WO2008146259A2 - Malt1 specific cleavage in assay and screening method - Google Patents

Abstract

The present invention is based on the surprising finding of a proteolytic cleavage function of human Malt1. More particularly, the present invention relates to enzyme and screening assays, methods for assessing cleaving activity, methods for screening, isolated polypeptides, antibodies and inhibitors of Malt1. The present invention also relates to the use of Malt1 as a proteolytic enzyme and the use of compounds comprising a peptide comprising an amino acid sequence according to any one of SEQ ID NO: 1-47 as substrates susceptible for specific proteolytic cleavage.

Description

MALTI SPECIFIC CLEAVAGE IN ASSAY AND SCREENING METHOD

Filed of the Invention

The present invention relates to assays and screening methods, a method for detecting Maltl - specific cleavage, isolated polypeptides, antibodies and inhibitors. The present invention also relates to the use of Maltl as a proteolytic enzyme and the use of compounds comprising a peptide comprising an amino acid sequence according to any one of SEQ ID NO: 1-47 as substrates susceptible for specific proteolytic cleavage.

Background of the Invention and problems to be Solved

Triggering of the antigen receptor of B or T cells leads to the initiation of multiple signalling pathways that regulate cellular proliferation and surviyal of immature and naive lymphocytes, and the effector functions of mature B and T cells. The signalling pathway that leads from antigen-receptor triggering to the activation of transcription factors of the nuclear factor-κB (NF-icB) family has a crucial role in these processes, and is controlled by highly similar molecular events in B and T cells. Genetic deficiencies in NF-κB-family members or signalling components that act upstream of NF-κB have been linked to immune deficiencies. whereas aberrant constitutive NF-K-B activation has been associated with the development of autoimmune and neoplastic disorders. The understanding of the molecular mechanisms that control NF-κB activation in lymphocytes is therefore the focus of intense investigation. Recent studies have identified Carmal (caspasc recruitment domain, CARD, membrane- associated guanylate kinase, MAGUK, protein 1), Bcl-10 (B-cell lymphoma 10) and Maltl (mucosa-associated lymphoid tissue lymphoma translocation protein 1) as signalling compounds that have crucial and specific roles in T-cell receptor (TCR)- and B-cell receptor (BCRVinduced NF-κB activation.

Maltl -deficient lymphocytes show impaired antigen receptor-induced activation of the transcription factor nuclear factor kappa B (NF-KB) and, as a consequence, impaired cytokine production and proliferation. Moreover, chromosomal translocation and abnormal expression or activity of Maltl has been associated with formation of B-cell lymphomas of the mucosa- associated tissue.

More recently, the role of Bcl-10 and Maltl has been extended to a role in lymphocyte adhesion, to activating signalling functions in other leukocytes such as phagocytes, mast cells and natural killer cells and to a signalling function downstream of G-protein coupled receptors, such as the receptors for lysophosphatidic acid or angiotensin II in non immune cells. This suggests that these proteins may have relevant immunomodulatory functions, but also be relevant for example in inflammation, tumour promotion and blood pressure regulation.

Dhaval N. Ghosalia at al., "Functional phenotyping of human plasma using a 361-fluorogenic substrate biosensing microarray", Biotechnology and Bioengineering, 94, 6, p. 1099-10 (2006) report enzymatic activity of 10 different plasma proteases on as many as 361 different fluorogenic substrates. The goal behind this study is to provide a microarray that gives quick information on enzymatic activity of plasma for diagnostic purposes, for example. In Table 1 a peptide substrate Ac-ARSR-ACC is disclosed. This reference does not concern enzymatic activity of hMaltl, the latter occurring inside cells in nature.

WO2002/33058 reports the nucleotide sequences encoding hMaltl, which is indicated as BAA83099. This reference mentions cysteine proteases, including hMaltl, and a ligand binding to the cysteine protease and inhibiting its protease activity. However, this reference does not disclose the nature of the protease activity of hMaltl and, therefore, does not disclose a real inhibitor either.

In needs to be noted that Maltl is known to comprise a putative active-site cysteine residue in the caspase-like domain of Maltl, as established by sequence alignments with other caspases. However, attempts to demonstrate caspase-like proteolytic activity led to the conclusion that Maltl does not have aspartate-specifϊc proteolytic activity (Snipas, SJ. et al., "Characteristics of the caspase-like catalytic domain of human paracaspase", Biol. Chem. 385, 1093-1098 (2004)).

In other words, while an enzymatic activity for Maltl was hypothesised on the basis of sequence analysis, extensive experimental efforts failed to demonstrate such a cleaving activity, which is why presently the role of hMaltl in signalling pathways is obscure.

In view of the above, it is an objective of the present invention to elucidate the role of Carmal, Bcl-10, and/or Maltl signalling pathways, for example inB or T cells.

It is a further objective to determine the mechanism by way of which these cell components function in the signalling pathways.

It is a still further objective to provide a possibility of interfering in this pathway so as to be able to remedy the consequences and symptoms of abnormal expression or activity of Maltl, and/or other components of this or other signalling pathways. It is, more particularly, an objective of the present invention to provide means, such as enzyme assays or screening methods, that permit the testing or screening for bioactive principles putatively interfering in a signalling pathway of cells, such as leukocytes, and in particular lymphocytes such as B and T cells.

It is also an objective to provide research tools for assisting the search and development of bioactive principles that remedy disorders and/or diseases, such as immune deficiencies, autoimmune or neoplastic disorders. Preferably, such principles are capable of affecting the cellular proliferation and survival of lymphocytes.

