WO2015032841A2 - Methods and pharmaceutical compositions for prevention or treatment of th17 mediated disease - Google Patents

Abstract

The present invention relates to methods and compositions for the prevention or treatment of Th 17 mediated disease.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR PREVENTION OR TREATMENT OF TH17 MEDIATED DISEASE

FIELD OF THE INVENTION:

The present invention relates to methods and compositions for the prevention or treatment of Thl7 mediated disease.

BACKGROUND OF THE INVENTION:

There is a need to develop new drugs that will be suitable for preventing or treating

Thl7 mediated disease. In this way, it has been suggested that characterisation of new therapeutic targets in Thl7 related disease may be highly desirable.

Calpains are cytosolic, calcium- activated neutral cysteine proteases (1, 2). Two ubiquitously expressed iso forms, calpain μ or 1 and m or 2, require micromolar and millimolar Ca2+ concentrations, respectively, for an activity which is tightly controlled by calpastatin (3). Calpains play an important pro -inflammatory role (4). They are involved in the NF-KB-dependent expression of pro -inflammatory cytokines and adhesion molecules. Calpains are also critical for inflammatory cell adhesion and chemotaxis, and inflammatory mediator processing. It was previously demonstrated that calpains are implicated in the cleavage of the heat shock protein 90, which is required to maintain glucocorticoid receptor in a ligand binding conformation and, thereby, to settle anti- inflammatory effects of glucocorticoids (5). Calpains participate in immune responses as well. T lymphocyte activation after T cell receptor (TCR) / CD3 complex engagement increases calpain expression (6, 7). In turn, calpain activity is involved in T cell migration (8), secretion of interleukin-2 (IL-2) and cell surface expression of IL-2 receptor subunit alpha (CD25) (9), secretion of interferon-γ (IFN- γ) and TH1 commitment (10), and secretion of IL-17 and TH17 commitment (11).

Although calpains are considered as intracellular enzymes, a few studies show that they are partly externalized. Calpains are secreted by lymphocytes, endothelial cells, chondrocytes, and osteoblasts, among other cells (6, 12). Calpain secretion is thought to be in an unconventional way due to the lack of N-terminal classic secretion signal peptide. Interestingly, a novel hypothesis proposes that unconventional secretion provides a mechanism through which the consequences of a single enzymatic activity differ dramatically according to intracellular or extracellular localization (13). This notion is strengthened by the observation that, when externalized, calpains seem to promote resolution and tissue repair instead of inflammation/immunity development. For instance, externalized calpains activate anti- inflammatory cytokines (TGF-β) (14) and inactivate proinflammatory proteins (chemerins) (15). In addition, it was demonstrated that extracellular calpains participate in both epithelium and endothelium regeneration after ischemia/inflammation (16, 17). The opposite activities of intra- and extra-cellular calpains raise questions regarding the mechanisms of calpain secretion.

There is no disclosure in the art showing that calpain externalization protects against inflammation by inhibiting IL-17 expression through the cleavage of TLR2 and limiting Thl7 commitment, nor the use of ABCA1 agonist for treatment of Thl7 related diseases, nor the use of ABCAl agonist for inducing calpain externalization, nor the use of compound that enhance calpain externalization for the treatment of Thl7 mediated diseases.

SUMMARY OF THE INVENTION:

The present invention relates to a compound which is selected from the group consisting of ABCAl agonists or antagonists, ABCAl expression activators or inhibitors, exogenous calpain or modified calpain, exogenous calpastatin or calpain inhibitors for use in the prevention or treatment of Thl7 mediated disease in a subject in need thereof.

DETAILED DESCRIPTION OF THE INVENTION:

The role of calpain externalization in inflammation was investigated by the inventors using human and mice purified T lymphocytes, ABCAl inhibitory drugs and ABCAl agonists. The inventors surprisingly found that calpain externalization protect against inflammation by inhibiting IL-17 expression and limiting Thl7 commitment. The regulatory role of extracellular calpains on IL-17 expression involves a cleavage of TLR2 extracellular domain. The inventors also demonstrate that T lymphocytes secrete calpain through an ABCA-1 -dependent pathway. The inventors also demonstrated that T lymphocytes secrete main calpain subunits in micro vesicles. Therapeutic methods and uses

Accordingly the present invention relates to a compound which is selected from the group consisting of ABCAl agonists or antagonists, ABCAl expression activators or inhibitors, exogenous calpain or modified calpain, exogenous calpastatin or calpain inhibitors for use in the prevention or treatment of Thl7 mediated disease in a subject in need thereof.

In one embodiment, the present invention relates to a compound which is selected from the group consisting of ABCAl agonists, ABCAl expression activators, exogenous calpain or modified calpain for use in the prevention or treatment of Thl7 mediated disease in a subject in need thereof, wherein said subject has been diagnosed with Thl7 cells bad prognosis in said disease.

Thl7 cells and IL17 have been shown to be critical in host defense against extracellular bacteria and fungi, especially at mucosal and barrier sites (Miossec P, Kolls JK. Targeting IL-17 and TH17 cells in chronic inflammation. Nat Rev Drug Discov. 2012 Oct;l l(10):763-76; Kim JS, Jordan MS. Diversity of IL-17-producing T lymphocytes. Cell Mol Life Sci. 2013 Jul;70(13):2271-90). Accordingly, in another embodiment, the present invention relates to a compound which is selected from the group consisting of ABCAl antagonists, ABCAl expression inhibitors, exogenous calpastatin or calpain inhibitors for use in the prevention or treatment of Thl7 mediated disease in a subject in need thereof, wherein said subject has been diagnosed with Thl7 cells good prognosis in said disease.

Exogenous calpain or modified calpain, exogenous calpastatin or calpain inhibitors of the present invention are compounds that remain in the extracellular milieu.

As used herein, the term "subject" denotes a mammal. In a preferred embodiment of the invention, a subject according to the invention refers to any subject (preferably human) afflicted or at risk to be afflicted with Thl7 mediated diseases. In another preferred embodiment, a subject according to the invention refers to any subject (preferably human) afflicted or at risk to be afflicted with TLR2 mediated diseases. The method of the invention may be performed for any type of Thl7 mediated disease such as inflammation, inflammatory-related diseases, cancer, autoimmune diseases, graft- versus-host-disease, graft rejection and atherosclerosis.

