WO2017212018A1 - Methods and pharmaceutical compositions for the treatment of autoimmune inflammatory diseases - Google Patents

Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of autoimmune inflammatory diseases. The Src-family kinase Fyn was the crucial effector for inhibition of SHP-1, while Lyn/Lck were required for its activation downstream of Fc, B-cell and T-cell antigen receptors. The inventors demonstrated that while Lyn-deficient mice aggravate inflammatory diseases, Fyn-deficient mice were protected. Similarly, they showed that subjects with lupus nephritis exhibited a Fyn-associated activatory signature, contrasting with a Lyn-associated inhibitory signature in healthy subjects. In particular, the present invention relates to a method of treating an autoimmune inflammatory disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a Fyn inhibitor.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF AUTOIMMUNE INFLAMMATORY DISEASES

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of autoimmune inflammatory diseases.

BACKGROUND OF THE INVENTION:

Autoimmune and inflammatory diseases present as a major public health problem worldwide. In particular, autoimmune and inflammatory renal diseases, mostly glomerular diseases, are the leading cause of renal transplantation in France and the third leading cause of renal replacement therapy by dialysis after hypertension and diabetes (REIN, Rapport Annuel 2010 (Rein rapports 201 1 , 2012, www.agence-biomedecine.fr)). The most prevalent glomerulonephritis (GN) are linked to dysfunctional immune system such as well-characterized IgA nephropathy (IgA-N or Berger's disease), lupus nephritis (LN) and renal-associated vasculitis such as Anti-Neutrophil Cytoplasmic Antibodies (ANCA) associated vasculitis (AAV). Patients present with a relative silent evolution and late appearance of symptoms, independent of the type of immune involvement. To date, diagnosis is only possible through invasive investigations, mainly renal histology comprising evaluation of the presence of immunoglobulins and complement deposits in the glomeruli. There is thus a medical need for the treatment of autoimmune and inflammatory diseases. To maintain homeostasis the immune system is controlled by a finely tuned network of regulatory mechanism (1). Among these, certains immunoreceptors have been shown to exert inhibitory and activating signal through ITAM motif (Yxx[L/I]x6-sYxx[L/I]) (2) depending on the valency of their respective ligand. Low valency interactions were shown to induce anergy and an inhibitory crosstalk with heterologous receptors thereby reducing the susceptibility to autoimmune and inflammatory diseases (3-10). By contrast, high valency ligand interactions promote an activating signal launching inflammatory and immune cascades to fight the inflammatory insult and restore homeostasis, but in case of ill-regulation or chronic stimulation can also result in autoimmune and inflammatory diseases (1, 10). The ITAM motif is found in the cytoplasmic domain of several transmembrane adapter molecules, such as the common γ subunit of FcR (FcRy), the Iga and Ig subunits of the BCR, and the γ, δ, ε and ζ subunits of the TCR-associated CD3 complex (1, 2), and in the cytoplasmic tail of other receptors, such as the FcyRIIA (11). Src- family kinases (SFKs) phosphorylate the ITAM motifs upon stimulus-induced receptor clustering leading to downstream effector recruitments and cell activation. However, how ITAM-bearing immunoreceptors translate ligand valency into opposite signals remains elusive. It also remains unclear why these receptors are associated with several SFKs, notably Lyn and Fyn (12-17).

SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of autoimmune inflammatory diseases. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Immunoreceptors play a crucial role in the regulation of immune homeostasis and inflammation. Depending on ligand valency they can promote either activating or inhibitory signals. The inventors investigated how these receptors translate outside ligand interactions into opposite signals. The Src-family kinase Fyn was the crucial effector for inhibition of SHP-1 , while Lyn/Lck were required for its activation downstream of Fc, B-cell and T-cell antigen receptors. Mechanistically, a Fyn-PI3K-PKC axis turns off Lyn-mediated SHP-1 activation by shifting tyrosine into serine phosphorylation. Accordingly, while Lyn-deficient mice aggravate inflammatory diseases, Fyn-deficient mice were protected. Similarly, subjects with lupus nephritis exhibited a Fyn-associated activatory signature, contrasting with a Lyn-associated inhibitory signature in healthy subjects. These findings demonstrate the distinct roles of antigen receptor-associated Src family kinases in regulating homeostatic and inflammatory conditions.

Accordingly, the first object of the present invention relates to a method of treating an autoimmune inflammatory disease in a subject in thereof comprising administering to the subject a therapeutically effective amount of a Fyn inhibitor. As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

