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    • 专利摘要:
    The invention provides an adjuvant for use in the prevention and / or treatment of an autoimmune disease.
    公开号:BE1023966B1
    申请号:E2016/5935
    申请日:2016-12-15
    公开日:2017-09-26
    发明作者:Sandra Morel；Charlotte Véronique Pouchy；Benoit Laurent Salomon；Nathalie Raoul Liliane Vanerheyde
    申请人:Glaxosmithkline Biologicals Sa；Universite Pierre Et Marie Curie (Paris 6)；Institut National De La Sante Et De La Recherche Medicale；
    IPC主号:
    专利说明:

    USE OF ADJUVANTS FOR THE PREVENTION AND / OR TREATMENT OF AUTOIMMUNE DISEASES
    Technical area
    The present invention relates to adjuvants, in particular the use of adjuvants in the prevention and / or treatment of autoimmune diseases.
    Technical background of the invention
    In many vaccines, the antigens are in the form of purified or subunit proteins to improve their safety, but this is detrimental to the diminishing immunogenicity. Therefore, effective adaptive immune responses require the addition of adjuvants to elicit early inflammation and activate antigen-presenting cells. Aluminum (alum) is widely used as an adjunct to promote antibody responses by inducing a Th2 response. Squalene-based MF59 adjuvants and the adjuvant (AS) 03 system promote both Th1 and Th2 responses by their ability to induce chemokine and cytokine release, leading to massive recruitment and activation of immune cells. . As for AS04 and AS01 adjuvants based on Toll receptor ligands, they induce Th1 and Th2 responses by activating the innate immunity cells directly.
    Beyond the effects on effector responses, some studies have revealed intriguing and paradoxical observations of the immunomodulatory impact of certain adjuvants on autoimmune diseases. Administration of Freund's complete adjuvant (CFA), used only in veterinary vaccines, has induced protection and remission of type 1 diabetes in non-obese diabetic mice (Sadelain et al (1990) Diabetes 39: 583). In addition, administration of lipopolysaccharide (LPS), a natural TLR4 ligand used in research trials for veterinary vaccines, has been shown to protect against experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. (Buenafe et al (2007) J. Neuroimmunol 182: 32). Although these observations are of interest, CFA and LPS are not suitable for use in humans because of their toxicity.
    Autoimmune diseases are caused by an immune response against constituents of the body's own tissues. More than 80 autoimmune diseases are known. Examples of autoimmune diseases include, for example, rheumatoid arthritis (RA), systemic lupus erythematosus (lupus), inflammatory bowel disease (IBD), multiple sclerosis (MS), type 1 diabetes mellitus , Guillain-Barré syndrome, Crohn's disease and psoriasis. These diseases are mostly chronic and can cause significant morbidity and disability. The treatment of autoimmune diseases is usually based on immunosuppression. Although significant progress has been made in the treatment of autoimmune diseases, there is still a need for improved products and methods that increase efficacy, reduce side effects, are easy to administer and safe, and can be used for a long time or even for the chronic treatment of these diseases. Summary of 11 invention
    It has now been unexpectedly discovered that AS01 and AS03, two non-alum-containing adjuvants suitable for and authorized for use in humans, can induce a nearly complete prevention of autoimmune disease in an animal model, which justifies their use in the prevention and / or treatment of autoimmune diseases in humans.
    Therefore, in a first aspect, the invention relates to an adjuvant for use in the prevention and / or treatment of an autoimmune disease, wherein the adjuvant does not include aluminum and is suitable for use in a human subject.
    In a further aspect, there is provided a method of preventing and / or treating an autoimmune disease, comprising administering an adjuvant to a subject, wherein the adjuvant does not include aluminum and is suitable for use in a human subject.
    In yet another aspect, the invention relates to the use of an adjuvant in the preparation of a medicament for the prevention and / or treatment of an autoimmune disease, wherein the adjuvant does not comprise aluminum and is suitable for use in a human subject.
    Brief description of the figures
    Figure 1: a representative image of popliteal drainage lymph node (DLN) 4 days after subcutaneous injection (legs) of vaccine adjuvants, b, conventional flow cytometric analysis of Treg cells. c, increased percentage of Foxp3 + cells among CD4 + cells, compared to mice injected with PBS, in pLNs 4 days and 7 days after subcutaneous injection of vaccine adjuvants. Cumulative data from 3 independent experiments, CD4 + Foxp3-CD90.1 + cells were adoptively transferred before adjuvant injection and Foxp3 induction on donor cells was assessed at day 4 as a marker of pTreg lymphocytes. An example of a Treg induction analysis after AS01 treatment is presented. e, f, ex vivo suppressive activity of purified Treg lymphocytes from dLN of AS01-treated mice 4 days earlier. Representative data at a Treg / Tconv lymphocyte ratio of 1 to 2 (e) and mean ± SEM at different Treg / Tconv lymphocyte ratios from 4 independent experiments (f) Lymphocyte "Treg" means regulatory T lymphocyte and "Tconv" lymphocyte means conventional T lymphocyte.
    Figure 2: a, absolute number of CD45 + lymphocytes in dLN 4 days after subcutaneous injection of vaccine adjuvants. Cumulative data from 3 independent experiments, b, representative expression of CD44, ICOS and KI-67 by Treg cells from dLN 4 days after subcutaneous injection of ASO1. c, in vitro suppressive activity of Treg cells purified from dLN 3 days after subcutaneous injection of vaccine adjuvants. Representative expression of Foxp3 by Treg lymphocytes from dLN 3 days after subcutaneous injection of ASO1. e, in vitro suppressive activity of Treg cells purified from dLN 7 days after the subcutaneous injection of ASO1. Lymphocyte "Treg" means regulatory T lymphocyte.