The method of the invention may be performed for any type of inflammation or inflammatory-related diseases such as inflammation due to trauma, toxins, neoplasia or microbial invasion, diabetes, obesity, atherosclerosis, infectious diseases, viral diseases, cataracts, reperfusion injury, cancer, sarcoidosis, post-infectious meningitis, rheumatic fever; rheumatic diseases comprising systemic lupus erythematosus, osteoarthritis and rheumatoid arthritis; skin diseases comprising various forms of acne, psoriasis, cicatrisation and rosacea, autoimmune encephalitis, uveitis, thyroiditis, myasthenia, plus non-autoimmune diseases, such as asthma, allergy, colitis and stroke, and intestine inflammatory disorders including irritable bowel syndrome and Crohn's disease, and CNS disorders including epilepsy, brain trauma, multiple sclerosis, Parkinson's disease and Alzheimer's disease. According to the present invention, Thl7 mediated disease with Thl7 cells bad prognosis is selected but not limited to inflammation, autoimmune disease, chronic inflammatory disease such as psoriasis, psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitid, and inflammatory bowel diseases, Crohn's disease, multiple sclerosis, Vasculitis, atherosclerosis, Lung disorders, asthma, chronic obstructive pulmonary disease, lupus nephritis, Helicobacter pylori-associated gastritis, gastric cancer, autoimmune myositis, vascular diseases such as giant cell arteritis and Wegener's granulomatosis, and uveitis (Miossec P, Kolls JK. Targeting IL-17 and TH17 cells in chronic inflammation. Nat Rev Drug Discov. 2012 Oct;l l(10):763-76; Kim JS, Jordan MS. Diversity of IL-17-producing T lymphocytes. Cell Mol Life Sci. 2013 Jul;70(13):2271-90).

According to the present invention, Thl7 mediated disease with Thl7 cells good prognosis is selected but not limited to extracellular microbial infectious diseases, viral diseases, bacterial diseases, fungal diseases, pneumonia induced by Klebsiella pseumoniae, systemic and oropharyngeal candidiasis, cutaneous Staphylococcus aureus infection, Candida albicans infection, mycoses, and chronic mucocutaneous candidiasis (Miossec P, Kolls JK. Targeting IL-17 and TH17 cells in chronic inflammation. Nat Rev Drug Discov. 2012 Oct;l l(10):763-76; Kim JS, Jordan MS. Diversity of IL-17-producing T lymphocytes. Cell Mol Life Sci. 2013 Jul;70(13):2271-90). The term "calpain" has its general meaning in the art and refers to Ca²⁺-dependent cysteine proteases expressed in all mammalian systems. Calpain 1 (μ-calpain) and calpain 2 (m-calpain) are the most widely distributed calpain iso forms in mammalian cells and are referred to as conventional calpains (Donkor, 2011).

The term "calpastatin" has its general meaning in the art and refers to an endogenous protease inhibitor that acts specifically on calpain. Calpastatin consists of four repetitive sequences of 120 to 140 amino acid residues (domain I, II, III and IV), and a non- inhibitory N-terminal sequence (domain L) (Donkor, 2011).

The term "ABCAl" or "ABCAl transporter" has its general meaning in the art and refers to ATP-binding cassette transporter Al . ABCAl is the major regulator of plasma high density lipoprotein (HDL) cholesterol responsible for the removal of excess cholesterol from peripheral cells and tissues.

As used herein, the term "Thl7" has its general meaning in the art and refers to a subset of T helper cells producing interleukin 17 (IL-17) (Steinman L (February 2007). "A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell mediated tissue damage". Nat. Med. 13 (2): 139-145.). The term "IL-17" has its general meaning in the art and refers to the interleukin- 17A protein. Typically, Thl7 cells are 15 characterized by classical expression of Th cell markers at their cell surface such as CD4, and by the expression of IL17. Typically, as referenced herein, a Thl7 cell is an IL-17+ cell.

In a further aspect, the present invention relates to a compound which is selected from the group consisting of ABCAl agonists, ABCAl expression activators, exogenous calpain or modified calpain for use in a method for reducing Thl7-mediated immune responses in a subject in need thereof.

In a further aspect, the present invention relates to a compound which is selected from the group consisting of ABCAl antagonists, ABCAl expression inhibitors, exogenous calpastatin or calpain inhibitors for use in a method for enhancing Thl7-mediated immune responses in a subject in need thereof. In a further aspect, the present invention relates to a compound which is selected from the group consisting of ABCAl agonists, ABCAl expression activators, exogenous calpain or modified calpain for use in a method of limiting Thl7 commitment in a subject in need thereof.

In a further aspect, the present invention relates to a compound which is selected from the group consisting of ABCAl antagonists, ABCAl expression inhibitors, exogenous calpastatin or calpain inhibitors for use in a method of enhancing Thl7 commitment in a subject in need thereof.

In a further aspect , the present invention relates to a compound which is selected from the group consisting of ABCAl agonists, ABCAl expression activators, exogenous calpain or modified calpain for use in the prevention or treatment of TLR2 mediated disease in a subject in need thereof.

The method of the invention may be performed for any type of TLR2 mediated disease such as inflammatory diseases, autoimmune diseases, sepsis, cancer, rheumatoid arthritis, inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), diabetes, Alzheimer's disease, myocardial ischemia, ischemia/reperfusion injury associated with organ transplantation, stroke, kidney and myocardial infarction (Kanzler H, Barrat FJ, Hessel EM, Coffman RL. Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists. Nat Med. 2007 May;13(5):552-9 ; Hennessy EJ, Parker AE, O'Neill LA. Targeting Toll- like receptors: emerging therapeutics? Nat Rev Drug Discov. 2010 Apr;9(4):293-307; Connolly DJ, O'Neill LA. New developments in Toll-like receptor targeted therapeutics. Curr Opin Pharmacol. 2012 Aug;12(4):510-8).