In some embodiments, the autoimmune inflammatory disease is selected from the group consisting of arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma, systemic scleroderma, sclerosis, systemic sclerosis, multiple sclerosis (MS), spino-optical MS, primary progressive MS (PPMS), relapsing remitting MS (R MS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, and ataxic sclerosis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, transmural colitis, autoimmune inflammatory bowel disease, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis, respiratory distress syndrome, adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, sudden hearing loss, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis, Rasmussen's encephalitis, limbic and/or brainstem encephalitis, uveitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, autoimmune uveitis, glomerulonephritis (GN), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), rapidly progressive GN, allergic conditions, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) or systemic lupus erythematodes such as cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), lupus erythematosus disseminatus, lupus (including nephritis, cerebritis, pediatric, non-renal, extra-renal, discoid, alopecia), juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, including vasculitis, large vessel vasculitis, polymyalgia rheumatica, giant cell (Takayasu's) arteritis, medium vessel vasculitis, Kawasaki's disease, polyarteritis nodosa, microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous, hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospho lipid antibody syndrome, allergic neuritis, Bechet's or Behcet's disease, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens- Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus, optionally pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, pemphigus erythematosus, autoimmune polyendocrinopathies, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathies, chronic neuropathy, IgM polyneuropathies, IgM-mediated neuropathy, thrombocytopenia, thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis); subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis, allergic encephalomyelitis, experimental allergic encephalomyelitis (EAE), myasthenia gravis, thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis, bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease, Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AGED), autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS), polychondritis such as refractory or relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis, optionally benign monoclonal gammopathy or monoclonal gammopathy of undetermined significance, MGUS, peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases, diabetic nephropathy, Dressler's syndrome, alopecia greata, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), and telangiectasia), male and female autoimmune infertility, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis, or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrine ophthamopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia- reperfusion injury, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman- Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired splenic atrophy, infertility due to antispermatozoan antobodies, non-malignant thymoma, vitiligo, SCID and Epstein-Barr virus-associated diseases, acquired immune deficiency syndrome (AIDS), parasitic diseases such as Lesihmania, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex- mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, peripheral neuropathy, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, ischemic re-perfusion disorder, reduction in blood pressure response, vascular dysfunction, antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, reperfusion injury of myocardial or other tissues, dermatoses with acute inflammatory components, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, acute serious inflammation, chronic intractable inflammation, pyelitis, pneumonocirrhosis, diabetic retinopathy, diabetic large-artery disorder, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis.

In some embodiments, the subject suffers from a renal autoimmune inflammatory disease.

In some embodiments, the subject suffers from an allergic disorder. As used herein,

"allergic disorder" refers to any disorder resulting from antigen activation of mast cells that results in an "allergic reaction" or state of hypersensitivity and influx of inflammatory and immune cells. Those disorders include without limitation: systemic allergic reactions, systemic anaphylaxis or hypersensitivity responses, anaphylactic shock, drug allergies, and insect sting allergies; respiratory allergic diseases, such asthma, hypersensitivity lung diseases, hypersensitivity pneumonitis and interstitial lung diseases (ILD) (e.g. idiopathic pulmonary fibrosis, ILD associated with rheumatoid arthritis, or other autoimmune conditions); rhinitis, hay fever, conjunctivitis, allergic rhinoconjunctivitis and vaginitis; skin and dermatological disorders, including psoriasis and inflammatory dermatoses, such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, dermatitis herpetiforms, linear IgA disease, acute and chronic urticaria and scleroderma; vasculitis (e.g. necrotizing, cutaneous, and hypersensitivity vasculitis); spondyloarthropathies; and intestinal reactions of the gastrointestinal system (e.g., inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, ileitis, enteritis, nontropical sprue and celiac disease).

In some embodiments, the subject suffers from asthma. As used herein, the term "asthma" refers to an inflammatory disease of the respiratory airways that is characterized by airway obstruction, wheezing, and shortness of breath.

In some embodiments, the subject suffers from anaphylaxis. As used herein, the term "anaphylaxis" refers to a life threatening allergic reaction characterized by decreased blood pressure, respiratory failure with bronchoconstriction, and skin rash due to release of mediators from cells such as mast cells.

In some embodiments, the autoimmune inflammatory diseases is secondary to therapeutic treatment, in particular a treatment with an immune checkpoint inhibitor. As used herein, the term "immune checkpoint inhibitor" has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein. Inhibition includes reduction of function and full blockade. Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. In some embodiments, the immune checkpoint inhibitor is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-PD-L2 antibodies anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies.

As used herein, the term "Fyn" has its general meaning in the art and refers to FYN proto-oncogene, Src family tyrosine kinase encoded by the FYN gene (Gene ID: 2534) also known as SLK; SYN; or p59-FYN. An exemplary human nucleic acid sequence is accessible in GenBank under the accessible number NM 002037.5 (isoform a), NM l 53047.3 (isoform b) or NM 153048.3 (isoform c). An exemplary human amino acid sequence is accessible in GenBank under the accessible number NP 002028.1 (isoform a), NP 694592.1 (isoform b) or NP 694593.1 (isoform c). Accordingly a "Fyn inhibitor" refers to any compound that is capable of inhibiting the activity or expression of Fyn. As used herein, the term "Fyn activity" includes any biological activity mediated by Fyn such as described in the EXAMPLE. In particular, the Fyn inhibitor of the present invention is particular suitable for abrogating kinase activity. Examples of Fyn inhibitors include but are not limited to polypeptides such as dominant- negative protein mutants, peptidomimetics, antibodies, ribozymes, antisense oligonucleotides, or other small molecules which specifically inhibit the activity or expression of Fyn.