    Figure 3. a, clinical score of EAE (experimental autoimmune encephalomyelitis) in mice immunized to induce EAE at day 0 and treated with adjuvants at days -3 and 0. Cumulative data from 3 independent experiments, b representative (from 2 independent experiments) proliferation of 2D2-specific T cells MOG35-55 3 days after transfer into mice immunized to induce EAE (day 0) and treated with adjuvants (day -3). c, production of INF-γ and IL-17 by MOG-sensitive T cells from dLN of mice immunized 10 days earlier to induce EAE (day 0) and treated with AS01 or AS03 (day -3) . d, Clinical score of EAE in mice immunized to induce EAE at day 0 and transferred the day before with purified Treg cells from mice treated with PBS, AS01 or AS03. e, f, percentage of Treg (e) and integrin αL + and αM + cells among Treg and Tconv (f) lymphocytes in dLNs at day 10 in mice immunized to induce EAE at day 0 and treated with AS03 at day -3. Lymphocyte "Treg" means regulatory T lymphocyte and "Tconv" lymphocyte means conventional T lymphocyte.
    Figure 4: a, b, clinical score of EAE (experimental autoimmune encephalomyelitis) in mice immunized to induce EAE at day 0 and treated with AS01 or AS03 at day -3 (a) and day 0 (b ). Cumulative data from 3 independent experiments, c, d, proportions of CCR6 + cells and CXCR3 + (c) and integrin α4 + cells and integrin αL + (d) cells among Treg and Tconv lymphocytes in dLNs at 10 days in mice immunized to induce EAE at day 0 and treated with AS03 on days 0 and -3. Lymphocyte "Treg" means regulatory T lymphocyte and "Tconv" lymphocyte means conventional T lymphocyte.
    detailed description
    As described above, in a first aspect, the invention relates to an adjuvant for use in the prevention and / or treatment of an autoimmune disease, wherein the adjuvant does not include aluminum and is suitable for use in a human subject.
    Typically, the purpose of the use of the invention, or the method of the invention, is to prevent an autoimmune disease, including the delayed onset of the disease, and / or to treat such a disease, that is, to reduce the severity of such a disease, for example by reducing the cause of the autoimmune disease and / or reducing its symptoms. In one embodiment, a symptom reduction of more than 50%, such as more than 75%, determined according to the examples presented herein, is achieved. Adjuvants for use in the invention
    In some embodiments, the adjuvant comprises an immunologically active saponin, for example QS21. Adjuvants comprising saponins have been described in the art. Saponins are described, for example, in Lacaille-Dubois and Wagner (1996) (A review of the biological and pharmacological activities of saponins, Phytomedicine Vol.2: 363). Saponins are known as adjuvants in vaccines. For example, Quil A (derived from the bark of the Quillaja Saponaria Molina tree from South America) has been described by Dalsgaard et al. (1974) in "Saponin Adjuvants" (Archiv für die gesamte Virusforschung, Vol 44, Springer Verlag, Berlin, 243) for adjuvant activity. HPLC purified Quil A fractions which retain adjuvant activity without the quil A-associated toxicity have been isolated by HPLC (Kensil et al., (1991) J. Immuno 1. 146: 431). Quil A fractions are also described in US 5,057,540 and "Saponins as vaccinia adjuvants" (Kensil, CR, Crit Rev. Ther., Drug Carrier System, 1996, 12 (1-2): 1- 55).
    Two of these moieties, suitable for use in the present invention, are QS7 and QS21 (also known as QA-7 and QA-21). QS21 is a preferred immunologically active saponin moiety for use in the present invention. QS21 has been reviewed in "Adjuvant Vaccine: Preparation Methods and Research Protocols" (Humana Press, Totowa, New Jersey, edited by Derek T. O'Hagan, Chapter 15: "QS21 Adjuvant"). Particulate adjuvant systems comprising Quil A moieties, such as QS21 and QS7, are described, for example, in WO 96/33739, WO 96/11711 and WO 07/068907.
    In addition to the saponin component, the adjuvant preferably comprises a sterol. The presence of a sterol can further reduce the reactogenicity of compositions comprising saponins, see for example EP 0 822 831. Suitable sterols include beta-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. Cholesterol is particularly appropriate. Suitably, the immunologically active saponin fraction is QS21 and the ratio QS21 / sterol is 1/100 to 1/1 w / w, such as 1/10 and 1/1 w / w, for example 1 1/5 to 1/1 in weight / weight.
    In some embodiments, the adjuvant used in the invention comprises a TLR agonist (Toll-like receptor), such as a TLR4 agonist, for example 3D-MPL. The use of TLR agonists in adjuvants is well known in the art and has been described in detail, for example, by Lahiri et al. (2008, Vaccine 26: 6777). TLRs that can be stimulated to achieve adjuvant effect include TLR2, TLR4, TLR5, TLR7, TLR8 and TLR9. TLR2, TLR4, TLR7 and TLR8 agonists, particularly TLR4 agonists, are preferred.
    Suitable TLR4 agonists include lipopolysaccharides, such as monophosphoryl lipid A (MPL) and 3-0-deacylated monophosphoryl lipid A (3D-MPL). US 4,436,727 discloses MPL and its preparation. US 4,912,094 and Reexamination Certificate B1,4912,094 disclose 3D-MPL and a process for preparing it. Another TLR4 agonist is glucopyranosyl lipid adjuvant (GLA), a lipid A synthetic molecule (see, e.g., Fox et al (2012) Clin Vaccine Immunol 19: 1633). In a further embodiment, the TLR4 agonist may be a synthetic TLR4 agonist, such as a synthetic disaccharide molecule, similar to MPL and 3D-MPL in structure, or may be a synthetic monosaccharide molecule, such as compounds aminoalkyl glucosaminide phosphate (AGP) disclosed, for example, in WO 98/50399, WO 01/34617, WO 02/12258, WO 03/065806, WO 04/062 599, WO 06/016 997, WO 06/12425, WO 03/066 065 and WO 01/90129. Such molecules have also been described in the scientific and patent literature as lipid A mimetics. Lipid A mimetics share appropriately some functional and / or structural activities with lipid A, and in one aspect are recognized by TLR4 receptors. AGPs, as described herein, are sometimes referred to as lipid A mimetics in the art. In a preferred embodiment, the TLR4 agonist is 3D-MPL. TLR4 agonists, such as 3-O-deacylated monophosphoryl lipid A (3D-MPL), and their use as adjuvants in vaccines have been described, for example, in WO 96/33739 and WO 07/068907. and reviewed in Alving et al. (2012, Curr Opin., Immunol., 24: 310).