The term "TLR2" has its general meaning in the art and refers to Toll-like receptor 2 also designated as CD282, a member of the Toll-like receptor (TLR) family. The Toll-like receptor (TLR) recognizes conserved structures of microbes and endogenous molecules. TLR2 is expressed on monocytes, mature macrophages and dendritic cells, and mast cells. It specifically recognizes components from Gram-positive bacteria, including lipoteichoic acid (LTA) with the assistance of the scavenger receptor CD36. TLR2 can form a heterodimer with either TLRl to recognize triacylated lipopeptides, or TLR6 to recognize diacylated lipopeptides (Kanzler H, Barrat FJ, Hessel EM, Coffman RL. Therapeutic targeting of innate immunity with Toll- like receptor agonists and antagonists. Nat Med. 2007 May;13(5):552-9 ; Hennessy EJ, Parker AE, O'Neill LA. Targeting Toll- like receptors: emerging therapeutics? Nat Rev Drug Discov. 2010 Apr;9(4):293-307; Connolly DJ, O'Neill LA. New developments in Toll-like receptor targeted therapeutics. Curr Opin Pharmacol. 2012 Aug;12(4):510-8).

The term "expression" when used in the context of expression of a gene or nucleic acid refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA. Gene products also include messenger RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins (e.g., calpain or calpastatin) modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, myristilation, and glycosylation. An "activator of expression" refers to a natural or synthetic compound that has a biological effect to activate the expression of a gene.

An "inhibitor of expression" refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene.

The term "ABCA1 agonist" refers to a compound that selectively activates the ABCA1 transporter. As used herein, the term "selectively activates" refers to a compound that preferentially activates ABCA1 transporter with a greater affinity and potency, respectively, than its interaction with the other sub-types or isoforms of the ABC transporter family. ABCA1 transporter agonist refers also to a compound that increases the ABCA1 activity level, whether this increase occurs by modulating the activity of the ABCA1 protein directly, by reducing ABCA1 degradation (e.g. ABCA1 stabilizer described in the US. Pat. No. 2007/0161702), or by any other means that result in an increase of ABCAl activity. Typically, an ABCAl transporter agonist is a small organic molecule, a peptide or a polypeptide.

In one embodiment of the invention, an ABCAl agonist is selected from chemicals increasing ABCAl activity, such as apolipoprotein A-I (apoA-I), apoA-I mimetic peptide 4F, diphenoquinone and ABCAl stabilizer described in the US. Pat. No. 2007/0161702 such as probucol spiroquinone, probucol diphenoquinone and probucol bisphenol.

In one embodiment of the invention, an ABCAl agonists include but are not limited to the ABCAl protein, active peptides and fragments thereof, and ABCAl expression activators, such as PvXR and LXR agonists (e.g., retinoic acid and oxysterols, including 22(R)- hydroxy cholesterol and 24-hydroxy cholesterol) (see Fukumoto et al. (2002) J. Biol. Chem., 277(5):4850813), cAMP, cAMP analogs (e.g., cpt-cAMP), vanadate, protein kinase A, okadaic acid, prostaglandins and PDE inhibitors.

The term "ABCAl antagonist" refers to a compound that selectively inactivates the ABCAl transporter. As used herein, the term "selectively inactivates" refers to a compound that preferentially inactivates ABCAl transporter with a greater affinity and potency, respectively, than its interaction with the other sub-types or iso forms of the ABC transporter family. ABCAl transporter antagonist refers also to a compound that decrease the ABCAl activity level, whether this decrease occurs by modulating the activity of the ABCAl protein directly, by enhancing ABCAl degradation, or by any other means that results in a decrease of ABCAl activity. Typically, an ABCAl transporter antagonist is a small organic molecule, a peptide, a polypeptide, an aptamer or an antibody.

In one embodiment of the invention, an ABCAl antagonist is selected from Cyclosporine A, FK506, pimecrolimus, PSC833a and rapamycin (Nagao et al, 2013), glibenclamide, sulfobromophthalein, flufenamic acid, diphenylamine-2-carboxylic acid, DIDS, bumetianide, and furosemide described in the EP. No. EP2001/0301000, and antibodies described in WO2005/116057.

In a further aspect, the present invention relates to a method of screening a candidate compound for use as a drug for the prevention or treatment of Thl7 mediated disease in a subject in need thereof, wherein the method comprises the step of selecting candidate compounds that activate ABCAl expression, induce ABCAl transporter activity, inhibit ABCAl expression or decrease ABCAl transporter activity. In a further aspect, the present invention relates to a method of screening a candidate compound for use as a drug for the prevention or treatment of Thl7 mediated disease in a subject in need thereof, wherein the method comprises the steps of:

providing an ABCAl transporter, providing a cell, tissue sample or organism expressing the ABCAl transporter, or providing an ABCAl transporter comprised in a lipid membrane separating two volumes comprising an aqueous medium, - providing a candidate compound such as small organic molecule, peptide or polypeptide,

measuring the activity of the ABCAl transporter,

and selecting positively candidate compounds that induce ABCAl transporter activity or decrease ABCAl transporter activity.

In one embodiment, the method of screening of the present invention is performed in vitro.

Preferably, measuring the activity of the ABCAl transporter involves determining the expression of the ABCAl transporter, and/or providing calpain, a substrate for the ABCAl transporter to said cell, tissue sample or organism and measuring the transport of the substrate by the ABCAl transporter across a lipid membrane of said cell, tissue sample or organism. Determining the expression the ABCAl transporter preferably involves determining the transcriptional activity of the nucleic acid molecule and/or determining the amount of the ABCAl transporter protein.

Measuring the activity of the ABCAl transporter in a lipid membrane separating two volumes comprising an aqueous medium involves providing calpain, a substrate for the ABCAl transporter to at least one of said volumes and measuring the transport of the substrate across said lipid membrane.

The activity of the ABCAl transporter is preferably measured by determining the rate of transport of calpain by the ABCAl transporter or measuring the calpain concentration in the intracellular and extracellular milieu or in the two separated volumes before and after providing the candidate compound. In one embodiment, the present invention relates to a method of screening ABCAl transporter agonists or antagonists for use in the prevention or treatment of Thl7 mediated disease in a subject in need thereof. Tests and assays for screening and determining whether a candidate compound is an

ABCAl transporter agonist or antagonist are well known in the art (US. Pat. No. 2008/0132447; US. Pat. No. 2002/0173004; US. Pat. No. 6,830,913). In vitro and in vivo assays may be used to assess the potency and selectivity of the candidate compounds to induce or decrease ABCAl transporter activity.