It is also an objective of the present invention to provide new peptide sequences, preferably useful in research and/or medicine.

Background knowledge on the field of the present invention is found in the review publications of Thome "Carmal, BcI- 10 and Maltl in Lymphocyte Development and Activation", Nat. Rev. Immunol, 4, 348-359 (2004); and Rawlings et al, "The CARMAl signalosome links the signalling machinery of adaptive and innate immunity in lymphocytes", Nat. Rev. Immunol, 6, 799-812 (2006).

Summary of the Invention

Remarkably, the present inventors report a proteolytic activity for human Maltl. Interestingly, this activity is induced by T cell receptor (TCR) stimulation. It is also remarkable that the cleaving sequence recognized by Maltl represents a new peptide cleaving sequence. There is currently no protease known having the same cleaving sequence specificity. These findings are useful in new enzyme assays and screening methods. They are also useful for in silico screening of potential Maltl- substrates.

Accordingly, the present invention provides, in a first embodiment, a method for detecting the occurrence of Maltl -specific cleavage, the method comprising the steps of assessing the consumption of a substrate cleaved by Maltl enzymatic cleaving activity and/or the presence of a product obtained by Maltl enzymatic cleaving activity. The invention also provides an assay for conducting this method.

The present invention further provides, in a second embodiment, an enzyme assay suitable to assess the consumption of a substrate and/or the production of a product, said assay comprising a substrate comprising a peptide comprising an amino acid sequence selected from any one of SEQ ID NO: 1 -47, preferably 4-47, wherein said substrate is intended to be enzymatically cleaved after the C-terminal Arginine of said amino acid sequence.

In a third embodiment, the present invention provides a screening assay comprising a proteolytic enzyme and a substrate, said substrate comprising a peptide comprising an amino acid sequence selected from any one of SEQ ID NO: 1-47, preferably 4-47, said substrate being susceptible of being cleaved by said enzyme.

In a fourth embodiment, the present invention provides a method of assessing cleaving activity on a substrate having a bond to be cleaved following an amino acid sequence according to SEQ ID NO: 1 - 47, preferably 4-47, said method comprising the steps of contacting the substrate with a sample for which cleaving activity is to be assessed.

In a third embodiment, the present invention provides a screening assay comprising a proteolytic enzyme and a substrate, said substrate comprising a peptide comprising an amino acid sequence selected from any one of SEQ ID NO: 1-47, preferably 4-47, said substrate being susceptible of being cleaved by said enzyme.

In a fourth embodiment, the present invention provides a method of screening for a bioactive principle, the method comprising the steps of: providing a system comprising an enzyme selected from: human Maltl (SEQ ID NO:48), a non-human homologue thereof or a functional variant of any of the foregoing; exposing and/or contacting the system to a principle to be screened, measuring a proteolytic activity of the enzyme; and, optionally, - selecting a particular principle on the basis on an effect the principle exerts on the proteolytic activity.

In a fifth embodiment, the present invention provides a compound capable of inhibiting the proteolytic cleavage catalysed by Maltl . The invention also provides a compound that is capable of inhibiting the proteolytic cleavage of human BcIlO or a homologue thereof at a position corresponding to Arg228 of SEQ ID NO: 50.

In an embodiment, the present invention provides a compound compound comprising a peptide sequence according to any one of SEQ ID NO. 1-47, preferably 4-47, linked at the C- terminal end of the respective sequence to a group selected from chloromethylketone (cmk), fluoromethylketone (fink), aldehyde (-CHO), and, said compound being optionally further substituted at the N-terminal end of said peptide. In a sixth embodiment, the present invention provides a compound capable of inhibiting the proteolytic cleavage of a substrate peptide comprising an amino acid sequence selected from any one of SEQ ID NO: 1 - 47, for example 4-47, said inhibited proteolytic cleavage being at the carboxyl end of the carboxy terminal Arginine amino acid residue of said amino acid sequence. Similarly, the present invention provides a compound capable of inhibiting an enzyme selected from human Maltl (SEQ ID NO: 48), an animal homologue of human Maltl or a functional variant of any of the two aforementioned.

In a seventh embodiment, the present invention provides an isolated polypeptide selected from:

(a) a polypeptide having an amino acid sequence SEQ ID. NO 50 (BcIlO large cleaved fragment);

(b) a homologue of SEQ ID. NO 50, or,

(c) a variant of SEQ ID NO: SEQ ID. NO 50. Preferably, the polypeptide of (a), (b) and/or (c) has a C-terminal -S-R amino acid sequence at the end. More preferably, the C-terminal ending of the polypeptide corresponds to the amino acid sequence according to any one selected from SEQ ID NO: 1-47, preferably 4-47. More particularly, the C-terminal sequence is selected from the groups of preferred Maltl target sequences mentioned herein. The present invention also provides the use of such polypeptides for detecting the occurrence of enzymatic activity of Maltl .

In a further embodiment, the present invention provides a peptide having an amino acid sequence according to SEQ ID NO: 49.

In still a further embodiment, the present invention provides an isolated polypeptide selected from (a) a polypeptide having amino acid sequence SEQ ID. NO 48 including isoforms; (b) a functional homologue of SEQ ID. NO 48; and, a variant of any of the polypeptides defined under (a) or (b), wherein said polypetide has proteolytic activity, in particular human Maltl - specific proteolytic activity.