    In a preferred embodiment of the invention, the adjuvant comprises both an immunologically active saponin and a TLR4 agonist, for example QS21 and 3D-MPL.
    In another preferred embodiment, the adjuvant comprises an immunologically active saponin is a TLR4 agonist, for example QS21 and 3D-MPL, in a liposomal formulation.
    The term "liposome", when used herein, refers to single or multi-lamellar lipid structures enclosing an aqueous inner portion. Liposomes and liposome formulations are well known in the art. Liposomal presentations are described, for example, in WO 96/33739 and WO 07/068907. Lipids that are capable of forming liposomes include all substances having fat or fat-like properties.
    The size of the liposomes can vary from 30 nm to several μm, depending on the composition of phospholipids and the process used for their preparation. In particular embodiments of the invention, the size of the liposomes will be in the range of 50 nm to 500 nm, and in other embodiments, in the range of 50 nm to 200 nm. Dynamic scattering of laser light is a method used to measure the size of liposomes well known to those skilled in the art.
    In a particularly suitable embodiment, the liposomes used in the invention comprise DOPC and a sterol, in particular cholesterol. Thus, in a particular embodiment, the adjuvants of the invention comprise QS21 in any amount described herein as a liposome, said liposome comprising DOPC and a sterol, particularly cholesterol.
    In one embodiment, the adjuvant comprises between 5 and 100, such as between 10 and 75, for example 25 or 50 μg of QS21 per dose and between 5 and 100, as between 10 and 75, for example 25 or 50 μg of 3D-MPL per dose.
    It is well known that for parenteral administration, the solutions must be physiologically isotonic (i.e. have a pharmaceutically acceptable osmolality) to prevent cell deformation or lysis. An "isotonic agent" is a compound that is physiologically tolerated and that imparts appropriate tonicity to a formulation (eg, the immunogenic compositions of the invention) to prevent the net flow of water through cell membranes that are in contact with the formulation. Aqueous adjuvant compositions are known which contain 100 mM sodium chloride or more, for example the adjuvant system A (AS) of WO 05/112 991 and WO 08/142 133 or the liposomal adjuvants disclosed in WO 07/068 907.
    In some embodiments, the isotonic agent used for the composition is a salt. In other embodiments, however, the composition comprises a nonionic isotonic agent and the concentration of sodium chloride or the ionic strength in the composition is less than 100 mM, as less than 80 mM, for example less than 30 mM as less than 10 mM or less than 5 mM. In a preferred embodiment, the nonionic isotonic agent is a polyol, such as sorbitol. The concentration of sorbitol may be, for example, from about 3% to about 15% (w / v), such as from about 4% to about 10% (w / v). Adjuvants comprising an immunologically active saponin fraction and a TLR4 agonist, in which the isotonic agent is a salt or a polyol, have been described in WO 10/142 685, see for example Examples 1 and 2 of the document WO 10/142 685.
    In one embodiment, the adjuvant used in the invention comprises an oil-in-water emulsion. Suitably, said emulsion comprises a metabolizable oil in an amount of 0.5% to 20% based on the total volume. The meaning of the term metabolizable oil is well known in the art. Metabolizable can be defined as "being able to be metabolized" (Dorland's Illustrated Medical Dictionary, W. B. Sanders Company, 25th Edition, (1974)). The oil can be any vegetable oil, fish oil, animal oil or synthetic oil, which is not toxic to the recipient and is able to be metabolized. Nuts, seeds and grains are classic sources of vegetable oils. Synthetic oils are also part of the present invention and may include commercially available oils, such as NEOBEE® and others. A particularly suitable metabolizable oil is squalene. Squalene (2,6,10,15,19,23-hexamethyl-2, 6, 10, 14, 18,22-tetracosahexaene) is an unsaturated oil found in large quantities in the liver oil. shark, and in smaller amounts in olive oil, wheat germ oil, rice bran oil, and yeast, and it is a particularly suitable oil for use in the present invention . Squalene is a metabolizable oil because it is an intermediate in the biosynthesis of cholesterol (Merck Index, 10th Edition, entry No. 8619).
    Oil-in-water emulsions per se are well known in the art, and it has been suggested that they may be useful as adjunctive compositions (EP 399,843; WO 95/17210). Suitably, the metabolizable oil is present in an amount of 0.5% to 20% (final concentration) based on the total volume of the immunogenic composition, suitably 1.0% to 10% relative to to the total volume, suitably in an amount of 0.2% to 6.0% based on the total volume.
    In a specific embodiment, the metabolizable oil is present in a final amount of about 0.5%, 1%, 3.5% or 5% based on the total volume of the immunogenic composition. In another specific embodiment, the metabolizable oil is present in a final amount of 0.5%, 1%, 3.57% or 5% based on the total volume of the immunogenic composition. An appropriate amount of squalene is about 10.7 mg per dose, suitably 10.4 to 11.0 mg per dose.