The ability of the candidate compounds to activate or inactivate the ABCAl transporter may be tested using isolated cells expressing ABCAl transporter, CHO cell line cloned and transfected in a stable manner by the ABCAl transporter or T cells.

The ability of the candidate compounds to activate or inactivate the ABCAl transporter may be assessed by the determination of a calpain transport across the ABCAl transporter cloned and transfected in a stable manner into a CHO cell line or T cells and determining the expression of the ABCAl transporter in the present or absence of the candidate compound.

Cells expressing another ABC transporter than ABCAl may be used to assess selectivity of the candidate compounds.

The term "calpain inhibitors" refers to any compound able to prevent the action of calpain. The calpain inhibitor of the present invention is a compound that inhibits or reduces the activity of calpain only in the extracellular milieu. However, decreasing and/or reducing the activity of calpain in the extracellular milieu can also be obtained by inhibiting calpain externalization. Calpain inhibitors of the present invention may consist but are not limited to inhibitor of calpain activity or calpastatin activator.

In one embodiment, the calpain inhibitor of the invention is an inhibitor of calpain activity. Said inhibitor of calpain activity may be selected from the group consisting of small organic molecules, peptides, polypeptides, aptamers or antibodies.

The inhibitors of calpain activity are well-known in the art as illustrated by Donkor, (2011), Pietch et al, (2010), Neffe and Abell, (2005). In one embodiment of the invention, the inhibitor of calpain activity may be calpastatin or truncated forms of calpastatin such as CP1B, PCP1B, 7-mer-PCPlB, 11R-CS, CPlB-[4-23] as described in U.S. Pat. No. 6,015,787; U.S. Pat. No. 6,294,518; U.S. Pat. No. 6,867,186.

In one embodiment of the invention, the inhibitor of calpain activity may be a peptidomimetic calpain inhibitor, a non-peptide calpain inhibitor such as disclosed in Donkor, (2011), and in the international patent application publication W092/11850. In one embodiment of the invention, the inhibitor of calpain activity is selected from the group consisting of SJA-6017, BDA-410, SNJ-1757, SNJ-1945, A-705253, MDL-28170, SC488, NS-398, SC-560, AK275, E64, calpeptin, calpastatin, acetyl-calpastatin, leupeptin, AK295, AK275, N-acetyl-leucyl-leucylmethional (ALLM or calpain inhibitor II), N-acetyl- leucylleucyl-norleucinal (ALLN or calpain inhibitor 1), calpain inhibitor III (carbobenzoxy- valyl-phenylalanal; Z-Val-PheCHO), calpain inhibitor IV (Z-LLY-FMK; Z-LLY-CH.sub.2 F where Z=benzyloxycarbonyl), calpain inhibitor V (MuVal-HPh-FMK where Mu is morphlinoureidyl and Hph is homophenylalanyl), mimetics thereof and compounds disclosed in U.S. Pat. Nos. 12,921,366; U.S. Pat. Nos. 5,716,980; 5,714,471; 5,693,617; 5,691,368; 5,679, 680; 5,663,294,5,661,150; 5,658,906; 5,654,146; 5,639,783; 5,635,178; 5,629,165; 5,622,981; 5,622,967; 5,621,101; 5,554,767; 5,550,108; 5,541,290; 5,506,243; 5,498,728; 5,498,616; 5,461,146; 5,444,042; 5,424,325; 5,422,359; 5,416,117; 5,395,958; 5,340,922; 5,336,783; 5,328,909; 5,135,916.

In one embodiment of the invention, the inhibitor of calpain activity is selected from synthetic calpain inhibitors such as PD- 150606 ((2Z)-3-(4-iodophenyl)-2-mercapto-2- Propenoic acid, 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid) and alpha- mercaptoacrylic acid derivatives such as disclosed in Wang et al, 1996.

The present invention also relates to the compound according to the invention in combination with or in place of IL2 for use in the prevention or treatment of Thl7 mediated disease in a subject in need thereof. In a particular embodiment, present invention relates to the compound according to the invention in combination with or in place of low doses IL2 for use in the prevention or treatment of Thl7 mediated disease in a subject in need thereof. Typically, the term "low doses IL2" refers to IL2 concentration in contact with T lymphocytes between 1 and 50 pg/ml, even more preferably between 1 and 10 pg/ml. Plasma concentrations of IL-2 are maintained at these values (15 pg/ml) when human patients receive 1.5 to 3.0 x 10⁶ IU per day (Matsuoka K, Koreth J, Kim HT, Bascug G, McDonough S, Kawano Y, Murase K, Cutler C, Ho VT, Alyea EP, Armand P, Blazar BR, Antin JH, Soiffer RJ, Ritz J. Low-dose interleukin-2 therapy restores regulatory T cell homeostasis in patients with chronic graft-versus-host disease. Sci Transl Med. 2013 Apr 3;5(179): 179ra43).

Thl7 cells present a controversial role in cancer. Several studies reports that Thl7 cells have the capability to eradicate advanced metastatic malignancies, otherwise another studies described that Thl7 cells are involved in tumor-induced immune tolerance required for cancer cell survival and expansion (Martin et al., 2012; Tosolini et al., 2011).

Accordingly, in another embodiment, the present invention relates to a compound which is selected from the group consisting of ABCA1 agonists, ABCA1 expression activators, exogenous calpain or modified calpain for use in the prevention or treatment of cancer in a subject in need thereof wherein said subject has been diagnosed with Thl7 cells bad prognosis in said cancer.

In another embodiment, the present invention relates to a compound which is selected from the group consisting of ABCA1 antagonists, ABCA1 expression inhibitors, exogenous calpastatin or calpain inhibitors for use in the prevention or treatment of cancer in a subject in need thereof wherein said subject has been diagnosed with Thl7 cells good prognosis in said cancer. Accordingly, in another embodiment, the present invention relates to a method of treating cancer in a subject in need thereof comprising the steps of:

i) determining if the Thl7 cells are a good or a bad prognosis in cancer, and ii) treating said subject having a cancer with a compound which is selected from the group consisting of ABCA1 agonists, ABCA1 expression activators, exogenous calpain or modified calpain wherein said subject has been diagnosed with Thl7 cells bad prognosis in said cancer, or

iii) treating said subject having a cancer with a compound which is selected from the group consisting of ABCAl antagonists, ABCAl expression inhibitors, exogenous calpastatin or calpain inhibitors wherein said subject has been diagnosed with Thl7 cells good prognosis in said cancer.