In particular, the Fyn inhibitor is particularly suitable for inhibiting the activation downstream of Fc, B-cell and T-cell antigen receptors. More particularly, the Fyn inhibitor is particularly suitable for restoring and maintaining homeostasis of the immune system.

Small molecule inhibitors of Fyn activity include PP1 (4-Amino-5-(4-methylphenyl)-7- (t-butyl)pyrazo lo [3 ,4-d]pyrimidine) .

In some embodiments, the inhibitor is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of Fyn. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide- long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene. Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siR A, shR A or ribozyme nucleic acid to the cells and typically mast cells. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

In some embodiments, the inhibitor is an intrabody having specificity for Fyn. As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In another embodiment, the antibody according to the invention is a single domain antibody. The term "single domain antibody" (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called "nanobody®". According to the invention, sdAb can particularly be llama sdAb.

By a "therapeutically effective amount" of the inhibitor as above described is meant a sufficient amount to provide a therapeutic effect. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day. According to the invention, the inhibitor is administered to the subject in the form of a pharmaceutical composition. Typically, the inhibitor may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer 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. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain 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 pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The inhibitor can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

A further object of the present invention relates to a method of screening a drug suitable for the treatment of an autoimmune inflammatory diseases comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression or activity of Fyn.

Any biological assay well known in the art could be suitable for determining the ability of the test compound to inhibit the activity or expression of Fyn. In some embodiments, the assay fist comprises determining the ability of the test compound to bind to Fyn. In some embodiments, a population of immune cells (mastocytes, monocytes, B cells, T cells...) is then contacted and activated so as to determine the ability of the test compound to inhibit the activation downstream of Fc, B-cell and T-cell antigen receptors. In some embodiments, the SHP-1 activation is determined and in particular the identification of serine into tyrosine phosphorylation. In particular, the effect triggered by the test compound is determined relative to that of a population of immune cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity or expression. Assays for determining the test compound to dampen the immune signal response are well known in the art and are typically described in the EXAMPLE. It is to be understood that test compounds capable of inhibiting immune response, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is selected from the group consisting of peptides, petptidomimetics, small organic molecules, antibodies (e.g. intraantibodies), aptamers or nucleic acids. For example the test compound according to the invention 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. In some embodiments, the test compound may be selected form small organic molecules. As used herein, the term "small organic molecule" refers to a molecule of size comparable to those organic molecules generally sued in pharmaceuticals. The term excludes biological macromolecules (e.g.; proteins, nucleic acids, etc.); preferred small organic molecules range in size up to 2000da, and most preferably up to about 1000 Da.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

Figure 1. Fyn aggravates spontaneous autoimmune nephritis development in FcyRIIA^T mice. (A) Body weight. (B) Serum BUN. (C) Representative photographs of one- year-old mice demonstrating that the absence of Fyn prevents FcyRIIA-mediated glomerulopathy with marked reduction in IgG deposits and fibrosis (H&E and Masson's stain). Scale Bars: 200 μπι. (D) Detection of CD1 lb⁺, F4/80⁺and CD3⁺ cells in kidney sections of WT, hFcyRIIA^Tg and Fyn^_/" hFcyRIIA^Tg mice by IHC. Quantification of positive cells is indicated on right panels. Scale Bars: 200 μιη. (*P<.01; Mann- Whitney test). Sections of five animals/group were automatically quantified with the software CaloPix piloted by an independent pathologist. (E) Relative mRNA expression of the indicated cytokines assessed by q-PCR on independent kidney tissue RNA samples (n = 5). *P< 05, **P< 01, ***P< 001; Mann- Whitney test, ns, non significant. Data from at least five mice per group. Data are presented as the mean ± s.e.m. (F) Analysis of phosphorylated ITAM effectors in blood leukocyte samples obtained from four lupus nephritis patients (LN) (1 and 2 are class IV; 3 and 4 are class V) and four healthy individuals (HI). Left panel, cell lysates were immunoblotted using indicated Abs. Right panel, FcyRII immunoprecipitation (IP) with an anti-FcyRIIA monoclonal antibody (IV.3) followed by immunob lotting using indicated antibodies. These results were confirmed in two additional LN patients and four HI.

EXAMPLE:

Material & Methods

Study subjects.

Fourteen individuals (eight HI and six LN patients) were studied. The LN group was composed of 6 patients attending or referred to the Bichat' s Hospital specialist nephrology unit between July 2014 and January 2016 meeting at least four ACR systemic lupus erythematosus criteria (32) presenting with active disease with nephritis proven by kidney biopsy (2 at class IV and 4 at class V) and in whom peripheral blood by venepuncture was obtained immediately prior to immunosuppressive therapy administration. All patients were female with age varying between 25 and 42. Ethical approval for this study was obtained from the Bichat Hospital Local Research. Ethics Committee and informed consent was obtained from all subjects enrolled.

