WO2014060392A1

WO2014060392A1 - Caspase-6 inhibitors for treating t cell activation and/or proliferation disorders

Info

Publication number: WO2014060392A1
Application number: PCT/EP2013/071486
Authority: WO
Inventors: Jérôme ESTAQUIER
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale)
Priority date: 2012-10-16
Filing date: 2013-10-15
Publication date: 2014-04-24

Abstract

Caspase-6 inhibitors for treating T cell activation and/or proliferation disorders. The invention relates to caspase-6 inhibitors for use in therapy, in particular for treating T cell activation and/or proliferation disorders.

Description

CASPASE-6 INHIBITORS FOR TREATING T CELL ACTIVATION AND/OR PROLIFERATION DISORDERS

FIELD OF THE INVENTION

The invention relates to methods for treating T cell activation and/or proliferation disorders.

BACKGROUND OF THE INVENTION Rejection of transplanted organs is the main barrier of transplantation today. It occurs as a result of humoral and cell-mediated responses by the recipient to specific antigens present in the donor tissue. These antigens are known as major histocompatibility complex (MHC) molecules. In humans, this group of molecules is referred to as human leukocyte antigen (HLA) complex molecules in humans. The recognition of these foreign MHC antigens initiates rejection, which occurs in two stages. During the first stage, known as sensitization, lymphocytes are alerted and respond to the foreign MHC molecules. Rapid proliferation occurs in this stage. In the second "effector" stage, the graft is destroyed by several cellular and molecular mechanisms.

If a bone marrow transplant can be performed, the transplant recipient's immune system can be replaced with the donor's immune system, thus enabling the recipient's body to accept the new organ without risk of rejection. This requires that the bone marrow, which produces the immune cells, be from the same person as the organ donation (or an identical twin or a clone). There is a risk of graft versus host disease (GVHD) in which the lymphoid cells co-injected with the bone marrow transplant recognize the host tissues as foreign and attack and destroy them accordingly.

In order to prevent graft rejection, immunosuppressive treatment is often used to inhibit the immune response. However, the organism then becomes vulnerable to infection and the risk of uncontrolled cell proliferation resulting in cancer is increased. In order to minimize the individual specific side-effects of the three effective agents used in clinical practice, namely azathioprine, corticosteroids and cyclosporin, small doses of each are used in combination "triple therapy". Of the three agents currently used in such triple therapy, cyclosporin is the most powerful, but has the unsatisfactory side-effect of nephrotoxicity in man which can lead to structural renal damage. Increased corticosteroid dosage and antilymphocyte antibody preparations, poly- or monoclonal, are used for the treatment of rejection crises. A number of studies have been taken to investigate other potentially effective compounds for use as immunosuppressive agents and transplant rejection inhibitors. For example, immunophilins that bind cyclosporine (cyclophilin) and FK-506/rapamycin (FK- BP12) derivatives have been developed as immunosuppressive agent. However to date, none have been found to be relevant in clinical settings because of side-effects, such as toxicity, the lack of efficacy or a combination of these factors. Thus, there is still a need in the art for a selective and effective immunosuppressive agent for treating T cell activation and/or proliferation disorders.

SUMMARY OF THE INVENTION The invention relates to caspase-6 inhibitors for use in therapy.

The invention relates to caspase-6 inhibitors for use as an immunosuppressive agent.

In particular the invention relates to caspase-6 inhibitors for use in treating a T cell activation and/or proliferation disorder.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed a novel class of immunosuppressive agents which selectively inhibit the activation and proliferation of T cells.

A first object of the invention is the use of caspase-6 inhibitors for therapy. The expression "caspase-6 inhibitors" refers to any compound which is able to inhibit signaling through caspase-6, i.e. able to diminish reversibly or irreversibly, totally or partially, the cleavage of target proteins by caspase-6.