According to the present invention, cancer with Thl7 cells bad prognosis is selected but not limited to hepatocellular cancer, colorectal cancer, pancreatic cancer, bile duct cancer, lung cancer, and cancer with Thl7 cells good prognosis is selected but not limited to esophageal cancer, gastric cancer, and ovarian cancer.

In one embodiment, the present invention relates to a method for preventing or treating Thl7 mediated disease in a subject in need thereof, wherein said subject has been diagnosed with Thl7 cells bad prognosis in said disease comprising the step of administering to said subject a compound which is selected from the group consisting of ABCAl agonists, ABCAl expression activators, exogenous calpain or modified calpain.

In another embodiment, the present invention relates to a method for preventing or treating Thl7 mediated disease in a subject in need thereof, wherein said subject has been diagnosed with Thl7 cells good prognosis in said disease comprising the step of administering to said subject a compound which is selected from the group consisting of ABCAl antagonists, ABCAl expression inhibitors, exogenous calpastatin or calpain inhibitors.

In a further aspect, the present invention relates to a method for limiting Thl7 commitment in a subject in need thereof comprising the step of administering to said subject a compound which is selected from the group consisting of ABCAl agonists, ABCAl expression activators, exogenous calpain or modified calpain.

In a further aspect, the present invention relates to a method for enhancing Thl7 commitment in a subject in need thereof comprising the step of administering to said subject a compound which is selected from the group consisting of ABCAl antagonists, ABCAl expression inhibitors, exogenous calpastatin or calpain inhibitors. The present invention also relates to the method according to the invention comprising the step of administering to said subject a compound which is selected from the group consisting of ABCAl agonists, ABCAl expression activators, exogenous calpain or modified calpain in combination with low doses IL2.

Pharmaceutical composition

The compound of the invention may be used or prepared in a pharmaceutical composition.

In one embodiment, the invention relates to a pharmaceutical composition comprising the compound of the invention and a pharmaceutical acceptable carrier for use in the prevention or treatment of Thl7 mediated disease in a subject of need thereof.

Typically, the compound of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" 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.

Preferably, 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 compound of the invention 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 preferred 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.

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.

In addition to the compounds of the invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used.

In a further aspect, the invention relates to a pharmaceutical composition comprising the compound of the invention and a pharmaceutical acceptable carrier for use in the prevention or treatment of TLR2 mediated disease in a subject of need thereof. 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: T lymphocytes secrete calpain activity through an ABC family transporter dependent pathway.

CD3+ T cells isolated from the spleen of C57BL/6 mice (A) and peripheral blood mononuclear cells (PBMC) isolated from healthy human subjects (B) were stimulated for 4 h with or without 1 μg/ml eCD3 mAb together with chemicals inhibiting ABCAl (glyburide 100 μΜ), ABCB1 (Cyclosporine A 10 μΜ) or ABCC1 (MK571 50 μΜ). Calpain activity was determined in both intracellular (■) and extracellular (□) milieu by measuring the calpain- specific cleavage of fluorescent AMC. (n = 3; * p <0.05 vs control without drug). Figure 2: T lymphocytes secrete calpain activity through an ABCA-l-dependent pathway.

CD3+ T cells isolated from the spleen of C57BL/6 mice were stimulated for 4 h with 1 μg/ml eCD3 mAb together with the indicated concentrations of chemicals increasing ABCAl expression or activity, including ApoA-I (A), ApoA-I mimetic peptide 4F (B), and diphenoquinone (C). Calpain activity was measured in both intracellular (■) and extracellular (□) milieu. Results are expressed as relative calpain activity, i.e. as % of control (n = 4-6; */?<0.05 and ** /?<0.01 vs control without drug).

Figure 3: T lymphocytes secrete main calpain subunits in microvesicles.

Spleen CD3+ T cells were stimulated for 4 h with or without 1 μg/ml eCD3 mAb. (A)

Proteins of cells and conditioned medium concentrated 10 x were subjected to immunoblot analysis using antibody against calpain 1 , 2, and 4. A representative experiment is shown. (B) Microvesicles isolated from conditioned medium by ultracentrifugation were analyzed by electron microscopy and for presence of calpain 4 by immunoblot. A representative experiment is shown.

Figure 4: Set of cytokines produced by T lymphocytes in response to exogenous calpain challenge. Spleen CD3+ T cells were stimulated for 4 h without (■) or with (□) μ-calpain (2 μ§/ι 1), and cytokine generation was analyzed in supernatants by ELISA. Results are expressed as pg/ml (n = 4. */?<0.05 and ** /?<0.01 vs control without drug). Figure 5: Activation of ABCA1 transporter limits IL-17 production by T lymphocytes.

Spleen CD3+ T cells were stimulated for 4 h with 1 μg/ml a CD3 mAb + 10 ng/ml IL- 6 in the presence of ApoA-I or apoA-I mimetic peptide 4F, together with or without calpastatin and IL-17 concentration was analyzed in supernatants by ELISA. Results are expressed as pg/ml (n = 4. */?<0.05 vs control).

Figure 6: Exogenous calpains limit IL-17 release expression.

A. Spleen CD3+ T cells were stimulated for 24 h with 1 μg/ml a CD3 mAb and 10 ng/ml IL-6 together with the indicated concentrations of μ-calpain. Then IL-17 generation and T cell proliferation were analyzed by ELISA and BrdU incorporation assay, respectively (n = 3-4). B. Spleen CD3+ T cells were stimulated for the indicated periods of time with or without 2 μg/ml calpain 1. Then Q-PCR was carried out to assess the expression of Rorc and IL-21 as compared to two housekeeping genes, (n = 6. */?<0.05 and ** /?<0.005 vs control). Figure 7: Identification of calpain targets at the surface of T lymphocytes.

Mouse spleen CD3+ T cells were stimulated for 4 h with 1 μg/ml a CD3 mAb together with or without μ-calpain (4 μg/ml), calpastatin (10 μg/ml), or neutralizing anti-TLR2 antibody (). Proteins isolated from plasma membranes after cell surface biotinylation and from conditioned medium after concentration (10 x) were subjected to SDS PAGE analysis (left panel) and immunoblot analysis using antibodies against gp96 and TLR2 (right panels). Shown gel and blots are representative of three independent experiments with similar results.