In further embodiments, the present invention provides the use of human Maltl, functional homologues or variants thereof as a specific protease, and a method of cleaving a substrate, the method comprising the step of contacting the substrate with human Maltl, a functional homologue and/or a variant thereof. The present invention also encompasses the use of human Maltl in enzyme assays. In still further embodiments, the present invention provides the use of substances comprising an amino acid sequence selected from any one of SEQ ID NO: 1 - 47, for example 4-47 as cleavable substrates, for example in enzyme assays and/or screening methods.

In yet another embodiment the present invention provides an antibody specifically binding to a product of MaIt-I specific cleavage. For example, the antibody binds to BcIlO, a homologue or variant thereof as cleaved by Maltl . Preferably, the antibody does not bind to BcIlO not cleaved by Maltl, the antibody thus specifically detecting only the cleaved specimen.

In another embodiment, the present invention provides a method of preparing and/or designing an inhibitor of Maltl proteolytic cleaving activity, the method comprising the steps of: (a) providing a peptide comprising an amino acid sequence according to any one of SEQ ID NO: 1-47, preferably 4-47 or any of the preferred sequences selected from SEQ ID NO: 1- 47 as defined in the present specification; (b) modifying said peptide, preferably by using peptidomimetics approaches, thereby obtaining a modified peptide; (c) exposing said modified peptide to enzymatically active Maltl, a homologue and or functional variant thereof; (d) determining the presence or absence of inhibition of Maltl proteolytic cleaving activity; and, if no inhibition is found, (e) repeating the previous steps, whereby in step (b) modification of the peptide is such that a modified peptide is obtained that is structurally different from any modified peptide obtained previously.

In yet another embodiment the present invention provides a method for in silico screening for putative substrates of Maltl, a homologue or variant thereof, the method comprising the steps of systematically looking for any of SEQ ID NO: 1 - 47, for example 4-47 or for the nucleotide sequences encoding any of SEQ ID NO: 1 - 47, for example 4-47 in a database comprising amino acid or nucleotide sequences of an organism, for example the genome of an organism. For example, the open reading frames of the genome of the organism may be screened.

Further aspects and embodiments of the present invention are provided in the appended claims.

Brief Description of the Drawings

Figures 1 a - c show western blots of lysates of a T cell- line (Jurkat), human cytotoxic T cell clones and a B-cell line (Raji) as indicated following stimulation of the cells with various TCR stimulants or reagents mimicking T- or B-cell activation. The stained proteins are involved in signalling and are used here to assess efficient lymphocyte activation. In all Figures, black arrowheads indicate the position of unmodified BcIlO, while white arrowheads indicate a faster migrating form of BcIlO.

Figures 2 a - e show western blots of lysates of different T cell-lines as indicated following stimulation of the cells with various TCR stimulants. In Figure 2a, cells were pre-incubated with different protein kinase inhibitors before lysis. In Figures 2b-e, cells transfected or transduced as indicated or with the expression of specific cellular proteins silenced by shRNA are compared.

Figure 3 a shows a western blot of BcIlO in a 2 dimensional SDS PAGE gel, revealing the presence of a faster migrating BcIlO species at a pi more acidic than the wildtype form of BcIlO.

Figure 3 b shows an alignment of the C-terminal amino acid sequence of human and murine BcIlO with previously identified metacaspase cleavage sites identified in Serpinl and metacaspase9 of A. thaliana (AtSerpinl and Atmc9) and in metacaspase type II of P. abies (mcIIa-Pa).

Figures 3 c and d show western blots of lysates of transduced and transfected cells. In Figure 3c, Jurkat T cells expressing either a wild-type form of BcIlO or a point mutated, non- cleavable form of BcIlO are compared following stimulation of the cells with the cell stimulants PMA (phorbol myristate acetate) and ionomycin. In Figure 3d, 293T cells expressing various FLAG- or VSV-tagged Maltl -mutated or BcllO-mutated cells are compared.

Figure 4 shows BcIlO C-terminal sequences of BcIlO obtained by immunoprecipitation of FLAG-tagged BcIlO from 293T cells cotransfected with FLAG-BcIlO and Maltl and digestion of isolated FLAG-BcIlO bands by GIu-C, followed by mass spectroscopy of digests.

Figures 5 a - c show lack of an effect of various protease inhibitors targeting lysosomal proteases (NHVleupeptin), proteasomal activities (MG 132) or caspases (Z-VAD-fmk) on the appearance of a faster migrating BcIlO species.

Figures 6 a and b show Maltl -dependent cleavage activity of lysates of T cells following T- cell activation and unstimulated control cells. For assessing cleavage activity, LRSR-peptide bound fluorogenic compound, Ac- LRSR-amc, was used. In Figure 6a, cleavage activity after various stimulation times are shown, whereas in Figure 6b incubation with T-cell stimulants was for 20 minutes and cleavage of LRSR-amc over time is compared for T cells transduced with control or Malt 1 -specific shRNA.

Figure 7 shows relative NF-KB activity as determined by dual luciferase assays of 293T cells co-transfected with various Maltl or BcIlO expression constructs and a NF-κB-luciferase construct. Reduced NF-KB activity is found in cells with non-functional, proteolytically inactive Maltl (Δcasp, H415/A, C464/A).