Caspase-6 is a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis. Caspases exist as inactive proenzymes which undergo proteolytic processing at conserved aspartic residues to produce two subunits, large and small, that dimerize to form the active enzyme. This protein is processed by caspases 7, 8 and 10, and is thought to function as a downstream enzyme in the caspase activation cascade.

The human pre-procaspase 6 gene encodes, through alternative splicing, two isoforms: the Genbank nucleotide sequence NM_001226.3 which encodes a an alpha isoform (NP_001217.2) and the Genbank nucleotide sequence NM_032992.2 which encodes the beta isoform (NP_ 116787.1).

Caspase-6 inhibitors have already been described in the literature. They include synthetic compounds such as the synthetic peptides commercialized by Merck as Z- Val-Glu(OMe)-Ile-Asp(OMe)-CH2F, also known as Z-VEID-FMK, and Ac-Ala- Ala- Val- Ala-Leu-Leu-Pro-Ala- Val-Leu-Leu- Ala- Leu-Leu-Ala-Pro-Val-Glu-Ile- Asp- CHO. These inhibitors are derivatives of peptides that serve as substrates. (Gregoli and Bondurant, 1999, J Cell Physiol, 178, 133-43). The derivatives benzyloxycarbonyl (z-) fluoromethyl-ketone (FMK or CH2F) and difluorophenoxyl (OPH) serve as stabilizing functions. The synthetic caspase-6 inhibitors of the invention can be reversible inhibitors (which is usually the case of aldehyde inhibitors of VEID peptides) or irreversible inhibitors (most conjugates of VEID with chloromethyl, fluoromethyl or acyloxymethyl groups). A caspase-6 inhibitor can also be small interfering RNAs which lower the expression level of the caspase-6 gene. Examples of siRNA against caspase-6 include the following nucleotide sequences described in the Examples section: j-004406-06 : CAUGGUACAUUCAAGAUUU (SEQ ID NO: l);

j-004406-07 : CUGACUUCCUCAUGUGUUA (SEQ ID NO:2);

J-004406-08 : GACAUUAACUGGCUUGUUC (SEQ ID NO:3);

1-004406-09: GCAGAUGCCGAUUGCUUUG (SEQ ID NO:4).

Antisense compounds which inhibit the expression of caspase-6 are described in document WO02/29066.

A caspase-6 inhibitor according to the invention can also be dominant a negative caspase-6 protein, i.e., a mutated caspase-6 molecule which competes with the endogenous caspase-6.

According to the invention, the caspase-6 inhibitor can also be a vector comprising a nucleotide sequence encoding the above mentioned siRNA or dominant negative caspase-6. For a given siRNA or a given dominant negative protein, those skilled in the art will be able to identify which nucleotide sequence(s) encode(s) such a siRNA or dominant negative protein, on the basis of the genetic code, the degeneracy of said code, and codon adaptation according to species. Methods for determining whether a given compound is a caspase-6 inhibitor can include for example determining whether the amount of caspase-6 protein in a given sample is decreased upon exposure to said compound, as described below in Figure IBa). The term "therapy", "treatment" or "treat" as used herein means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such a disorder or condition.

The expression "immunosuppressive agent" as used herein refers to an agent that inhibits or prevents the activity of the immune system. Immunosuppressive agents are used to prevent the rejection of transplanted organs and tissues (e.g. bone marrow, heart, kidney, liver). The expression "T cell activation and/or proliferation disorder" as used herein, refers to diseases or disorders due to an increased activation and/or proliferation of T cells. Typically, a T cell activation and/or proliferation disorder is selected from the group consisting of graft versus host disease, organ transplant rejection, T cell lymphoma etc. The expression also includes HIV-1 infection, since it has been demonstrated that such infection is associated with a proliferation of T cells (Hurtrel CDD 2005, Cumont J Virol 2007, Golstein J Virol 2006, Silvestri G, Immunity. 2003 Mar;18(3):441-52).

In a preferred embodiment, said T cell activation and/or proliferation disorder is graft versus host disease.