    Suitably, the oil-in-water emulsion systems used in the present invention have a small submicron oil droplet size. Suitably, the sizes of the droplets will be in the range of from 120 to 750 nm, suitably diameters of from 120 to 600 nm. Traditionally, the oil-in-water emulsion contains oil droplets of which at least 70% in terms of intensity have a diameter of less than 500 nm, in particular at least 80% in terms of intensity have a smaller diameter. at 300 nm, suitably at least 90% in intensity have a diameter in the range of 120 to 200 nm. The oil-in-water emulsion according to the invention may comprise a sterol or a tocopherol, such as alpha-tocopherol. Sterols are well known in the art, for example cholesterol is well known and is, for example, described in the Merck Index, 11th Edition, page 341, as a naturally occurring sterol found in animal fats. Other suitable sterols include β-sitosterol, stigmasterol, ergosterol and ergocalciferol. Suitably, alpha-tocopherol or a derivative thereof, such as alpha-tocopherol succinate, is present. Suitably, alpha-tocopherol is present in an amount of between 0.2% and 5.0% (v / v) based on the total volume of the immunogenic composition, suitably in an amount of 2, 5% (v / v) in a dose volume of 0.5 ml, or 0.5% (v / v) in a dose volume of 0.5 ml or 1.7 to 1.9 % (v / v), suitably 1.8% in a dose volume of 0.7 ml. For clarification purposes, volume / volume concentrations can be converted to concentration by weight / volume by application of the following conversion factor: an alpha-tocopherol concentration of 5% (by volume / volume) is equivalent at an alpha-tocopherol concentration of 4.8% (w / v). An appropriate amount of alpha-tocopherol is about 11.9 mg per dose, suitably 11.6 to 12.2 mg per dose. The oil-in-water emulsion may comprise an emulsifying agent. The emulsifier may be present in an amount of from 0.01 to 5.0% by weight of the immunogenic composition (w / w), suitably present in an amount of 0.1 to 2.0% by weight. (in weight / weight). A suitable concentration is 0.5 to 1.5% by weight (w / w) based on the total composition. The emulsifying agent may suitably be a polyoxyethylene sorbitan monooleate (polysorbate 80 or Tween 80). In a specific embodiment, a 0.5 ml dose volume contains 1% (w / w) Tween 80, and a 0.7 ml dose volume contains 0.7% (w / w). In another specific embodiment, the concentration of Tween 80 is 0.2% (w / w). A suitable amount of polysorbate 80 is about 4.9 mg per dose, suitably 4.6 to 5.2 mg per dose.
    Span 85 (polyoxyethylenesorbitan trioleate) may also be present, for example at a level of 1%. An exemplary oil-in-water emulsion additive comprising Span 85 for use in the invention is given and detailed in EP 0 399 843 B, also known as MF59.
    In a preferred embodiment, the adjuvant is an oil-in-water emulsion comprising squalene, alpha-tocopherol and a surfactant, i.e., the emulsion comprises squalene, alpha-tocopherol and a surfactant, for example polysorbate 80, in an aqueous phase. The preparation of such adjuvants is described, for example, in WO 95/17210 and WO 06/100109. In one embodiment, the emulsion comprises 2 to 10% (v / v) of squalene, 2 to 10% (v / v) alpha-tocopherol and 0.3 to 3% (v / v) Tween 80. Preferably, the emulsion comprises 2.5% squalene (by volume / volume), 2.5% alpha-tocopherol (v / v), 0.9% polyoxyethylene (volume / volume) sorbitan monoleate (Tween 80).
    Autoimmune diseases to prevent and / or treat
    As explained above, the invention relates to the use of adjuvants for the prevention and / or treatment of autoimmune diseases. The following diseases have been classified as autoimmune diseases: acute disseminated encephalomyelitis, Addison's disease, agammaglobulinemia, alopecia, amyloidosis, ankylosing spondylitis, anti-GBM / anti-nephritis. TBM, antiphospholipid syndrome (APS), autoimmune hepatitis, autoimmune inner ear disease (AIED), axonal neuropathy & neuronal (AMAN), Behcet's disease, bullous pemphigoid, Castleman's disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid / mucous membrane pemphigoid, Cogan syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis Herpetiformis, Dermatomyositis, Devia Disease (Acute Optic Neuromyelitis), Discoid Lupus, Dressier's Syndrome, Endometriosis, Eosinophilic Esophagitis (EoE), Eosinophilic Fasciitis, Erythema nodosum, Cryoglobulinaemia essential syndrome, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura (HSP), herpes gestationis, or pemphigoid gestationis (PG), hypogamma-globulinemia, IgA nephropathy, IgG4-associated sclerosing disease, inclusion myositis (IBM), interstitial cystitis (IC), juvenile rheumatoid arthritis, juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, woody conjunctivitis, linear IgA disease (LAD), lupus, chronic Lyme disease, Meniere, microscopic polyangiitis (MPA), mixed connectivity (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis (MS), myasthenia gravis, myositis, narcolepsy, optical neuromyelitis acute, neutropenia, cicatric pemphigoid ocular, optic neuritis, palindromic rheumatism (PR), PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococci), paraneoplastic cerebellar degeneration (PCD), paroxystic nocturnal hemoglobinuria (PNH), Parry Romberg syndrome , pars planitis (peripheral uveitis), Parsonnage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, modifications cutaneous), polyarteritis nodosa, rheumatic polymyalgia, polymyositis, post-myocardial infarction syndrome, post-commissurotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, erythroblastopenia (PRCA), phagedenic pyoderma, Raynaud's phenomenon, reactive arthritis e, reflex sympathetic algodystrophy, Reiter's syndrome, atrophic polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis (RA), sarcoidosis, Schmidt syndrome , scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff man syndrome (SPS), subacute bacterial endocarditis (SBE), Susac's syndrome, ophthalmia sympathetic (SO), Takayasu arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (HRT), transverse myelitis, type 1 diabetes mellitus, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vascularized, vitiligo and Wegener granulomatosis (now called granulomatosis with polyangiitis (GPA).