Figure 8 shows the adhesion on fϊbronectin-coated culture dishes of BcllO-silenced Jurkat T cells transduced with expression vectors for with wild-type BcIlO or a mutant BcIlO R228G, which cannot be cleaved by Maltl. It can be seen that cells expressing wildtype BcIlO, stimulated with anti-human CD3 and anti-CD28 or with PMA, show better adherence to fϊbronectin than BcIlO R228G mutants. These results show the implication of hMaltl - mediated enzymatic cleavage of BcIlO for the adhesion of activated T cells.

Figure 9 shows the presence of cleaved BcIlO in lymphoma samples. BcIlO cleavage was assessed in Western blot using lymphoma tissue lysates from patients with mucosa-associated lymphoid tissue (MALT) lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular cell lymphoma (FCL) or mantle cell lymphoma (MCL). Controls include lysates of non- stimulated (-) and PMA/ionomycin-stimulated (+) CD8⁺ T cell clones.

Figure 10 a and b show sequence alignments of amino acid sequences originating from different species and also from a virus. The sequences are aligned on a target cleaving sequence that can be cleaved by hMaltl . Fig. 1OA shows experimentally established cleaving sites of natural substrates in stimulated lymphocytes. Fig. 1OB shows cleaving sites identified upon artificial co-expression of hMaltl, the substrate comprising the indicated sequence, and hBcllO in 293T cells. In the case of Maltl auto-cleavage (SEQ ID NO: 61 in Fig. 10B), the wild type hMaltl is co-expressed with hBcllO. In this experiment, respective R-mutants of the proteins listed in Fig. 1OB are no longer cleaved. Data obtained with SEQ ID NO: 59 (vElO) is not definite.

Figures 11 a-f illustrate the inhibition of hMaltl cleaving activity by Maltl inhibitors of the present invention. In Fig. 11a recombinant purified GST-Maltl protein, oligomerized by binding to glutathione sepharose, was tested for LRSR-amc clavage activity in the absence (black bars) or presence (bright bars) of increasing concentrations of the inhibitor z-VRPR- fmk described herein, resulting in reduction of cleavage activity in dependence of the concentration. Figs, b and c show western blots of lysates of Jurkat and CTL cells, respectively, showing BcIlO of cells activated by PMA and ionomycin for the indicated times and exposed or not to the inhibitor z- VRPR- fink. Figs, d and e show the effect of the same inhibitor on the NF-kB and IL-2 response in Jurkat T cells activated as indicated, and Fig. f shows the effect of the presence of the inhibitor on adhesion to fϊbronectin of Jurkat T-cells following activation or not, as indicated.

Figure 12 shows immunoblot analysis of lysates of human cytotoxic T lymphocytes (CTLs) that were left unstimulated or stimulated for the indicated times with the phorbolester PMA and ionomycin. The anti-Bel 10 blot (left panel) detects BcIlO (black arrowhead), cleaved BcIlO (white arrowhead) and phosphorylated isoforms of BcIlO with a higher apparent molecular weight. The antibodies specific for cleaved BcIlO (middle panel) detect cleaved BcIlO (and higher migrating phosphorylated isoforms of cleaved BcIlO) in samples of stimulated cells, but does not cross-react with uncleaved BcIlO, since it does not detect BcIlO in lysates of unstimulated cells.

The attached sequence listing is integral part of the present specification. SEQ. ID. NOs: 1-47 show target sequences that are specifically recognized and cleaved at the C-terminal Arginine residue by Maltl. SEQ ID NO: 48 is the amino acid sequence encoding hMaltl, which was described previously. SEQ ID NO: 49 is the shorter of two proteolytic cleavage products of Maltl on hBcllO, while SEQ ID NO: 50 shows the larger of the two peptide products. SEQ ID NO: 51 is the amino acid sequence encoding hC ARMAl as previously described. SEQ ID NOs: 52-61 are extracts of sequences of various organisms, aligned for comparison and for the determination of a consensus sequence in Figures 1OA and B. SEQ. ID. NOs: 62-64 are sequence extracts of polypeptides, which sequence extracts can also be aligned with SEQ ID NOs: 52-61.

Detailed Description of the Preferred Embodiments

The embodiments of the present invention are based on the finding of a proteolytic activity of human Maltl (hMaltl) and on the finding of so far unknown peptide target cleavage sequences for which hMaltl is specific. The present inventors further found that the proteolytic activity of hMaltl plays an important role in the adaptive immune response through its contribution in signalling pathways of the B-cell and T-cell receptors. The proteolytic activity of Maltl described herein is equally important for other receptors that signal via Maltl. For example, these include receptors that signal via ITAM (immunoreceptor tyrosine-based activation motifs), such as for example receptors of the FcR family and pathogen receptors similar to Dectin-1, and G-protein coupled receptors such as for example the receptors for lysophosphatidic acid or for angiotensin II, which has pro -inflammatory effects and effects on blood pressure. The involvement of Maltl in these pathways is reviewed in an article by Wegener and Krappmarm: "CARD-BcllO-Maltl signalosomes: Missing link to NF-kB", SciSTKE2007, pe21 (2007).

For the purpose of the present invention, the expression "Malt 1 -specific cleavage" refers to cleavage resulting from enzymatic cleaving activity of the enzyme hMaltl, a homologous protein of hMaltl of another species and from a functional variant of any of the foregoing. The expression "hMaltl" encompasses different isoforms of the protein according to SEQ ID NO: 48 found in humans.