Interestingly, the inventors have observed that the caspase-6 inhibitors of the invention are very effective and specific immunosuppressive agents. Indeed, they are useful for the specific suppression of an undesirable response of the innate immune system such as a T cell activation and/or proliferation. Advantageously, they have no effect on inflammation or on interferon secretion. Thus, these two components of the anti-viral response are preserved. This can help avoid many of the complications observed during classical immunosuppressive therapy.

The invention also relates to methods and compositions for treating a T cell activation and/or proliferation disorder in a patient.

As used herein, the term "patient" denotes a mammal, such as a rodent, a feline, a canine and a primate. Preferably, a patient according to the invention is a human.

The caspase-6 inhibitor may be administered in the form of pharmaceutical composition. Preferably, said caspase-6 inhibitor is administered in a therapeutically effective amount. By a "therapeutically effective amount" is meant a sufficient amount of the serotonin reuptake inhibitor to treat and/or to prevent the above mentioned disorders at a reasonable benefit/risk ratio applicable to any medical treatment. It is understood 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 patient 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, gender and diet of the patient; 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 compound 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. The caspase-6 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" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semisolid 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 caspase-6 inhibitor 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 polypeptides in the required amount in the appropriate solvent with various 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. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 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 preferred embodiment, the caspase-6 inhibitor is administered in combination with another active agent.

In one embodiment, the caspase-6 inhibitor can be administered in combination with an immunosuppressive agent.

Suitable immunosuppressive agents include, but are not limited to glucocorticoids; cytostatics such as alkylating agents, antimetabolites, cytotoxic antibiotics; T cell receptor directed antibodies, IL-2 receptor directed antibodies; agents acting on immunophilins such as cyclosporine, tacrolimus and sirolimus; TNFalpha binding proteins such as infliximab, etanercept and adalimumab; mycophenolate and the synthetic drugs FTY720 and myriocin.

In another embodiment, the caspase-6 inhibitor can be administered in combination with an anti-cancer agent.

Suitable anti-cancer agents include, but are not limited to fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, and lavendustin.

In another embodiment, the caspase-6 inhibitor can be administered in combination with an anti- viral agent. Suitable anti-viral agents include, but are not limited to non- nucleoside reverse transcriptase inhibitors such as nevirapine, delavirdine and efavirenz; nucleoside reverse transcriptase inhibitors such as zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, and emtricitabine; protease inhibitors such as amprenavir, fosamprenavir, indinavir, Iopinavir/ritonavir, ritonavir, saquinavir and nelfinavir; nucleotide analog reverse transcriptase inhibitors such as tenofovir and adefovir, integrase inhibitors such as Raltegravir, and entry inhibitors such as Maraviroc and Enfuvirtide.

When used in combination with another active agent, the therapeutically effective dose of each agent can be lowered compared to the therapeutically effective dose of one active agent alone. Thus, side-effects can be significantly reduced. The invention also relates to a kit comprising a caspase-6 inhibitor and an active agent as described above, such as an immunosuppressive agent.

The invention will be further described by the following figures and examples, which are not intended to limit the scope of the protection defined by the claims.

FIGURE LEGENDS

Figure 1: Effect of caspase inhibitors on T cell proliferation

A) T lymphocytes from healthy donors were activated in the presence of ConA and IL-2 in the absence or presence of the different synthetic peptide inhibitors (30 μΜ). Only the broad caspase inhibitors (zVAD-fmk, and Boc-D-fmk) and the specific caspase-8 and -6 inhibitors prevented cell proliferation as determined by the incorporation of ³H-TdR. Results are expressed as the percentage of cell inhibition compared to the control (DMSO alone).

B) (a) siRNA either unrelated (Unr) or specific to the caspase-3, -6 and -8 were used to treat PBMC before cell activation. Western blots show specific inhibition of caspase-6 expression; (b) cell proliferation was measured using WST-1 assay after conA and IL-2 stimulation; (c) cell cycle was determined by flow cytometry using Propidium Iodide. Percentages show cells into S/G2/M phases after conA and IL-2 stimulation.