Figure 8: Regulatory role of extracellular calpains on IL-17 expression involves

TLR2.

CD3+ T cells isolated from the spleen of C57BL/6 WT or TLR2-/- mice (upper panel) and peripheral blood mononuclear cells (PBMC) isolated from healthy human subjects (lower panel) were incubated for 24 h with 1 μg/ml a CD3 mAb and 10 ng/ml IL-6 together with μ- calpain (4 μg/ml), calpastatin (10 μg/ml), Pam3Cys (10 ng/ml), isotype-matched control IgGl (4 μ /ηι1), or neutralizing anti-TLR2 antibody (4 μ /ηι1). Then IL-17 generation was analyzed by ELISA (n = 3-6. */?<0.05 vs control; § /?<0.05 vs Pam3Cys; # /?<0.05 vs IgG).

Figure 9: Low doses IL-2 decrease IL-17 expression partly through calpain exteriorization.

(A) CD3+ T cells isolated from the spleen of C57BL/6 mice were stimulated for 24 h with 1 μg/ml eCD3 mAb together with the indicated concentrations of IL-2. Calpain activity was measured in both intracellular (·) and extracellular (o) milieu (n = 4; */?<0.01 and *** /?<0.002 vs control without IL-2). (B) CD3+ T cells isolated from the spleen of C57BL/6 mice were stimulated for 24 h with 1 μg/ml eCD3 mAb together with or without 0.01 ng/ml IL-2. Proteins isolated from plasma membranes after cell surface biotinylation were subjected to immunoblot analysis using anti-ABCAl antibody. (C) CD3+ T cells isolated from the spleen of C57BL/6 mice were stimulated for 72 h with 1 μg/ml eCD3 mAb and the indicated concentrations of IL-2 together with (□) or without (■) calpastatin. Then IL-17 generation was analyzed by ELISA. The IL-2 dependent decrease in IL-17 production is expressed as percentage (n = 5; */?<0.05 vs control without calpastatin).

EXAMPLE:

Calpains are secreted by T lymphocytes through an ABCAl-dependent pathway and function as a negative regulator of IL-17 expression

Material and Methods

Cell isolation.

Blood was collected from healthy subjects and PBMC were isolated by density centrifugation on Ficoll-Paque (GE Healthcare). T lymphocytes were isolated from mouse spleen cells of C57BL/6J mice using the mouse CD3+ T cell enrichment kit (Stem Cell Technologies). Microvesicles were isolated from conditioned medium of these cells using ultracentrifugation, as previously described (18).

Calpain activity assay.

Intracellular calpain activity was determined in spleen CD3+ T cells (5-10.106), as previously described (19). For measuring calpain activity in extracellular milieu, isolated T lymphocytes were cultured in 24-well tissue culture dishes, in RPMI medium (5-10.106 cells in 500 μΐ). After the indicated culture period, cell conditioned medium was diluted (1/1) in KRB solution (pH 7.4) containing 2 mM CaC12 with or without ΙΟΟμΜ calpain inhibitor 1 (Sigma) and incubated 15 min before the addition of the calpain substrate (50 mM). After a 90 min incubation period, fluorescence was detected at 360 nm excitation and 460 nm emission, using the FLX800 spectrofluorimeter (Bio-Tek Instruments). Calpain activity was determined as the difference between fluorescences measured without and with calpeptin, and expressed as μΜ AMC using a standard curve (0 to 25 μΜ) constructed for each assay.

Cell proliferation.

T cell proliferation was determined with the Bromodeoxyuridine (BrdU) labeling solution (Roche Diagnostic).

Cytokine measurement.

Spleen CD3+ T cells (6.106/ml) were incubated for 4 h in the presence or absence of μ-calpain (1 μg/ml). Cytokines (IFN-g, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, and TNF-a) were measured in the supematants using the Mouse Inflammatory Cytokines & Chemokines Multi-Analyte ELISArray Kit (SABiosciences).

Western blot analysis.

The primary antibodies used were anti-calpain 1 (H-65), anti-calpain 2 (C-19), and anti-calpain 4 (P-l), all from Santa Cruz, anti-GP96 (AP4899a from Abgent), anti-TLR2 (T2.5 from Invivogen), and anti-ABCAl (PA1- 16789 from Pierce).

Quantitative real-time PCR.

Total cellular RNA was collected from mouse T cells using using RNA Miniprep (Bio Basic Inc.), reverse transcripted into cDNA with Superscript II (Life Technologies BRL), and amplified by PCR using a LightCycler 480 (Roche Diagnostic) with SYBR Green (Fast Start DNA Master SYBR Green I; Roche Diagnostic) and specific primers for mouse RORgt, IL- 21 and two housekeeping genes (beta-actin and glucuronidase beta, Gusb), as previously described (20). The two reference genes were used to normalize the Q-PCR results, using Roche LightCycler 2.0 software (Roche Diagnostic).

Results

T lymphocytes secrete calpains through an ABCA-l-dependent pathway. There are two potential explanations for finding calpains in the extracellular milieu of lymphocytes: passive release due to cell death and active transport through cell membrane. Previous studies excluded a defect in lymphocyte viability, thus suggesting a secretion process (6). For unconventional secretion of proteins which lack an amino -terminal signal- peptide sequence such as calpains (1), different pathways are potentially involved, including in particular ATPbinding cassette (ABC) family transporter (21). To address whether T lymphocytes secrete calpains through an ABC-dependent transport, we analyzed the effects of ABC inhibitory drugs on calpain activity in both intra- and extra-cellular milieu (Fig. l).The release of calpain activity from either mouse or human purified T lymphocytes was reduced by an ABCA1 transporter inhibitor, glyburide, but not by a p-glycoprotein (ABCB1) inhibitor, cyclosporine A, nor by a multidrug resistance-related protein (MRP / ABCC), MK571. The extracellular differences between glyburide-treated and untreated cells were reflected in the retention of calpain activity in intracellular compartment. Chemicals increasing ABCA1 expression or activity, including apoA-I, apoA-I mimetic peptide 4F, and diphenoquinone displayed dosedependent stimulatory effects on calpain secretion while decreasing cytosolic activity (Fig. 2). These compounds exerted no toxic effects on T lymphocytes, as assessed by annexin V / propidium iodide staining (> 98% cell viability).