    Preferred diseases to prevent and / or treat according to the invention include: • Rheumatoid arthritis
    In people with rheumatoid arthritis, the immune system primarily targets the lining (synovial membrane) that covers various joints. Inflammation of the synovial membrane is usually symmetrical (appearing in the same way on both sides of the body) and causes pain, swelling and stiffness of the joints. These features distinguish rheumatoid arthritis from osteoarthritis, which is more common and degenerative "wear" arthritis. Currently, available treatment focuses on reducing joint inflammation with anti-inflammatory and immunosuppressive drugs. Sometimes the immune system can also target the lungs, blood vessels or eyes; occasionally, patients may also develop symptoms of other autoimmune diseases, such as Sjögren's inflammation, pruritus and desquamation. • Multiple sclerosis
    Multiple sclerosis is a disease in which the immune system targets the nervous tissues of the central nervous system. Very frequently, damage to the central nervous system occurs intermittently, allowing a person to lead a relatively normal life. At the other extreme, symptoms may become constant, leading to progressive disease with possible blindness, paralysis, and premature death. Some medications, such as interferon beta, are useful for people suffering from the intermittent form of multiple sclerosis. In young adults, multiple sclerosis is the most common disabling disease of the nervous system. • Diabetes mellitus type 1 or immune-mediated
    Type 1 diabetes mellitus is a result of autoimmune destruction of pancreatic insulin producing cells. Insulin is required by the body to keep the sugar (glucose) level in the blood under control. High glucose levels are responsible for the symptoms and complications of the disease. However, most insulin-producing cells are destroyed before the patient develops the symptoms of diabetes. Symptoms include fatigue, frequent urination, increased thirst, and possible sudden confusion. Type 1 diabetes mellitus is usually diagnosed before the age of 30 and can be diagnosed in the first month of life. In combination with type 2 diabetes (which is not considered an autoimmune disease), diabetes mellitus is the leading cause of kidney damage, loss of vision and amputation of the legs. Strict regulation of sugar levels decreases the frequency with which these events occur. • Inflammatory bowel diseases
    This medical term is used for both Crohn's disease and ulcerative colitis, two diseases in which the immune system attacks the intestine. Patients may experience diarrhea, nausea, vomiting, abdominal cramps, and pain that can be difficult to relieve. The disease in affected people can result from intestinal inflammation and side effects of the drugs used for the disease. For example, the daily use of a high dose of corticosteroid (prednisone), which is necessary to fight the severe symptoms of Crohn's disease, can predispose patients to infections, thinning bones (osteoporosis) and fractures. • Systemic lupus erythematosus
    Patients with systemic lupus erythematosus very often experience deep fatigue, rash and joint pain. In severe cases, the immune system can attack and damage many organs, such as the kidneys, brain or lungs. For many individuals, the symptoms and damage caused by the disease can be controlled with the available anti-inflammatory drugs. • Psoriasis
    Psoriasis is an immune system disorder that affects the skin, and occasionally the eyes, nails and joints. Psoriasis can affect very small areas of skin or cover the entire body with an accumulation of red crusts called plaques. The plates are of different sizes, shapes and gravities and can be painful as well as aesthetic. Bacterial infections and pressure or trauma to the skin can aggravate psoriasis. Most treatments focus on topical skin care to relieve inflammation, pruritus and scabs. • Scleroderma
    Autoimmune disease causes thickening of the skin and blood vessels. Virtually every scleroderma patient suffers from Raynaud's syndrome, which is a spasm of the blood vessels of the fingers and toes. Symptoms of Raynaud's syndrome include increased sensitivity of the fingers and toes to cold, a change in skin color, pain and occasionally ulcers at the end of the fingers or toes. In people with scleroderma, thickening of the skin and blood vessels can lead to loss of movement and shortness of breath or, more rarely, kidney, heart or lung failure. • Autoimmune thyroid diseases
    Hashimoto's thyroiditis and Graves' disease result from the destruction or stimulation of the thyroid tissue by the immune system. Symptoms of low (hypo-) or hyperactive (hyper-) function of the thyroid are nonspecific and may develop slowly or suddenly; these include fatigue, nervousness, intolerance to cold or heat, weakness, changes in texture or amount of hair, and weight gain or loss. The diagnosis of thyroid disease is easy to make with appropriate laboratory tests. The symptoms of hypothyroidism are controlled with thyroid hormone replacement pills; however, complications from over- or under-replacement of the hormone may occur. Treatment of hyperthyroidism requires long-term therapy with an antithyroid drug or destruction of the thyroid gland with radioactive iodine or surgery. These two treatment approaches have certain risks and long-term side effects.
    In a preferred embodiment, the autoimmune disease to be prevented and / or treated is a disease affecting the nervous system, such as the central nervous system. In a more preferred embodiment, the autoimmune disease is multiple sclerosis. In an even more preferred embodiment, the autoimmune disease is type 1 diabetes. In a more preferred embodiment, the autoimmune disease is acute disseminated encephalomyelitis.
    In particular, adjuvants comprising an oil-in-water emulsion, for example comprising squalene, alpha-tocopherol and polysorbate 80 in an aqueous phase, are preferred for the prevention and / or treatment of diseases affecting the system. nervous, such as the central nervous system, for example multiple sclerosis.
    Treatment options
    In preferred embodiments, the method of the invention, or the use of the invention, comprises multiple administrations of the adjuvant, for example at least 2, at least 3, at least 4, at least 5 or at least 10 administrations of the adjuvant. In one embodiment, the time interval between each of the administrations is between 1 day and 6 months, for example the interval can be from 1 week to 1 month between each administration.
    In a preferred embodiment, the subject is a human subject. The human subject to be treated using the method of the invention can be of any age. However, in one embodiment, the human subject is over 18 years old when the treatment is initiated. In additional embodiments, the subject is over 40 years old, as is over 50 years old, for example over 60 years old. The subject can be a man or a woman.
    In one embodiment, the adjuvant is for use in the prevention of autoimmune disease in a human subject who is predisposed to the development of an autoimmune disease, for example a subject who is predisposed to the development of an autoimmune disease. multiple sclerosis or a subject who is predisposed to the development of type 1 diabetes. A human subject who is predisposed to the development of an autoimmune disease may be, for example, a subject who exhibits early clinical symptoms of autoimmune disease. -immune, a subject who presents a risk of developing an autoimmune disease or a subject with specific autoantibodies.
    A person predisposed to the development of multiple sclerosis is, for example, a person suffering from CIS (clinically isolated syndrome) - a first and only neurological episode of inflammation or demyelination in the central nervous system lasting at least 24 hours, especially when lesions compatible with multiple sclerosis are observed by MRI.