In vivo, "Malt 1 -specific cleavage" refers to intracellular enzymatic cleavage activity. Of course, enzyme assays using isolated and/or recombinant hMaltl, a functional homologue or variant thereof may be provided, so that the hMaltl specific cleavage is conducted in a system, which is free of cells and/or in the extracellular matrix.

"Malt 1 -specific cleavage" is preferably characterised by the specificity of suitable peptide target sites where cleavage can occur. Accordingly, "Maltl -specific cleavage" refers to the cleavage of molecules comprising the specific target cleaving sites mentioned herein. In particular, it refers to cleavage of molecules comprising any one of SEQ ID NO: 1-47, in particular 4-47, and/or molecules comprising any one of SEQ ID NO 52-61 (Sequence alignment of Fig. 11), preferably, comprising any one of preferred peptide target sequences of suitable substrates mentioned herein.

Accordingly, the present invention provides a method, an assay and a screening method in which the Maltl -specific proteolytic activity is assessed.

The term "assess" or "assessment" used with respect to the enzyme or screening assay of the invention and also with respect to the methods of the invention, refers to one or more activities related to the qualitative finding of presence or absence of an enzymatic activity as specified, the quantitative approximation of such activity, for example in terms of rate of product consumption, relative comparison of activities, and/or also the quantitative measurement of such activity, for example.

Proteolytic activity or proteolytic cleavage, in the context of the present invention, preferably refers to the cleavage of a covalent bond of one or more compounds of formula (I) to (IV) below,

(I) (H)

(III) (IV) wherein the covalent bond being cleaved is indicated as a dashed line and wherein Ri and R₂ refer to organic residues including organometallic compounds and complexes, and wherein R₂ can also be H. Preferably, the compounds of formula (I)-(IV) comprise an amino acid sequence selected from any one of SEQ ID NO: 1-47, in particular 4-47, and/or any one of the amino acid sequences which are underlined in Fig. 11 (from SEQ ID NOs 1, 2 and/or from SEQ ID NO: 52-61).

For example, R₂ is -Leu-R3, with R3 being an organic residue including organometallic compounds or -H. Preferably, Ri is -NH-R3. An organic residue is a hydrocarbon optionally substituted and optionally comprising one or more heteroatoms. For example, Ri and R₂ comprise and/or consist of peptides such as mono-, di-, tripeptides, oligopeptides (3-10 amino acids) and/or polypeptides (>10 amino acids). Preferably, these peptides are peptides comprising and/or consisting of L-amino acids, more preferably amino acids selected from the 20 standard proteinogenic amino acids. Ri and R₂ may be the same or different. Ri and/or R₂ may be proteins having a specific enzymatic activity. Furthermore, Ri and R₂ may be compounds having any specific biologic activity or fluorescence or phosphorescence, for example.

The covalent bond to be cleaved preferably is a C-N bond, for example a peptide bond. It may also be a C-C bond, or a C-O bond, for example an ester or ether bond. In compounds (I)-(IV), the amino acid moieties Arg, Ser and/or Thr may be chemically modified, for example in order to improve cell permeability of the substrate. For example, any one or both of the free amino group of the Arg-residues may be Cl-C4-alkylated, preferably methylated.

Herein below, compounds (I)-(IV), the principle substrates of the enzyme assay and screening method of the present invention, will also be referred to as Rj-R-S-R-R₂ and R₁-R-T-R-R₂, with R, S and T standing for Arginine, Serine, and Threonine. For the purpose of the present specification, if peptide sequences are indicated with amino acids abbreviated by the one- or three letter code from left to right, the N-terminal end of the sequence is indicated left and the C-terminal end of the sequence is indicated right, as is conventional.

Accordingly, the term "substrate" for the present invention encompasses dipeptides, tripeptides, oligopeptides and polypeptides as well as derivatives thereof. In this sense, the term substrate includes compounds comprising a peptide comprising an amino acid sequence as defined in any one selected from SEQ ID NO: 1-47, in particular 4-47. Preferably, the substrate comprises and/or consists of a tri- or an oligopeptide having 3 to 10 amino acids.

The sequences of SEQU ID NO: 1-47, preferably 4-47, are also referred herein as the target cleaving sequence.

According to a preferred embodiment, the substrate comprises a peptide comprising an amino acid sequence of Xaa-R- (SEQ ID NO: 1), wherein Xaa stands for the any one amino acid selected from the groups of S, T, V, and G. Preferably, the substrate comprises a peptide comprising an amino acid sequence of -S/T-R- (selected from the group of SEQ ID NO: 2 and 3). More preferably, the substrate comprises a peptide comprising an amino acid sequence of - S-R- (SEQ ID NO: 2), wherein said substrates are cleaved by Maltl following the carboxyl terminus of the R (Arg) residue. For example, according to an embodiment, the substrate comprises a peptide comprising an amino acid sequence of -R-S/T-R- (selected from the group of SEQ ID NOs.: 4 and 5). For example, said amino acid sequences of said substrates are provided in a larger peptide comprising further amino acids or amino acid sequences at the C-terminal of the indicated target sequence.

Preferably, the substrate comprises a peptide that comprises an amino acid sequence selected from any one of SEQ ID NO: 4, 5, 8-47, in particular any one of 5, 4, 11, 13, 23, 31, 33 and/or 43. Preferably, the substrate comprises an amino acid sequence as defined by the general consensus sequences according to SEQ ID NO: 6 and/or 7, with Xaa standing for any amino acid but preferably L, P or H. According to a preferred embodiment, the substrate comprises an amino acid sequence selected from LRSR and LRTR.