Mice

C57BL/6 hFcyRIIA^Tg mice expressing the WT human FcyRIIA on CD 1 lb-positive cells were from Jackson Laboratory (JAX, Bar Harbor, ME, USA). Fyn ' hFcyRIIA¹⁸, Lyn^_/" hFcyRIIA^Tg were obtained by the intercross of hFcyRIIA^Tg mice with mice knockout for Fyn (JAX) or for Lyn (previously described in (15)). All mice carrying the hFcyRIIA transgene were used as heterozygous animals. Mice were bred and maintained at the mouse facilities of the Bichat Medical School campus. All experiments were performed in accordance with the French Council of Animal Care guidelines and national ethical guidelines of INSERM Animal Care Committee (Animal Use Protocol number 75-1596).

Nephrotoxic nephritis (NTN) mouse model.

NTN was induced by i.p. injection (200 μ1/20 g body weight) of rabbit anti-mouse glomerular basement membrane (GBM) in C57BL/6 hFcyRIIA^Tg, Lyn^_/", Fyn^_/", Fyn^_/" hFcyRIIA^Tg, Lyn ' hFcyRIIA^ mice (7 to 9 wk old). Briefly, mice were preimmunized i.p. with normal rabbit IgG (0.5 mg/20 g body weight) in CFA 5 days prior to i.p. administration of NTN serum. Blood samples were collected and animals were sacrificed at day 7 following NTN injection. Renal function parameters (urinary proteins and BUN), histological and immunohistological parameters were studied.

Collagen Antibody-Induced Arthritis (CAIA) model.

Arthritis was induced as described (9, 30) using the Arthrogen-CIA® Arthritogenic Monoclonal Antibody kit (Chondrex, Inc.). Mice were injected i.v. with anti-CII Ab cocktail (Day 0) followed by LPS (i.p.) 3 days later. Animals were injected i.p. with 10 mg/20 g body weight of 500 μg serum human IgA (purchased from Biomedicals)/20 g body weight or 100 μg AT-10 F(ab)'₂ or irrelevant mAb F(ab')₂ (clone 320) for 10 days at 2-day intervals. The first dose was administered 2 days prior to anti-CII Ab cocktail injection. Paw thickness was measured with a pocket thickness gauge. On day 10, animals were sacrificed and hind paws and knees were fixed in formalin or snap-frozen.

Cells and reagents.

Human blood samples (12 ml) were first submitted to red cell lysis and pellets of 10⁷ leukocytes were subjected RIPA buffer treatment (see below). BMM from 6- to 8-week-old mice were obtained after a 7-day culture with M-CSF (R&D systems). THP-1 and THP1- hFcyRIIA-R131⁺-CD14⁺ cell lines (kindly provided by Novimmune) (33) were maintained in RPMI-1640, 10% FCS and 50 μΜ β-mercaptoethanol or supplemented with 200 μg/ml Zeocin, 10 μg/ml blasticidin and 2 μg/ml puromycin (Invitrogen, France). Jurkat and Ramos human cell lines were maintained in RPMI-1640 supplemented with 10% FCS and antibiotics. FCS was removed from the culture medium immediately before stimulation as described (34). Mouse mAb anti-hFcyRII (clone AT-10), anti-hCD3 (clone HIT-3a) or anti-hCD79a (clone ZL7-4) were purchased from Santa Cruz and used in their F(ab')₂ fragment forms. Mouse mAb anti-hFcaRI (clone A77) and irrelevant control mAb (320) were purified in-house and were used as F(ab')₂, as previously described (3, 9). For biochemical studies, rabbit anti-Syk, anti- Zap70, anti-SHP-1 , anti-Lyn, anti-Lck, anti-Fyn, anti-ER (all from Santa Cruz Biotechnology), anti-SHPl (phospho-Y536) (ECM Biosciences), and anti-SHPl (phospho- S591) (Abeam) were used. Anti-pERK, anti-pAKT, anti-pPKCa, anti-AKT and anti-PKCa were from Cell Signaling.

Cell stimulation.

For ITAMi signaling, 5xl0⁶ of monocytic cell lines (THP-1 -CD 14⁺-FcyRIIA⁺ or THP-

1-CD14⁺), Jurkat T cell and Ramos B cell lines (transfected with different siRNA) were incubated for 30 min with 10 μg/mL of anti-hFcyRII (clone AT-10), anti-hCD3 (clone HIT-3a) or anti-hCD79a (clone ZL7-4) Abs F(ab')₂ fragments at 37°C, respectively. Cells were then incubated with or without LPS (10 ng/ml) and Flagellin (5 μ /ηι1) as described (35) or Pam3csk4 (1 μ /ηι1) for 1 hour for monocytic cell lines and 18 hours for Jurkat and Ramos. For ITAM signaling, cells were incubated with 10 μg/ml of anti-hFcyRII (clone AT- 10), anti-hCD3 (clone HIT-3a) or anti-hCD79a (clone ZL7-4) Abs F(ab')₂ at 4°C followed by an anti-κ light chain F(ab')₂ at 37° C for 18 h for cytokine measurement or 6 hours for intracellular IL-2 staining.