Figure 2: Effect of caspase 6 inhibitor on T cell proliferation

A) T lymphocytes from healthy donors were activated in the presence of CD3 mAbs and IL-2 in the absence or presence of the synthetic caspase-6 inhibitors (10 μΜ). Cell division was assessed by flow cytometry using CFSE dilution staining. Dose- response of caspase-6 inhibitor. Results show the percentages of cells dividing after 5 days.

B) Electron microscopic analysis of PBMC either unstimulated or stimulated with CD3 plus IL-2 in the absence or presence of caspase-6 inhibitor.

C) Western blots show the expression of cyclin D3 and RB phosphorylation of PBMC either unstimulated (Med) or stimulated with CD3 plus IL-2 in the absence or presence of casp6 inhibitor. Actin is used as control of loading. Caspase-6 inhibitor prevents cell cycle entry and cell division.

Figure 3: Cytokine detections after TLR stimulation.

PBMC isolated from healthy donors were stimulated overnight in the absence (No stimulation) or presence of the TLR agonists: LPS, 10 ng/ml (TLR4), and CL097, 1 g/ml (TLR7/8). Cells were treated in the absence (Med) or presence of specific caspase inhibitors. TNF-a and IL-12 expressions were analyzed among the HLA- DR⁺Lin^" subsets by flow cytometry. Similar data have been obtained with 3 healthy donors.

Figure 4: Cytokine detections after TLR stimulation. PBMC isolated from healthy donors were stimulated overnight in the absence (Medium) or presence of the TLR agonists: LPS, 10 ng/ml (TLR4), and CL097, 1 g/ml (TLR7/8). Cells were treated in the absence (DMSO, None) or presence of specific caspase inhibitors. Supernatants were collected and assessed for the detection of IL-8, IL6 and IL-10. Similar data have been obtained with 3 healthy donors.

Figure 5: Type I interferon detection after TLR stimulation.

PBMC were stimulated as in figure 3. Cells were stained for IFN-a2 and analyzed by flow cytometry among the different subsets CD11C⁺CD123^~ (dendritic cells) and CD123⁺CD11C^" (plasmacytoid cells) and including a Lineage marker (Lin). Similar data have been obtained with 2 healthy donors.

EXAMPLE

Materials and Methods

Cells and Treatments

Peripheral blood leukocytes (PBL) from normal healthy donors (Etablissement Francais du Sang) were isolated by Ficoll-Hypaque gradient centrifugation. PBLs were activated with 1 μg/ml concanavalin A (ConA) (Sigma-Aldrich) and 10 units/ml recombinant human interleukin 2 (IL-2) (Roussel-Uclaf, France). Cells were maintained as suspension cultures with RPMI 1640 (Gibco, Invitrogen) medium plus 10% FCS supplemented 100 U/mL of penicillin/streptomycin and 2 mM L-glutamine (Gibco, Invitrogen).

Pan- or specific fluoromethylketone (fmk)-conjugated caspase inhibitors (R&D) were prepared as a lOmM stock in DMSO. Caspases inhibitors zVAD-fmk (Pan- caspase), zDEVD-fmk (caspase-3), zVEID-fmk (caspase-6), zIETD-fmk (caspase-8), were all O-methylated and were use at 10μΜ one hour before T cell activation. Following a 24h rest after the siRNA transfection, the cells were activated or not with ConA and IL-2 in the presence or absence of caspase inhibitors. The cell were then analysed at day 4 after activation. RNAi Transfections

Transfections of Peripheral blood leukocytes cells were performed using the Amaxa transfection technology and the Amaxa unstimulated human T cell transfection kit (Amaxa Biosystems), according to the manufacturer's instructions. Specific suppression of caspase-3, caspase-6 and caspase-8 expression was accomplished using the transfection of a pooled siRNA targeting 4 regions of the transcript (SmartPool, Dharmacon) (0.75μg/ 10⁶ cells). Casp3 :

J-004307-06 : CCGACAAGCUUGAAUUUAU (SEQ ID NO:5);

J-004307-07 : CCACAGCACCUGGUUAUUA (SEQ ID NO:6);

J-004307-08 : GAAUUGAUGCGUGAUGUUU (SEQ ID NO:7);

J-004307-09 : GCGAAUCAAUGGACUCUGG (SEQ ID NO: 8).