    A person predisposed to the development of type 1 diabetes is, for example, a person with autoantibodies, such as GADA, IA-2A and / or mIAA autoantibodies (Sosenko et al (2013) Diabetes Care 36 : 2615). The adjuvant may be administered by a variety of appropriate routes, including parenterally, such as intramuscular, intradermal or subcutaneous administration. Suitably, the adjuvant compositions used in the present invention have a human dose volume of between 0.05 ml and 1 ml, such as between 0.1 and 0.5 ml, particularly a dose volume of about 0. , 5 ml, or 0.7 ml. The adjuvant can be given as monotherapy or in combination with other substances, for example other substances known to have a therapeutic or prophylactic effect on autoimmune diseases.
    The teachings of all references cited in this application, including patent applications and granted patents, are hereby incorporated by reference in their entirety. The terms "comprising", "understanding" and "comprehension" may, in this document, be optionally substituted by the terms "consisting of", "consisting of" and "consisting of", respectively. The invention will be described in more detail with reference to the following non-limiting examples.
    EXAMPLES
    Example 1 - Process
    Mouse
    WT mice were purchased from Janvier Labs (France). Transgenic 2D2 T cell receptor transgenic mice, specific for myelin oligodendrocyte glycoprotein, were purchased from Jackson Laboratory. Activated Foxp3-IRES-GFP (Foxp3GFP) mice were kindly provided by Pr. Bernard Malissen. 2D2 and Foxp3GFP mice were backcrossed with CD90.1 congenic animals. All the mice were on a C57B1 / 6 background.
    The mice were housed under specific conditions without pathogenic organisms and were studied at 7 to 14 weeks of age. All experimental protocols have been approved by the local ethics committee and are in compliance with EU directives. Reagents ASO1 is composed of MPL (1 mg / ml), QS-21 (1 mg / ml) and liposomes. AS03 is composed of alpha-tocopherol (23.72 mg / ml), squalene oil (21.38 mg / ml) and polysorbate 80 (9.72 mg / ml). AS04 is composed of MPL (0.1 mg / ml) and alum (1 mg / ml). Freund's adjuvants were purchased from Sigma-Aldrich. Incomplete form (IFA) contains 85% paraffin oil and 15% mannide monooleate. The complete form (CFA) additionally contains Mycobacterium tuberculosis (H37Ra) killed by heat and dried at a concentration of 1 mg / ml.
    Administration of adjuvants
    The mice received 30 μl of vaccine adjunct or PBS subcutaneously in the left hind paw. To analyze their effect, popliteal drainage lymph nodes (dLN) and non-draining right brachial lymph nodes were collected at different time points. Mice immunized to induce EAE (experimental autoimmune encephalomyelitis) received 100 μl of vaccine adjunct or PBS subcutaneously at the base of the tail and in the upper back 3 days before and during the day induction of EAE (experimental autoimmune encephalomyelitis).
    Flow Cytometry Analyzes
    Cells from the lymph nodes were mechanically dissociated and resuspended in 3% PBS FCS. They were first treated with 2.4G2 antibody to block the Fc receptor and stained with the following antibodies: anti-CD45 (30-F11), anti-CD3 (145-2C11), anti-CD4 (RM4- 5), anti-CD8 (53-6.7), anti-CD25 (PC61), anti-Foxp3 (MF23), anti-CTLA-4 (UC10-4F10-11), anti-GITR (DTA-1), anti- ICOS (17G9), anti-Ki-67 (B56). All antibodies were obtained from BD Biosciences. Intracellular staining was performed using the set of permeabilization and intracellular binding buffers available from eBioscience. The cells were acquired on a BD LSRII cytometer and analyzed using the FlowJo software.
    Evaluation of the peripheral induction of Tregs
    Lymph nodes (brachial, axillary, cervical and inguinal) and spleen cells of Foxp3GFP CD90.1 mice were isolated, treated as described above and stained with anti-CD4 antibody. CD4 + GFP-cells (Tconv) were purified using BD FACSAria II, then injected intravenously into WT mice (106 cells / mouse) The following day, the mice received 30 μl of vaccine adjuvant or PBS subcutaneously in the left hind paw Three days later, the dLN were removed and the cells were stained with anti-CD4, anti-CD90.1 (OX-7), CD90.2 (30-H12) and anti-Foxp3 The induction of Foxp3 in CD90.1 transferred mice was evaluated by flow cytometry.
    In vitro Treg suppression assay
    Foxp3GFP mice received 100 μl of vaccine adjunct or PBS subcutaneously at the base of the tail and in the upper back. Brachial and inguinal lymph nodes were collected at 3 and 7 days. The cells were isolated and stained as described above. The CD4 + GFP (Tconv) and CD4 + GFP + (Treg) cells were then sorted using BD FACSAria II Tconv cells were labeled with the CellTrace Kit Violet Proliferation Kit (Life Technologies) and placed in a 96-well plate. well at 2.5 x 104 cells / well with splenocytes from CD3 - / - mice at 7.5 x 104 cells / well The culture medium was supplemented with an anti-CD3 (KT3) available from BioXCell at 0 0.5 μg / ml The Treg cells were then added at various ratios ranging from 1/1 to 1/16 At day 3, the CellTrace dilution was evaluated by flow cytometry.
    Induction of EAE
    The mice were immunized by subcutaneous injection of 100 μg of MOG35-55 peptide (PolyPeptide) emulsified in 100 μl of CFA (Sigma-Aldrich) supplemented with 50 μg of heat-killed Mycobacterium tuberculosis H37Rα (BD Biosciences). The animals were further injected intravenously with 200 ng Bordetella pertussis toxin (Enzo) at the time of immunization and two days after immunization. The clinical evaluation was performed on a daily basis with a 5-point scale comprising 0, no clinical signs; 1, soft tail; 2, soft tail, altered straightening reflex, and paresis of a limb; 3, paralysis of the hind limbs; 4, paralysis of the hind limbs and forelegs; 5, moribund.
    Measurement of cytokines
    Ten days after induction of EAE, brachial and inguinal dLN were collected. The cells were cultured with 1 μg / ml of MOG35-55 peptide in a 96-well plate at 2 x 10 5 cells / well. On day 3, the supernatants were collected to measure the secretions of INF-γ and IL-17 by ELISA (eBioscience).