According to an embodiment, the substrate comprises an amino acid sequence of at least 5 amino acids, said five amino acids being an N-terminal Proline followed by an amino acid sequence comprising 4 amino acids according to any one of SEQ. ID. NO 8-47 (P-G-R-S-R, P-A-R-S-R, etc.), preferably, PLRSR, PHRSR, PPRSR, PLRTR, PHRTR, and/or PPRTR.

The substrate comprising an amino acid sequence according to any one of SEQ ID NO: 1- 47, preferably 4-47, may be provided in the form of a modified peptide. Modifications contemplated by the invention include, but are not limited to, pegylation (PEG linkage), glycosylation, amidation, carboxylation, phosphorylation, or addition of an acetyl, myristic, palmitic, stearic, or acidic group, creation of acid addition salts, amides, esters, in particular C-terminal esters, and N-acyl derivatives of the peptides of the invention. The peptides also can be modified to create peptide derivatives by forming covalent or noncovalent complexes with other moieties. Covalently-bound complexes can be prepared by linking the chemical moieties to functional groups on the side chains of amino acids comprising the peptides, or at the N- or C-terminus. These modifications may also occur at amino acids that form part of the target cleaving sequence according to any one of SEQ ID NO: 1- 47, preferably 4-47.

Accordingly, substrates according to the invention are BcIlO of Homo sapiens (NCBI accession number CAH71557), Mus musculus (NP 033870), Rattus norvegicus (NP 112618) or Bos Taurus (AAIl 8327), these proteins all being characterised by the target cleaving sequence LRSR. Another substrate is vElO of Equine herpesvirus 2, which contains a LRTR target sequence. Furthermore, Carmal of Homo sapiens (NP 115791), Mus musculus (NP 780571), Bos taurus (XP 593388) or Canis familiaris (XP 547005) are also potential substrates.

According to an embodiment, the substrate is (a) human BcIlO (hBcllO), (b) a non-human homologue thereof, or (c) a variant of any of (a) or (b). HBcllO corresponds to the peptide according to SEQ ID NO: 50 with the short peptide SEQ ID NO: 49 being added at the C- terminal end of SEQ ID NO: 50.

A homologue of a specific peptide of reference (the "original" peptide) mentioned herein is another peptide occurring in another species than the original peptide Homo sapiens but sharing the ancestry with the original peptide. Preferably, a homologue according to the present invention may or may not perform the same function in its natural environment as the original peptide reported herein. Rodent homologues of human BcIlO, for example, are indicated above.

A variant peptide is a peptide differing from an original peptide in that the original peptide has been chemically modified. For example, amino acid residues of the original peptide have been deleted, added or changed, but which still is capable of fulfilling substantially the same function as the original peptide. Variant polypeptides encompass peptides in which specific amino acid residues have been replaced by similar amino acids, such as amino acids having similar properties. For examples, a variant peptide would be a peptide in which an amino acid having a hydrophobic residue has been replaced by an amino acid having another hydrophobic residue, or in which an acidic amino acid by another, similarly acidic amino acid, and so on. Variant peptides also include compounds resulting from chemical modification of an original peptide, in particular original peptides comprising markers suitable to detect and/or quantify the peptide or a fragment thereof. A variant also encompasses a peptide obtained by gene shuffling using a nucleotide sequence encoding the original peptide. Gene shuffling is often used to improve the desired activity of an enzyme by creating mutated genes encoding a variant of the original enzyme and selecting those variants having improved characteristics.

Preferably, in a variant peptide, the domain or amino acid sequence corresponding to that of the original peptide (excluding added sequences e.g. with fusion peptide variants) has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 97% amino acid sequence identity with the original peptide as referred to herein.

The reference to a protein and/or peptide, for the purpose of the present invention, also includes a reference to naturally occurring isotypes. Preferably, these isotypes are able to fulfil the same function as required for the purpose of the present invention, or the isotype is provided in a variant form capable of fulfilling the function.

According to a preferred embodiment, the substrate comprises a marker molecule, for example marker peptides, suitable for assessing, preferably quantifying, the consumption of the substrate and/or the production of the product.

For example, suitable marking systems could be substrates in which cleavage induces the loss of fluorescence activated energy transfer (FRET) between suitable donor/acceptor pairs that would be added on suitable sites within the substrate which are preceding and following the cleavage site (for example the N- and C-terminus of a substrate peptide). This would allow to assess a loss of FRET upon cleavage because of the resulting separation of acceptor and donor moieties. A similar principle could be used to change (for example, destroy or obtain) any physical, chemical, or biological (e.g. enzymatical) property depending on proximity of two subunits/protein parts for functionality. For example, it is possible for a marker of hMaltl -specific proteolytic activity to obtain measurable physical, chemical or biological property, such as fluorescence, enzymatical or other biological activity through cleavage of the substrate.

It would also be possible to immobilise the substrate via one particular part of it (for example the free amino terminus of the peptide) and to assess proteolytic release of a measurable enzymatic or radioactive or fluorescent activity that has been covalently or not covalently linked to the substrate (for example to its C-terminus as part of Ri) and that would be released into the supernatant upon incubation with samples containing proteolytically active Maltl.

For example, the substrate comprises a fluorescent compound, for example 7-amino-4- methylcoumarin (AMC) or a fluorescent derivative thereof, linked to the C-terminal Arginine of the target cleaving sequence, and wherein substrate consumption or product production is assessed by measuring, approximating and/or comparing fluorescence intensity of cleaved AMC or the fluorescent derivative thereof.