Analysis of intracellular cytokine staining.

After washing, Jurkat cells were incubated with or without anti-CD3 F(ab')₂ fragment or with preformed complexes of anti-CD3 F(ab')₂ plus anti-kappa F(ab')₂ fragments. Cells were then stimulated or not with flagellin (1 μg) for 6 hours. PMA (40 nM) and ionomycin (InM) were used as positive stimuli for 6 hours. Brefeldin A was added after 2 hours stimulation and maintained for 4 hours. The stimulation was stopped by adding 1 ml cold PBS. Intracellular cytokine staining was performed on fixed/permeabilized cells in residual permeabilization wash buffer (Biolegend, USA) using a conjugated antibody (anti-IL-2 PE or appropriate isotype control) for 20 min in the dark at room temperature as described (36). Data acquisition was performed using a BD Biosciences LSR Fortessa cytometer, and results were analyzed using Flow Jo analysis software (Tree Star).

Immunoprecipitation and immunoblotting.

Cells (5 x 10⁶ to 10⁷) were solubilized in RIPA lysis buffer containing 1% Nonidet P- 40/0.1% sodium dodecyl sulfate (SDS) as described⁸. For immunoprecipitation, cell lysates were incubated with 2 μg/ml of AT- 10 anti-FcyRIIA, A77 anti-FcaRI, HIT-3a anti-CD3 or ZL7-4 anti-CD79a mAbs and immunoprecipitated overnight at 4°C with Protein G-Sepharose (GE Healthcare). Samples were resolved by SDS polyacrylamide gel electrophoresis (10%), transferred to nitrocellulose membranes and immunob lotted with rabbit antibodies followed by goat anti-rabbit IgG (GE Healthcare) coupled to horseradish peroxidase. Membranes were developed by enhanced chemical luminescence treatment (Amersham Biosciences).

Enzyme-linked immunosorbent assay (ELISA).

IL-8 and IL-2 were measured in the supernatants of stimulated cells using ELISA kits (R&D Systems) according to the manufacturer's instructions.

Histological analysis.

For the kidney, paraffin-embedded sections 4 μιη in thickness were stained with PAS for morphological analysis. For immunohistochemistry, frozen kidney sections were incubated with biotinylated antibodies against rabbit IgG or mAb anti-mouse CD l ib, -mouse F4/80, - mouse CD3, and -mouse Ly6G (Becton Dickinson) for 1 hour at room temperature. When necessary, the primary antibody incubation was followed by incubation with anti-rabbit IgG or anti-goat IgG (Southern Biotech Associates). Slides were mounted with the Eukitt mounting medium (Electron Microscopy Sciences) and read with an upright microscope (DM2000; Leica) using the IM50 software (Leica). For colocalization experiments, frozen kidney sections were incubated overnight successively with each antibody (anti-phospho Syk^Y525"526 AF488 (Cell Signaling) and anti-phalloidin AF568 (Life Technologies) followed by incubation with streptavidin-Alexa Fluor 488 (Life Technologies) at room temperature. Tissue sections were mounted with Vectashield (Vector Laboratories). Slides were read with a laser-scanning confocal microscope (LSM 510; Carl Zeiss) at 630 magnification using the LSM Image Browser (Carl Zeiss). For formalin- fixed hind paws and mouse knees, the samples were decalcified and embedded in paraffin. Sagittal sections were stained with haematoxylin and eosin. Histological score of peri-articular exudates, synovitis and tissue loss were graded as 0 (normal), 3 (mild), 6 (moderate), or 9 (severe). The cumulative score of all features was used as histological score to represent overall disease severity. Sections were incubated with rabbit primary antibodies against mouse phospho (p)-Syk (Y525-526)(J7), mouse p-SHPl (S591) (both from Abeam) or p-SHPl (Y536) (Assay Biotech). The presence of antibodies was detected with biotinylated anti-rabbit antibody (Southern Biotech) followed by peroxidase streptavidin and diaminobenzidine successive stains. Positive cells were quantified in hind paw sections with analysis software CaloPix (TRIBVN, Chatillon, France) after scanning with Aperio ScanScope CS (Leica Biosystems, Nanterre, France).

Gene expression and Real-time PCR.

RNA purification from ho mogen i zed_k i d ney s was performed by using RNAble (Eurobio). cDNA was obtained by reverse transcription using using Moloney murine leukaemia virus (Invitrogen). Samples were analyzed by real-time PCR with Taq Man® Gene Expression Master Mix (Applied Biosystem). Primers were purchased from Eurofms (Supplementary Table 1). Gene quantification was performed using a Chrom o4 Real-Time PCR Detection System (Bio-Rad Laboratories). Data were normalized to β-actin values.

siRNA transfections.

Experiments were performed using predesigned HP GenomeWide (Qiagen, Courtaboeuf, France) Single strand sense and antisense RNA nucleotides were annealed to generate an RNA duplex according to the manufacturer's instructions. Cells were incubated with 5 to 10 nM of each siRNA tested and 2 μΐ of Lipofectamine® RNAiMAX prepared according to the manufacturer's instructions (Invitrogen, Saint Aubin, France) for 48 or 72 hours at 37°C before use. Primers and probe sequences for RT-qPCR.