    Evaluation of T cell priming
    Lymph nodes and spleen cells from 2D2 CD90.1 mice were stained with the following biotin-labeled antibodies: anti-CD19 (6D5), anti-CD11b (Ml / 70), anti-CD11c (N418), anti - CD8 (53-6.7) and anti-CD25 (7D4), then coated with anti-biotin microbeads (Miltenyi Biotec). After magnetic stirring, the cells of the CD4 + enriched negative fraction were labeled with the CellTrace Violet Proliferation Kit and injected intravenously into naive mice (106 cells / mouse). The next day, cells were immunized with MOG35-55 peptide for induction of EAE. The CellTrace dilution of CD4 + CD90.1 + Vβll + cells (T cell receptor transgene) was evaluated by flow cytometry from the brachial and inguinal lymph nodes at day 3. The mice receiving the adjuvants received by injection 100 μl. vaccine adjunct or PBS subcutaneously at the base of the tail and in the upper back.
    Adoptive transfer of Treg lymphocytes
    Foxp3GFP CD90.1 mice received 100 μl of vaccine adjunct or PBS subcutaneously at the base of the tail and in the upper back. At day 3, the brachial and inguinal lymph nodes were removed and the cells were stained with anti-CD4 antibody. CD4 + GFP + (Treg) cells were then purified using BD FACSAria II and injected intravenously to WT mice (1 x 10 6 cells / mouse). The following day, the mice were immunized with the MOG35-55 peptide as previously described in induction of EAE.
    Statistical analysis
    Statistical analyzes were performed using the GraphPad Prism software. Statistical significance was determined using the non-parametric Mann-Whitney U-test. * p <0.05, ** p <0.01, *** p <0.001. The means ± SEM were used in all the figures.
    Example 2 - Vaccine adjuvants induce transient preferential expansion of Treg cells
    CFA, incomplete Freund's adjuvants (IFA), ASO1, AS03 and AS04 were administered subcutaneously to mice. After 4 days, very significant inflammation and swelling of the lymphatic drainage lymph nodes (dLN) was observed (Figure la). The cellularity of these dLNs increased by 11 to 20 fold (Figure 2a). In contrast, alum injection had no effect on the size and number of dLN cells.
    The Treg lymphocytes were then analyzed by flow cytometry (FIG. 1b). Except in the case of alum, we observed a rapid increase (day 4) in the proportion of Treg cells among CD4 + cells after the injection of the different adjuvants, up to 1.9 times compared to the mice that received PBS (Figure 1). This expansion of Treg lymphocytes was associated with an activated phenotype, as shown by the increased expression of CD44, ICOS, and Ki-67 (Figure 2b). This expansion and activation of Treg cells was transient because they were no longer present at day 7, except for IFA (Figure 1). The expansion of Treg lymphocytes induced by adjuvant injections was due to the accumulation of thymic Treg (tTreg) and not to the increased induction of peripheral Tregs. Indeed, Foxp3 expression was not induced by adjuvants in adoptively transferred CD4 + Foxp3-cells (FIG. 1d). These data revealed the in vivo activation of tTreg lymphocytes by new generation adjuvants.
    Next, we evaluated the effect of administration of vaccine adjuvants on Treg cell activity by a standard in vitro suppression assay. Alum, Freund's adjuvant, AS03 and AS04 had no effect on Treg cell function (FIG. 2c), whereas ASO1 partially reduced their suppressive activity and Foxp3 expression. (Figures 1a and 1f and 2d). This effect was observed at day 4, but no longer at day 7 (Figure 2e). Therefore, the administration of new generation adjuvants does not compromise, or only marginally, the suppressive function of Treg cells.
    EXAMPLE 3 Prevention of EAE by Administration of Adjuvants AS01 and AS03
    In order to evaluate the effects of vaccine adjuvants on the induction of tolerance in a physiopathological context, we tested adjuvant treatments with the autoimmune EAE model. Surprisingly, 2 injections of ASO1 or AS03, 3 days before the immunization and at the same time as the immunization, induced a quasi-complete prevention against the development of the disease. Mean clinical scores were less than 0.14-0.17, compared to 1.17 in controls. In contrast, AS04 and alum had no effect on EAE (Figure 3a). After only one injection of ASO1 or AS03 performed 3 days before immunization or at the time of immunization, EAE was significantly delayed or reduced, respectively (Figures 4a and 4b). Therefore, AS01 and AS03 have a powerful preventive effect against EAE.
    Next, we studied the initiation and polarization of cytokines of autoreactive Tconv lymphocytes. The proliferation of T lymphocytes was evaluated after adoptive transfer of 2D2 transgenic mice expressing a transgene T-cell receptor with specific I-Ab restriction of the MOG35-55 peptide (Bettelli et al (2003) J. Exp Med 197: 1073 ) before the induction of EAE. Administration of ASO1 slightly reduced the proliferation of MOG35-55-specific T cells at day 3 in the dLNs, whereas AS03 had no effect (Figure 3b). T cell polarization was assessed by measuring the release of IFNy and IL-17, 2 major pathogenic cytokines in EAE, at day 10 in dLNs after induction of EAE by self-reactive Tconv lymphocytes specific for the MOG35-55 immunizing peptide. Administration of ASO1 was associated with decreased production of IFNγ, whereas IFNγ and IL-17 were unaffected by administration of AS03 (Figure 3c). Therefore, although both ASO1 and AS03 administration prevented the development of EAE, only AS01 disrupted the priming and polarization of MOG35-55-reactive T cells.
    Next, we performed adoptive transfer experiments to further analyze the role of Treg cells in the prevention of adjuvant-directed EAE. Purified Treg cells derived from adjuvanted mouse dLN were injected into naive mice prior to induction of EAE. Surprisingly, the mice that received the Treg lymphocytes from the AS03-treated mice were totally protected against the disease. In contrast, Treg lymphocytes from AS01-treated mice had no effect because the recipient mice transferred with these cells developed the same EAE as the control mice transferred with PBS or Treg from naive mice (Figure 3d). . These data show that the administration of AS03 strongly enhances the ability of Treg cells to fight EAE.