Casp6 :

J-004406-06 : CAUGGUACAUUCAAGAUUU (SEQ ID NO: l);

J-004406-07 : CUGACUUCCUCAUGUGUUA (SEQ ID NO:2);

J-004406-08 : GACAUUAACUGGCUUGUUC (SEQ ID NO:3);

J-004406-09: GCAGAUGCCGAUUGCUUUG (SEQ ID NO:4).

Casp8 :

Casp8 : J-003466-13 : GGACAAAGUUUACCAAAUG (SEQ ID NO:9);

Casp8 : J-003466-14 : GCCCAAACUUCACAGCAUU (SEQ ID NO: 10);

Casp8 : J-003466-15 : GAUAAUCAACGACUAUGAA (SEQ ID NO: 11);

Casp8 : J-003466-16 : GUCAUGCUCUAUCAGAUUU (SEQ ID NO: 12).

Immunoblotting

Cells were lysed in 20mM Tris (pH 7.4), 0.5% SDS supplemented with the protease inhibitor mixture Complete (Roche Molecular Biochemicals), phosphatase inhibitor coktail 2 (Sigma- Aldrich) and in the presence of 10 units of Benzon nuclease (Novagen) for 5 min at room temperature.The samples were centrifuge 5 min at 10 000 x g and boiled for 5 min. Cell lysates were resolved by SDS-PAGE (NuPAGE 4-12% Bis-Tris gel; Novex) and transferred to nitrocellulose membranes. After blocking nonspecific sites for 1 h at room temperature with 5% nonfat milk and 0.1% Tween 20 in phosphate-buffered saline (pH 7.4), the membranes were incubated over night with rabbit polyclonal anti-caspase 6 (Cell Signaling) (1:1000). To confirm equal protein loading and transfer, membranes were subsequently reprobed with anti-actin (Sigma-Aldrich, clone AC-40) (1 : 5000). Immunoreactive proteins were detected using enhanced chemiluminescence (ECL; Amersham Biosciences) using using a DDC camera (G:Box Chemi, Biorad).

Cell Cycle Analysis and cell viability

Briefly, cells (0.5 x 10⁶) were washed in PBS and resuspended in 300 μΕ hypotonic fluorochrome solution (50 μg/mL propidium iodide in 0.1% sodium citrate plus 0.1 % Triton X-100; (Sigma-Aldrich). Samples were placed at 4°C in the dark before flow cytometric analysis of propidium iodide (PI) fluorescence of individual nuclei using a FACScan flow cytometer. The numbers refer to the percentages of cells in the S and G2/M phases. These experiments have been repeated twice, yielding similar results.

A colorimetric assay for quantification of cell viability based on the reduction of the tetrazolium salt WST-1 (Roche Diagnostics, Germany) was employed according to the manufacturer's instructions.

TLR activation

PBMCs were isolated by ficoll gradiant and then further incubated at 0.5x10⁶ cells (IFN-CC staining ) or 3xl0⁶ (TFN-CC and IL-12 staining) in the absence or presence of either LPS (TLR4 agonist: 10 ng/ml) or CL097 (TLR7/8 agonist: 1 g/ml), in the absence or presence of specific caspase inhibitors at 20 μΜ (caspase- 3: z-DEVD-fmk; caspase-6: z-VEID-fmk; and caspase-8: z-IETD-fmk) from R&D Systems.