Gene, accession number, sequence and location of primers and probes. F indicates a forward primer, R indicates a reverse primer, and P indicates a FAM-TAMRA probe.

Statistical analysis.

Statistical analyses were performed using GraphPad Prism software http://www.graphpad.com/scientific-software/prism/. All data were expressed as mean ± SEM. Statistical significance between two groups was examined by the Student's t-test or the Mann- Whitney test, while the one-way and two-way analysis of variance (ANOVA) with Bonferroni's, Holm-Sidak's or Newman-Keuls multiple comparisons test were used to evaluate multiple groups. -values of 0.05 were considered significant; values less than 0.05 are indicated in the figure legends.

Results and discussion:

To maintain homeostasis the immune system is controlled by a finely tuned network of regulatory mechanism (1). Among these, certains immunoreceptors have been shown to exert inhibitory and activating signal through IT AM motif (Yxx[L/I]x6-sYxx[L/I]) (2) depending on the valency of their respective ligand. Low valency interactions were shown to induce anergy and an inhibitory crosstalk with heterologous receptors thereby reducing the susceptibility to autoimmune and inflammatory diseases (3-10). By contrast, high valency ligand interactions promote an activating signal launching inflammatory and immune cascades to fight the inflammatory insult and restore homeostasis, but in case of ill-regulation or chronic stimulation can also result in autoimmune and inflammatory diseases (1, 10). The IT AM motif is found in the cytoplasmic domain of several transmembrane adapter molecules, such as the common γ subunit of FcR (FcRy), the Iga and Ig subunits of the BCR, and the γ, δ, ε and ζ subunits of the TCR-associated CD3 complex (1, 2), and in the cytoplasmic tail of other receptors, such as the FcyRIIA (11). Src-family kinases (SFKs) phosphorylate the ITAM motifs upon stimulus- induced receptor clustering leading to downstream effector recruitments and cell activation. However, how ITAM-bearing immunoreceptors translate ligand valency into opposite signals remains elusive. It also remains unclear why these receptors are associated with several SFKs, notably Lyn and Fyn (12-17). In order to address whether SFKs play a role in the induction of ITAMi or ITAM signaling, we downregulated Fyn or Lyn expression in representative human monocytic cell lines. These cells were either stimulated for ITAMi induction by divalent targeting, or for ITAM by multivalent crosslinking of FcyRIIA or FcaRI. ITAMi molecular signature was characterized by transient Syk recruitment followed by stable and prolonged SHP-1 recruitment. This signature required the presence of Lyn (but not Fyn), which were recruited to their respective immunoreceptors. In contrast, multivalent crosslinking of these receptors induced an IT AM molecular signature with a stable recruitment of Syk family kinases (but not SHP1), requiring the presence of recruited Fyn. Fyn silencing had no effect on ITAMi signaling but reversed the ITAM to an ITAMi signature, whereas Lyn silencing completely abolished ITAMi signaling without affecting ITAM. Functional consequences were then evaluated. Lyn but not Fyn is essential for the ITAMi inhibitory signals observed in FcR- mediated inhibition of LPS -mediated IL-8 production. By contrast, Fyn but not Lyn is essential for ITAM-mediated cell activation as measured by IL-8 production after multivalent engagement of FcyRIIA or FcaRI. To investigate whether other ITAM-bearing receptors could also deliver such opposite signals, divalent or multivalent targeting of TCR and BCR were performed using anti-CD3 or anti-CD79a F(ab')₂ fragments or complexed with anti-κ light chains in representative lymphocytic cell lines. Similarly to FcRs, while divalent targeting of TCR or BCR resulted in ITAMi signatures, multivalent crosslinking led to expected ITAM signature. As for FcR, Lck or Lyn was required for TCR- or BCR-mediated ITAMi signals, whereas Fyn was essential for ITAM configuration. Moreover, TCR-Fyn dissociation led to SHP-1 recruitment suggesting that Fyn could inhibit SHP-1 recruitment. We next evaluated the effect of such opposite signals on cytokine production. Whereas, TCR- and BCR-divalent targeting resulted in inhibition of IL-2 and IL-8 secretion induced by flagellin or Pam3csk4 which were dependent on Lck or Lyn, respectively, Fyn was required for these cytokine production induced upon TCR and BCR multivalent engagement. These results were confirmed by intracellular IL-2 detection on the Jurkat T cell line. Altogether, these data indicated that during ITAM signaling, Fyn would constrain the inhibitory signal. Strikingly, these results also extend to TCR and BCR that ITAM-bearing immunoreceptors undergo crosstalk with other receptors, thus dampening inflammatory responses through ITAMi signaling generated by low intensity signals (18). In addition, these results reveal that this effect is strictly regulated through the balance between non-redundant SFKs. These results mechanistically explain previous findings using weakly binding ligands or antibodies that trigger negative signals by TCR and BCR (19-23).

