in human peripheral blood. J. Immunol. 165:6037-6046.
  • Plasmacytoid dendritic cells sense hepatitis C virus-infected cells, produce interferon, and inhibit infection. Proc Natl Acad Sci U S A 107:7431-7436.

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Abstract

La présente invention concerne l'utilisation d'un antagoniste du récepteur oestrogénique des cellules dendritiques plasmacytoïdes (pDC) pour le traitement de maladies autoimmunes. L'invention concerne également l'utilisation d'un agoniste du récepteur oestrogénique des cellules dendritiques plasmacytoïdes (pDC) pour le traitement de cancers et de maladies infectieuses ou pour la stimulation de l'immunité dans une vaccinothérapie.
PCT/EP2010/062698 2009-08-31 2010-08-31 Modulateurs de récepteur oestrogénique pour le traitement de maladies impliquant les cellules dendritiques plasmacytoïdes WO2011023824A2 (fr)

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CN110337491A (zh) * 2017-01-31 2019-10-15 丘拉提斯股份有限公司 免疫耐受性浆细胞样树突状细胞及其制备方法
WO2019210179A1 (fr) * 2018-04-27 2019-10-31 Stemline Therapeutics, Inc. Méthodes de traitement de maladies auto-immunes à l'aide d'un conjugué d'interleukine-3 (il-3) humaine et de toxine diphtérique (dt-il3)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110337491A (zh) * 2017-01-31 2019-10-15 丘拉提斯股份有限公司 免疫耐受性浆细胞样树突状细胞及其制备方法
WO2019210179A1 (fr) * 2018-04-27 2019-10-31 Stemline Therapeutics, Inc. Méthodes de traitement de maladies auto-immunes à l'aide d'un conjugué d'interleukine-3 (il-3) humaine et de toxine diphtérique (dt-il3)

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