T lymphocytes secrete main calpain subunits in microvesicles.

Conditioned medium from mouse T lymphocytes stimulated for 4 h with or without eCD3 mAb was analyzed for the presence of calpain subunits by immunoblotting (Fig. 3A). Under the two conditions, T lymphocytes released calpain 1, 2, and 4. As expected, glyburide limited this release, thus promoting cell retention. We next examined the mode of release of calpain subunits, focusing on microvesicle shedding. To this aim, microvesicles were isolated from conditioned medium of mouse T lymphoctes by ultracentrifugation. They were characterized by transmission electron microscopy as vesicles less than 0.1 μιη (Fig. 3B). In particular calpain 4 was detectable by immunoblot analysis in these isolated microvesicles. Taken together, these results indicate that activated T lymphocytes secrete calpains through an ABCA1 -dependent process that involves, at least in part, microvesicle shedding.

Extracellular calpains are an important negative regulator of IL-17 production by T lymphocytes.

To gain insights into the effects of secreted calpains on T lymphocyte differentiation and function, we initially examined whether addition of exogenous calpains affected the production of cytokines by mouse T cells in vitro (Fig. 4). The levels of IFN-g (TH1), IL-4 (TH2), IL-6, IL-12, and TNF-a were not modified. In contrast, addition of μ-calpain decreased the levels of IL-2 (but these differences did not reach statistical significance), IL- 10, and even more markedly IL-17.

Given the effect of exogenous μ-calpain on IL-17 production, experiments next determined whether secretion of endogenous calpains through an ABCA1 -dependent process was limiting also IL-17 expression. To this aim, naive T lymphocytes were stimulated under minimal Thl7 polarizing conditions, consisting of 1 μg/ml soluble anti-CD3 Ab and 10 ng/ml IL-6. Chemicals increasing ABCA1 expression or activity, apoA-I and apoA-I mimetic peptide 4F, blunted IL-17 production in a dose-dependent manner (Fig. 5). As expected, addition of calpastatin to inactivate calpains specifically in the extracellular medium amplified IL-17 production and completely suppressed the inhibitory effect of apoA-I mimetic peptide 4F. Taken together this suggests that ABCA1 -dependent secretion of calpains play a role that fundamentally controls IL-17 expression.

Regulatory role of extracellular calpains on IL-17 expression involves a cleavage of TLR2 extracellular domain.

Having established that calpains, either added to the extracellular milieu or exteriorized from T lymphocytes, control mainly IL-17 expression, we sought to focus our study on IL-17. Inhibition of IL-17 expression by μ-calpain occurred rapidly, significant after 24h and its maximum effect was produced at a concentration of 4 μg/ml (Fig. 6A). It was not explained by a death of T cells since their viability even increased slightly under these conditions. To address whether extracellular calpains influence IL-17 expression by regulating gene transcription and/or mRNA stability, we carried out a quantitative RT-PCR assay. The activation of T lymphocytes in the presence of anti-CD3 and IL-6 resulted in a rapid induction of the mRNA coding for RORgt, the master regulator for Thl7 differentiation (Fig. 6B). This was almost completely blunted in the presence of extracellular μ-calpain, indicating a transcriptional control. Addition of μ-calpain blunted the expression of mRNA coding for 11-21 required to expand Thl7 cell population (Fig. 6C).

Next we sought to examine the molecular mechanisms whereby extracellular calpains control IL-17 expression. We hypothesized that they would cleave membrane receptor and/or bound ligand involved in the transcription of IL-17. To explore this possibility, we compared the profile of membranous proteins in mouse T lymphocytes treated with and without 2 μg/ml μ-calpain. The ID SDS-PAGE analysis of proteins isolated after cell surface biotinylation revealed that μ-calpain diminished mainly two proteins with an apparent molecular weight (MWapp.) of ~40 and ~20 kDa, respectively (Fig. 7). The main ~20 kDa band was excised from the gel and after in-gel tryptic digestion, the resulting peptide digests were analyzed by MALDI-TOF/TOF. The obtained MS and MS/MS data were used for a data search (NCBI), leading to the identification of the NH2 -terminus of the chaperone glucose-regulated protein 94 (GRP94 also known as gp96) with the highly significant score of 94. To confirm the relationship between the ~20 kDa band and gp96 fragment, we next performed Western blot experiments using an antibody directed against the NH2-terminus of gp96 (Fig. 7). In T lymphocyte membranes this antibody recognized an intact form of gp96 and NH2 -terminus fragments of ~72, ~52, ~40, and ~20 kDa. The intensity of either band was markedly decreased by exposure of T lymphocytes to μ-calpain, and, conversely, amplified by calpastatin. Interestingly, the disappearance of the main membrane-associated form of gp96 was concomitant with its appearance in extracellular medium (Fig. 7). These results strongly support that extracellular calpains, either exogenous or endogenous, limit the association of gp96 with T lymphocyte plasma membrane by increasing its detachment rather than its breakdown.

Even if gp96 is generally localized in the endoplasmic reticulum, where it is required for cell surface expression of TLRs, it may translocate to the cell surface and gain extracellular access after cell stress (22). When externalized, gp96 or its NH2 -terminus fragments eventually serve as endogenous ligands for TLR2 (23). Thus, we considered that extracellular calpains could affect the association of gp96 with T lymphocyte plasma membrane by limiting its binding to TLR2. To explore this hypothesis, we assessed the expression of gp96 at the surface of T lymphocytes in the presence of neutralizing antibody to TLR2 (Fig. 7). As expected, TLR2 inactivation reduced membrane association of gp96, markedly for NH2 -terminus fragments of ~52 and ~40 kDa, and moderately for its intact form. Such a defect could be explained by a calpain dependent cleavage of TLR2 ectodomain, leading to the appearance of soluble TLR2 (sTLR2) with the potential to act as a decoy receptor (24). We therefore assessed the release of sTLR2 in the extracellular milieu of T lymphocytes (Fig. 7). Western blot showed a major 75-kDa sTLR2 band whose intensity was dramatically increased by T lymphocyte exposure to μ-calpain and, conversely, blunted by calpastatin. Because TLR2 activation is known to enhance IL-17 production by both mouse and human T lymphocytes (25, 26), we finally investigated whether the decrease in IL-17 expression observed in response to extracellular calpains was explained by sTLR2 release. Treatment of mouse or human T lymphocytes with a TLR2-TLR1 ligand, Pam3Cys (10 ng/ml), induced an increase in IL-17 production, which was suppressed by μ-calpain (Fig. 8). Conversely, addition of a neutralizing antibody to TLR2 limited IL-17 production whose residual level was not affected by μ-calpain or calpastatin. Similar results were obtained by using T lymphocytes isolated from the spleen of Tlr2-I- mice. Taken together, our data demonstrate clearly that extracellular calpains limit IL-17 expression by cleaving TLR2 and thereby preventing T lymphocyte response to exogenous (e.g. Pam3Cys) or endogenous (gp96) TLR2 ligands.