To address the mechanism by which Fyn deletion reverses ITAM to ITAMi, we explored a possible link between SHP-1 and Fyn under ITAM configuration. The inhibition observed under ITAM conditions in the absence of Fyn was reversed when SHP-1 was silenced and LPS-mediated IL-8 production was restored, suggesting that Fyn abrogated an inhibitory signal via the inactivation of SHP-1. Given that Y⁵³⁶ and S⁵⁹¹ residues are essential for SHP-1 activation/inhibition (24, 25), we analyzed whether Lyn and Fyn controlled SHP-1 function through differential phosphorylation of these residues by taking advantage of bone marrow- derived macrophages (BMM) from transgenic mice with the pro-autoimmune R131 isoform of the FcyRIIA (26). Whereas Lyn was responsible for Y536 phosphorylation of SHP-1 during FcyRIIA-ITAMi signaling, Fyn was responsible for S⁵⁹¹ phosphorylation of SHP-1 during FcyRIIA-ITAM signaling. Moreover, regarding the latter, the absence of Fyn resulted in Lyn- dependent Y⁵³⁶ phosphorylation of SHP-1, thus mimicking an ITAMi signal. To understand how Fyn, a tyrosine kinase, promotes SHP-1 phosphorylation on serine, we performed FcyPJIA-ITAM stimulation in the presence or absence of ER , PKC and PI3K inhibitors (14, 27). Whereas PI3K and PKC inhibitors completely blocked both SHP-1^S591 and PKC phosphorylation, the ERK inhibitor had no effect. In addition, PI3K and PKC inhibitors favoured SHP-1 ^Y536 phosphorylation under conditions of FcyRIIA-ITAM signalling, and this preference required the presence of Lyn. Together, these results indicate that during IT AM signaling, Fyn-mediated inactivation of SHP-1 through phosphorylation of the serine residue 591 by a PI3K-PKCa axis, thereby blocking its activation by Lyn.

To investigate the functional role of Lyn and Fyn in the regulation of IT AM signals in vivo, we first used a mild immune-complex nephritis model, the nephrotoxic nephritis (NTN), via administration of a rabbit serum anti-mouse glomerular basement membrane. This mild model was used because FcyRIIA^Tg and Lyn^_/~ mice have a well-established autoimmune phenotype (26, 28, 29). Consistent with the above-described role of Lyn in maintaining ITAMi- mediated immune homeostasis, NTN serum induced severe acute nephritis associated with high mortality one week after i.p. injections into Lyn^"/_FcyRIIA^Tg recipients only, whereas Lyn^_/", Fyn ^1' or Fyn ^1' FcyRIIA^Tg mice did not develop significant disease despite glomerular Ab deposits. Renal disease development in Lyn^_/~ FcyRIIA^Tg mice was characterized by a marked increase in urinary protein and blood urea nitrogen concentration (BUN). Lyn^"/_FcyRIIA^Tg mice also exhibited severe renal injury involving extensive mesangial and capillary (subendothelial or even intracapillary) deposits associated with mild mesangial and endocapillary plus extracapillary proliferation. Glomerular lesions were characterized by an intense macrophage infiltrate and cytokine production. These effects involved activation of ITAM signaling, as demonstrated by in situ phosphorylation of the Y⁵²⁵ residue in Syk. The protective role of Lyn was confirmed in another autoimmune model, the collagen antibody-induced arthritis (CAIA) model. Here, targeting human FcyRIIA or FcaRI in Tg animals for ITAMi signaling (by AT- 10 F(ab')₂ or monomeric IgA (9, 30)) prevented disease development, and this protection required the presence of Lyn. In contrast, the aggravating role of Fyn was highlighted by the absence of lesions in both Fyn^FcyRIIA^ and Fyn ^/'FcoU ^Tg mice. Importantly, tissue analysis of phosphorylated kinases and phosphatases in joints of arthritic mice revealed that Lyn may protect the host against autoimmunity by constitutive phosphorylation of SHP-1^Y536 residue, whereas Fyn favours autoimmunity development by inactivation of SHP-1 through the phosphorylation of the SHP-1^S591 residue associated with induction of Syk^Y525~526 phosphorylation. Altogether, these results suggest that non-redundant SFKs determine the ITAMi/ITAM balance that governs the outcome of inflammatory and autoimmune responses.