An antibody specifically binding to a product of Maltl -specific cleavage is encompassed by the invention and may also be used as a marker for detecting MaIt-I cleaving activity, in particular to a product obtained from Maltl specific cleavage. Preferably, the antibody does not bind to a substrate from which said product is obtained by Maltl -specific cleavage. Preferably, the antibody of the invention specifically binds to cleaved BcIlO, such as the polypeptide of SEQ ID NO: 49, but does not bind to a BcIlO not having the C-terminal amino acid sequence of SEQ ID NO:49. This C-terminal amino acid sequence preferably involves a sequence of the at least 4, more preferably the at least 6 and most preferably the at least 8 amino acids of the C-terminus of SEQ ID NO: 49.

In the assay and the method of screening of the present invention, a substrate consumption and/or product production is preferably catalysed by human Maltl, a homologue thereof and/or a variant thereof. For example, a variant hMaltl comprising and/or consisting essentially of the caspase-like domain of hMaltl or of a homologue thereof may be suitable. The caspase-like domain of hMaltl is the sequence stretching from aa 336 - 572 of SEQ ID NO: 48. Depending the authors and sequence analysis program used, the caspase-like domain of hMaltl may comprise a few amino acids more or less on either side. These caspase-like domains provide the cleaving activity of hMaltl, homologues and/or functional variants thereof. Variants comprising all or part of the caspase-like domain are therefore examples of functional variants of hMaltl. Of course, in variants comprising all or part of aa 336-572 of SEQ ID NO: 48, individual amino acids may be exchanged and/or omitted, while the cleaving activity is retained. For example, amino acids may be classified as polar and apolar, as acidic, neutral and (weakly) basic. It is common to provide peptide variants in which amino acids having similar properties while retaining the activity. Such variants are also included herein.

Other enzymes, so far not described, may have the same cleaving activity as defined above and may be used instead. With the teaching of the present invention it is now possible to more specifically search and identify other enzymes having the same activity.

Accordingly, the enzymatic activity reported herein may be accomplished by hMaltl according to SEQ ID NO: 48. Various iso forms of hMaltl have been reported so far, for example under accession number BAA83099 in the NCBI protein database. Isoforms of proteins mentioned herein are also encompassed by the present invention.

The assay of the present invention may be provided in the form of a kit comprising various components, one of which being, for example, a substrate as defined above susceptible of hMaltl -specific cleavage. The term assay as used in this description also encompasses a kit.

The enzyme having hMaltl -specific cleaving properties may be a component of the assay, the assay being provided in the form of a kit comprising various components. Preferably, however, the enzyme may be included in a sample to be tested and is, in this case, provided by the user of the assay.

The assay may be used to assess the activity of an enzyme by exposing cells to a substrate as defined herein. Cells may be any cells of interest, including human and/or non-human cells. In particular, the cells may be human, animal, plant and/or bacterial cells. The cells may be provided in the form of a sample of human or animal, for example, rodent, tissue. The sample to be tested may be provided in the form of a slice of tissue of a human or an animal. The sample may contain blood cells, in particular lymphocytes, for example B- or T-lymphocytes. In particular, the sample may be a tumor tissue sample, for example a lymphoma sample.

Cells to be tested may or may not be pre-treated ("activated") before testing enzymatic hMaltl -specific cleaving activity. For example, cells may be exposed to specific substances for testing the effect of these compounds on the hMaltl -specific cleaving. Such substances may be substances activating TCR- or BCR-mediated cell signalling cascades, such as those mentioned in the examples below. Such substances may be part of the assay or may be separately obtained and applied by a user of the assay. Such activating substances may form part of the assay.

Preferably, cells are preferably destroyed, for example lysed, to provide a sample for which the occurrence of Malt 1 -specific cleavage can be detected. Since Maltl -specific cleavage occurs in the intracellular space, destruction of cells naturally expressing Maltl is preferred in order to assess the consumption of a substrate and/or the presence of a product of Maltl- specifϊc cleavage. Lysis facilitates the contact of the substrate with hM alt 1 -specific cleaving enzymes present in the cells.

Preferably, the enzymatic activity is assessed in presence of a specific buffer enabling hMalt- specifϊc enzymatic activity. Buffers suitable for assessing hMaltl -specific enzymatic activity are preferably buffers with physiological pH and ionic strength, for example 20 mM Tris/HCl and 150 mM NaCl, and preferably comprising a reducing agent, for example dithiothreitol or mercaptoethanol. Preferably, the buffer contains Ca²⁺ ions. Furthermore, buffers may contain inhibitors of proteases and/or phosphatases to avoid degradation of the substrate by other, non-specific proteases.

Suitable protease and/or phosphatase inhibitors not inhibiting hMaltl -specific cleavage and/or suitable buffers may be part of the assay.

The present invention also provides an assay, in particular a screening assay and/or a screening method. One goal of these aspects of the present invention is to provide a possibility of assessing the effect of a principle to be screened on hMaltl -specific cleaving.

In a step of the screening method, a system comprising an enzyme capable of hMaltl -specific cleaving is provided. For example, the system may comprise hMaltl, a homologue and/or derivative there from. The enzyme may, for example, be provided in the form of cells as defined above, said cells comprising the enzyme. For example, cells transfected or transduced to express increased amounts of hMaltl may be used. Instead of using cells, it is preferable to use purified and/or isolated enzyme, for example enzyme produced in a suitable expression system. Preferably, the enzyme is recombinant.