    In order to better understand the mechanism of prevention against EAE by AS03, we also studied the Treg lymphocytes and the molecules involved in the migration of T lymphocytes. Cells derived from dLN from mice immunized to induce EAE and treated with AS03 were analyzed on day 10. The proportion of Treg lymphocytes had significantly increased in mice treated with AS03 compared to the control (Figure 3e). The expression of CCR6 and CXCR3 chemokine receptors by Tconv lymphocytes, which play a critical role in the entry of pathogenic T cells into the central nervous system (CNS) during EAE (Reboldi et al (2009) Nat. Immunol., 10: 514; Sporici and Issekutz (2010) Eur J. Immunol., 40: 2751), was unaffected by AS03 treatment (Figure 4c). It is interesting to note that expression levels of integrins involved in CNS locating (Yednock et al., (1992) Nature 356: 63, Rothhammer et al (2011) J. Exp Med 208: 2465) significantly modified by the adjuvant. Treg lymphocytes expressed a higher level of α1 integrin, whereas Tconv lymphocytes exhibited a decrease in α 1 integrin level (Figure 3f). In addition, the levels of expression of the 4 and α1 integrins by Tconv lymphocytes were not affected (Figure 4d). These results suggest that administration of AS03 has an impact on the migration of Treg and Tconv lymphocytes in the CNS, which may explain its ability to suppress EAE.
    Conclusion
    This work demonstrates that some of the new generation vaccine adjuvants have potent immunoregulatory properties and affect the activation and function of Treg cells, thus demonstrating their potential in the treatment of autoimmune disorders. The administration of ASO1 and the administration of AS03 both induced protection against EAE. It is interesting to note that their suppression mechanism seems different. AS01 altered cytokine priming and polarization of encephalitogenic Tconv lymphocytes, whereas AS03 strongly increased the protective capacity of Treg cells in EAE and may alter Tconv and Treg cell migration.
    权利要求:
    Claims (21)
    [1]
    An adjuvant for use in the prevention and / or treatment of an autoimmune disease, wherein the adjuvant does not include aluminum and is suitable for use in a human subject.
    [2]
    The adjuvant of claim 1, wherein the adjuvant comprises an immunologically active saponin.
    [3]
    3. Adjuvant according to claim 2, wherein the adjuvant comprises QS21.
    [4]
    4. Adjuvant according to claim 2 or 3, wherein the adjuvant comprises a sterol.
    [5]
    An adjuvant according to any of the preceding claims, wherein the adjuvant comprises a TLR agonist, such as a TLR4 agonist.
    [6]
    An adjuvant according to any one of the preceding claims, wherein the adjuvant comprises 3D-MPL.
    [7]
    An adjuvant according to any one of the preceding claims, wherein the adjuvant comprises QS21 and 3D-MPL in a liposomal formulation.
    [8]
    An adjuvant according to any one of the preceding claims, wherein the adjuvant comprises an oil-in-water emulsion.
    [9]
    The adjuvant of claim 8, wherein the emulsion comprises squalene.
    [10]
    The adjuvant of claim 9, wherein the emulsion further comprises alpha-tocopherol and polysorbate 80 in an aqueous phase.
    [11]
    An adjuvant according to any one of the preceding claims, wherein the autoimmune disease is a disease selected from the group consisting of: multiple sclerosis, type 1 diabetes, rheumatoid arthritis, inflammatory bowel diseases, lupus erythematosus disseminated, psoriasis, scleroderma and autoimmune thyroid diseases.
    [12]
    An adjuvant according to any one of the preceding claims, wherein the autoimmune disease is a disease affecting the nervous system, such as the central nervous system.
    [13]
    An adjuvant according to any one of the preceding claims, wherein the autoimmune disease is multiple sclerosis.
    [14]
    An adjuvant according to any one of the preceding claims, wherein the autoimmune disease is acute disseminated encephalomyelitis.
    [15]
    15. Adjuvant according to any one of claims 1 to 11, wherein the autoimmune disease is type 1 diabetes.
    [16]
    An adjuvant according to any one of the preceding claims, wherein the adjuvant is for use in a human patient.
    [17]
    An adjuvant according to any one of the preceding claims, wherein the adjuvant is for use in the prevention of autoimmune disease in a human patient who is predisposed to the development of an autoimmune disease.
    [18]
    A method of preventing and / or treating an autoimmune disease comprising administering an adjuvant to a subject, wherein the adjuvant does not include aluminum and is suitable for use in a human subject .
    [19]
    The method of claim 18 including one or more of the additional features as recited in claims 2 to 17.
    [20]
    20. Use of an adjuvant in the preparation of a medicament for the prevention and / or treatment of an autoimmune disease, wherein the adjuvant does not comprise aluminum and is suitable for use in a human subject .
    [21]
    21. Use according to claim 20, comprising one or more of the additional characteristics as mentioned above in claims 2 to 17.
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    同族专利:
    公开号 | 公开日
    GB201522329D0|2016-02-03|
    CA3008333A1|2017-06-22|
    BR112018012126A2|2018-12-04|
    MX2018007408A|2018-11-09|
    BE1023966A1|2017-09-25|
    EP3389709A1|2018-10-24|
    US20200261569A1|2020-08-20|
    JP2019504020A|2019-02-14|
    WO2017102939A1|2017-06-22|
    CN109069619A|2018-12-21|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    GB0323965D0|2003-10-13|2003-11-19|Glaxosmithkline Biolog Sa|Immunogenic compositions|
    GB0329146D0|2003-12-16|2004-01-21|Glaxosmithkline Biolog Sa|Vaccine|CN111617107A|2020-04-10|2020-09-04|王全栋|Pharmaceutical composition for treating colitis|
    法律状态:
    2018-01-10| FG| Patent granted|Effective date: 20170926 |
    优先权:
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    GBGB1522329.0A|GB201522329D0|2015-12-17|2015-12-17|Use of adjuvants for the prevention and/or treatment of autoimmune diseases|
    GB1522329.0|2015-12-17|
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