To elucidate whether a naturally occurring inflammatory disease is influenced by Fyn, we first analyzed whether pro-autoimmune R131 FcyRIIA^Tg mice could develop overtime spontaneous nephritis as described for spontaneous autoimmune arthritis (26) and compared with animals deficient for Fyn. While 50-week-old FcyRIIA^Tg mice developed end-stage chronic kidney disease (with enhanced body weight, increased BUN levels, glomerular IgG deposits, sclerotic glomeruli, and fibrosis and kidney interstitial infiltration by CD l ib, F4/80 and CD3-positive cells), 50-week-old Fyn 'FcyBIIA⁷⁸ mice were protected (Fig. 1 , A to D). This protective effect was associated with decreased levels of chemokines and mRNA expression coding for proinflammatory cytokines (Fig. IE). Thus, the Fyn kinase is essential for R131 FcyRIIA-mediated autoimmune disease development. Finally, we analysed the steady-state activation of different IT AM signaling effectors in blood leukocytes from untreated lupus nephritis (LN) patients with severe disease clinically and morphologically classified as class IV (immune-active nephritis) and class V (less-active nephritis with membranous immune deposits only) compared with healthy individuals (HI) (Fig. IF). Consistent with the role of Lyn in homeostasis, Lyn was strongly associated with FcyRIIA in healthy individuals but weakly associated in patients, whereas Fyn and Syk were exclusively associated with FcyRIIA in LN patients. In contrast, SHP-1 and pSHP-l^Y536 were associated with FcyRIIA exclusively in cells from healthy individuals. Strikingly, pSHP- 1 ^S591 and pPKCa were exclusively observed in LN patient cell lysate samples, thus emphasizing an ITAM configuration. However, this inactive pSHP-l^S591 and pPKCa were not associated with FcyRIIA (Fig. IF, right panel). Taken together, our observations demonstrate a balance of SFK fulfilling opposite signaling functions in FcR, TCR and BCR

immunoreceptor signaling. Lyn or Lck are crucial to maintain ITAMi-mediated homeostasis, whereas Fyn is essential for ITAM-mediated cell activation by inducing the PI3K- PKC signaling axis that inactivates SHP-1 during inflammatory responses for most immunoreceptors. Under chronic pathogenic immunoreceptor triggering, this activating loop may amplify inflammatory processes aggravating autoimmune or inflammatory diseases.

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Claims

CLAIMS:

A method of treating an autoimmune inflammatory disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a Fyn inhibitor.

The method of claim 1 wherein the autoimmune inflammatory disease is selected from the group consisting of arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma, systemic scleroderma, sclerosis, systemic sclerosis, multiple sclerosis (MS), spino-optical MS, primary progressive MS (PPMS), relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, and ataxic sclerosis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, transmural colitis, autoimmune inflammatory bowel disease, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis, respiratory distress syndrome, adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, sudden hearing loss, IgE- mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis, Rasmussen's encephalitis, limbic and/or brainstem encephalitis, uveitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phaco antigenic uveitis, posterior uveitis, autoimmune uveitis, glomerulonephritis (GN), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), rapidly progressive GN, allergic conditions, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) or systemic lupus erythematodes such as cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), lupus erythematosus disseminatus, lupus (including nephritis, cerebritis, pediatric, non-renal, extra-renal, discoid, alopecia), juvenile onset (Type I) diabetes mellitus, including pediatric insulin- dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, including vasculitis, large vessel vasculitis, polymyalgia rheumatica, giant cell (Takayasu's) arteritis, medium vessel vasculitis, Kawasaki's disease, polyarteritis nodosa, microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous, hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen- antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospho lipid antibody syndrome, allergic neuritis, Bechet's or Behcet's disease, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens- Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus, optionally pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, pemphigus erythematosus, autoimmune polyendocrinopathies, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathies, chronic neuropathy, IgM polyneuropathies, IgM-mediated neuropathy, thrombocytopenia, thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis); subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis, allergic encephalomyelitis, experimental allergic encephalomyelitis (EAE), myasthenia gravis, thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis, bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease, Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AGED), autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS), polychondritis such as refractory or relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis, optionally benign monoclonal gammopathy or monoclonal garnmopathy of undetermined significance, MGUS, peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases, diabetic nephropathy, Dressler's syndrome, alopecia greata, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), and telangiectasia), male and female autoimmune infertility, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post- cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis, or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrine ophthamopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired splenic atrophy, infertility due to antispermatozoan antobodies, non-malignant thymoma, vitiligo, SCID and Epstein-Barr virus-associated diseases, acquired immune deficiency syndrome (AIDS), parasitic diseases such as Lesihmania, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, peripheral neuropathy, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil- related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia- myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott- Aldrich syndrome, ataxia telangiectasia, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, ischemic re-perfusion disorder, reduction in blood pressure response, vascular dysfunction, antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, reperfusion injury of myocardial or other tissues, dermatoses with acute inflammatory components, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, acute serious inflammation, chronic intractable inflammation, pyelitis, pneumonocirrhosis, diabetic retinopathy, diabetic large-artery disorder, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis.

3. The method of claim 1 wherein the subject suffers from a renal autoimmune inflammatory disease, an allergic disorder asthma, anaphylaxis or an autoimmune inflammatory disease which is secondary to therapeutic treatment, in particular a treatment with an immune checkpoint inhibitor.

4. The method of claim 1 wherein the Fyn inhibitor is used for inhibiting the activation downstream of Fc, B-cell and T-cell antigen receptors.

5. The method of claim 1 wherein the Fyn inhibitor is a small organic molecule.

6. The method of claim 1 wherein the Fyn inhibitor is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of Fyn.

7. A method of screening a drug suitable for the treatment of an autoimmune inflammatory diseases comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression or activity of Fyn.