After overnight culture, the cytokines were then quantified in the supernatants. Type I IFN was measured by ELISA (PBL Biomedical laboratories) that detects 14 out of the 15 identified human IFN-OC subtypes; IL-6, IL-8 and IL-10 was measured by using the inflammatory cytokine cytometric bead array (CBA) kit (BD Biosciences).

Concomitantly, the cells were analyzed by flow cytometry for intracellular cytokine expressions. In that case, two hours after activation, the cells were treated with BrefeldinA (BFA: 5 g/ml) a protein transport inhibitor commonly used to enhance intracellular cytokine staining signals by blocking transport processes during cell activation. In order to analyze the expression of type I IFN-a, the cells were harvested and stained using CD123-PE (clone 7G3), and CDl lc-APC (clone 5-HCL- 3) antibodies (BD Biosciences) at 4°C for 30 min. Cells were also stained for CD3- Percp, CD14-Percp and CD20-Percp for lineage negative cells (Lin ). After fixation and permeabilization, the cells were then incubated with FITC-labeled anti-IFN-a2 (clone 225.C) antibody (eBiosciences). The IFN-a staining was analyzed among the CD123⁺CD1 lCTLin^" and CD1 lc⁺CD1237Lin^" subset. In order to analyze TFN-a and IL-12 expressions, the cells were first stained with CD3-Percp, CD14-Percp and CD20-Percp for lineage negative cells (Lin ) and HLA-DR-FITC. Then the cells were fixed and permeabilized for TNF-a-PE (cloneMAbl 1, BD-Bioscience) and IL- 12-APC (clone 8.6, Miltenyi) staining. Cytokines were analyzed among the HLA- DR⁺/Lin^" subset. The acquisition was performed by a FACScalibur and the analysis of 10⁶ cells by the CellQuest software.

Results

The inventors have shown that broad spectrum caspase inhibitors (zVAD-fmk, and Boc-D-fmk) and the specific synthetic caspase- 8 and -6 inhibitors prevented T cell proliferation (Figure 1A). Caspase-6 siRNA effectively inhibited RNA expression and also prevented T cell proliferation (Figure IB), by preventing cell cycle entry and division (Figure 2).

The effect of caspase-6 inhibitor was specific to T cell activation and proliferation, contrary to the effect of caspase-3 and caspase-8 inhibitors (Figures 3 to 5). Indeed, the expression of TNF-a, IL-12 (Figure 3), IL-8, IL-6, IL-10 (Figure 4) and INF-a after stimulation were compared in the absence or presence of caspase-6 inhibitors compared to caspase-3 and caspase-8 inhibitors.

The caspase-6 inhibitor of the invention did not provoke the same undesirable effects on inflammation or on interferon secretion as caspase-3 and caspase-8 inhibitors and were thus specific.

These observations demonstrate that caspase-6 inhibitors are useful for the treatment of pathologies associated with increased T cell activation and/or proliferation.

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.

Claims

1. A caspase-6 inhibitor for use in therapy.

2. A caspase-6 inhibitor for use as an immunosuppressive agent.

3. A caspase-6 inhibitor for use in treating a T cell activation and/or proliferation disorder.

4. A caspase-6 inhibitor according to any one of claims 1 to 3, wherein said caspase- 6 inhibitor is a synthetic compound such as Z-VEID-FMK.

5. A caspase-6 inhibitor according to any one of claims 1 to 3, wherein said caspase- 6 inhibitor is a siRNA selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

6. A caspase-6 inhibitor according to any one of claims 3 to 5, wherein said T cell activation and/or proliferation disorder is selected from the group consisting of graft versus host disease, organ transplant rejection and T cell lymphoma.

7. A pharmaceutical composition for treating a T cell activation and/or proliferation disorder in a patient comprising a caspase-6 inhibitor as defined in any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

8. A kit for treating a T cell activation and/or proliferation disorder in a patient comprising a caspase-6 inhibitor as defined in any one of claims 1 to 5 and another active agent such as an immunosuppressive agent or an anti-cancer agent.