Low doses IL-2 decrease IL-17 expression partly through calpain exteriorization.

Next we sought to examine under which condition this novel pathway that limits Thl7 cell function might be operational. IL-2 limits Thl7 differentiation and IL-17 expression, but underlying mechanisms remain only partially understood (27). IL-2 could interfere with IL-6- dependent signalling events, e.g. by limiting IL-6 receptor expression and/or by triggering the replacement of STAT3 with STAT5 on the locus encoding IL-17 (27). To investigate whether calpain secretion could play a role as well, we first analyzed the effects of IL-2 on calpain activity in both intra- and extra-cellular milieu of mouse T lymphocytes. We were surprised to observe that low doses of IL-2 (0.001 to 0.01 ng/ml) increased calpain secretion, thus decreasing cytosolic activity, while higher doses did not (Fig. 9A). At effective IL-2 concentrations, plasma membranes of T lymphocytes showed increased amounts of ABCAl (Fig. 9B). Further work will be required to identify molecular mechanisms involved in this upregulation.

Consistent with previous reports, we found that IL-2 limited IL-17 production in a dose-dependent manner (Fig. 9C). More strikingly, inactivation of secreted calpains by the addition of calpastatin removed the inhibitory effect of IL-2 only at low concentrations. These data support the principle that low doses IL-2 limit IL-17 production, at least partly, through calpain exteriorization.

Discussion/Conclusion

This study reveals both the mode of calpain secretion by T lymphocytes and one of the autocrine / paracrine functions of externalized calpain. - Our studies demonstrate for the first time that calpain export from T lymphocytes involves mainly ABC A 1 transporter.

- Our studies demonstrate for the first time that calpains secreted by T lymphocytes through the ABCA1 pathway mainly reduce IL-17 generation, with a weaker effect on IL-2 and IL-10. Importantly, the pattern of cytokines expressed after T lymphocyte exposure to extracellular calpains mimics entirely that observed after inhibition of intracellular calpains by calpastatin transgene (11). Given the pro -inflammatory role of IL-17, our results strengthen the hypothesis that intra- and extra-cellular calpains have opposite effects on inflammatory process. The idea that the same proteins have distinct (even) opposing functions in intracellular and extracellular milieu is not without precedent (13).

- Our studies demonstrate for the first time that extracellular calpains are responsible for the shedding of TLR2, a proteolysis mechanism so far not identified (24). In addition, we demonstrate that TLR2 shedding participates in the control of IL-17 production by extracellular calpains. Our results also suggest that any pathway increasing ABCA1 expression or stability and, thereby, calpain exteriorisation would limit various functions of TLR2 besides IL-17 generation. This happens at the right time since there is a growing interest in the targeting of TLRs in various inflammatory and cancer diseases (28, 29).

- Finally, our studies demonstrate for the first time that extracellular calpains participate in the control of IL-17 production by low doses of IL-2. Again, this is at the opportune moment as low dose IL-2 as recently been shown efficient in immune diseases including vasculitis, GVH (30).

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method for preventing or treating Thl7 mediated disease in a subject in need thereof, wherein said subject has been diagnosed with Thl7 cells bad prognosis in said disease comprising the step of administering to said subject a compound which is selected from the group consisting of ABCAl agonists, ABCAl expression activators, exogenous calpain or modified calpain.

2. The method according to claim 1 for treating inflammation.

3. The method according to claim 1 for treating cancer in a subject in need thereof wherein said subject has been diagnosed with Thl7 cells bad prognosis in cancer.

4. The method according to claim 1 for treating autoimmune disease.

5. The method according to claim 1 for treating graft-versus-host disease.

6. A method for limiting Thl7 commitment in a subject in need thereof comprising the step of administering to said subject a compound which is selected from the group consisting of ABCAl agonists, ABCAl expression activators, exogenous calpain or modified calpain.

7. A method of screening a candidate compound for use as a drug for the prevention or treatment of Thl7 mediated disease in a subject in need thereof, wherein said method comprises the step of selecting candidate compounds that activate ABCAl expression, induce ABCAl transporter activity, inhibit ABCAl expression or decrease ABCAl transporter activity.

8. The method of screening a candidate compound according to claim 7 for use as a drug for the prevention or treatment of Thl7 mediated disease in a subject in need thereof, wherein said method comprises the steps of:

providing an ABCAl transporter, providing a cell, tissue sample or organism expressing the ABCAl transporter, or providing an ABCAl transporter comprised in a lipid membrane separating two volumes comprising an aqueous medium, - providing a candidate compound such as small organic molecule, peptide or polypeptide,

measuring the activity of the ABCAl transporter,

and selecting positively candidate compounds that induce ABCAl transporter activity or decrease ABCAl transporter activity.

9. The method according to any of claims 1 to 6 comprising the step of administering to said subject a compound which is selected from the group consisting of ABCAl agonists, ABCAl expression activators, exogenous calpain or modified calpain in combination with low doses IL2.

10. A method for preventing or treating Thl7 mediated disease in a subject in need thereof, wherein said subject has been diagnosed with Thl7 cells good prognosis in said disease comprising the step of administering to said subject a compound which is selected from the group consisting of ABCAl antagonists, ABCAl expression inhibitors, exogenous calpastatin or calpain inhibitors.

11. The method according to claim 10 for treating cancer in a subject in need thereof wherein said subject has been diagnosed with Thl7 cells good prognosis in cancer.