The system comprising the enzyme preferably comprises co-factors assisting the proteolytic cleavage. In particular, the system preferably comprises a peptide selected from human Carmal (SEQ ID NO: 51), a non-human homologue thereof and/or a functional variant thereof. It was shown in the present invention that Carmal, in particular its binding to BcIlO and its oligomerization, may play a role in optimal hMaltl -specific cleavage. As with the enzyme providing hMaltl -specific cleaving, Carmal may be provided in the form of cells expressing Carmal, or in a recombinant, preferably purified form.

The system also preferably comprises a peptide selected from BcIlO, a non-human homologue thereof and/or a functional variant thereof, since the capacity of Carmal to bind BcIlO (which mediates formation of a Carmal -BcI 10-Maltl complex) seems to be required for activation-induced BcIlO cleavage by Maltl.

Of course, isolated Carmal, hMaltl and further selected components may be provided together in a purified preparation. These components may be provided in any method and/or assay of the present invention. For example, Carmal and/or BcIlO may be provided in a purified preparation, or even all three together.

Preferably, if recombinant Maltl is used for the purpose of the present invention, for example in the screening method, a recombinant Maltl form is selected that contain oligomerization domains or that contain protein domains that can be inducibly oligomerized by addition of suitable bi-valent or multivalent compounds. The recombinant Maltl may also be chemically modified to allow inducible oligomerization. The necessity of oligomerization is explained by the fact that a role of Carmal seems to be the induction of an oligomerization of Maltl.

The components used for the screening method, such as the enzyme as well as the substrate and optional cofactors, buffers, and the like may be used as indicated with respect to the assay described above. For example, the system comprising the enzyme is an aqueous solution or suspension to which a purified form of the enzyme and, optionally cofactors have been added. Accordingly, the present invention also relates to a screening assay suitable for conducting the screening method. The screening assay may also be provided in the form of a kit.

Preferably, the screening method of the present invention comprises the step of comparing the measured proteolytic activity of the enzyme to a control proteolytic activity measured in absence of the principle to be screened.

Preferably, the screening method of the present invention comprises the step of comparing the measured proteolytic activity of the enzyme to a control proteolytic activity measured in presence of a control principle of which the effect (e.g. activation, inhibition or stabilisation) on the enzyme is known.

Generally, the screening method comprises the step of assessing if the principle to be screened exerts an effect on said proteolytivc activity by comparing the measured proteolytic activity with the control proteolytic activity and by finding an effect if said activity and said control activity differ from each other.

The effect that the principle to be screened exerts may be selected from an inhibitory effect or an activating effect, wherein an inhibitory effect is found if said proteolytic activity is lower than said control proteolytic activity and/or wherein an activating effect is found if said proteolytic activity is higher than said control proteolytic activity.

The present invention also provides an inhibitor, in particular (1) a compound capable of inhibiting the proteolytic cleavage of a substrate polypeptide at the carboxyl end of the

Arginine amino acid residue at a cleavage site having an amino acid sequence selected from any one of SEQ ID NO: 1-11 , or from any one of 4, 5, and 8-11; (2) a compound capable of inhibiting the proteolytic cleavage of human BcIlO or a homologue thereof at a position corresponding to Arg228 of SEQ ID NO: 49 (hBcllO, cleaved).

Such an inhibitor can be an antibody and/or a peptide comprising an amino acid sequence selected from any one of SEQ ID NO: 1^47, preferably 4-47.

The inhibitor may be a monoclonal or polyclonal antibody.

Preferably, the inhibitor is a compound capable to bind to the proteolytically active site of human Maltl, including compounds binding to or covering sites Cys464 and/or His415 of SEQ ID NO: 48. Alternatively, the inhibitor binds to or associates with other portions of Maltl, for example portions outside the caspase-like domain, said compound inhibiting proteolytic activity of Maltl by said binding or association to said other portions of Maltl, for example by inducing conformational changes affecting the proteolytically active site.

The present invention particularly relates to inhibitors comprising the target peptide sequence. These are substrate-mimetic inhibitors that comprise the Maltl recognition sequence (any one of SEQ ID NO: 1-47, in particular 4-47, such as SR and preferably RSR, RTR, in particular RPR) linked to reactive groups such as chloromethylketone (cmk) or fluoromethylketone (frnk), for example, that irreversibly inhibit; or aldehyde (CHO) reactive groups that reversibly inhibit the active site of Maltl. To increase cell permeability, the peptides can be N-terminally modified with Ac (acetyl) or Z (benzyloxycarbonyl) groups and the amino groups of the arginine residues can be alkylated, for example with a Cl-ClO, preferably Cl- C5 alkyl, preferably methylated, for example. Other ways of increasing cell permeability are also encompassed within the scope of the present invention. As another example, the peptides may be biotinylated, for example N-terminally. A biotinylated inhibitor is useful as a research tool to inhibit and subsequently isolate caspases, for example by pull-down assays using avidin- or streptavidin-coupled supports. According to yet another embodiment, the inhibitor comprises a fluorescent grouping. For example, fluorescent FITC (Fluorescein isothiocyanate) may be linked N-terminally to the peptide group of the inhibitor to yield FITC-RSR-fink, or FITC-VRPR-fmk, for example.

According to an embodiment, at least one amino group of an arginine residue of said peptide is alklyated, for example with an Cl-ClO, preferably a C1-C5 alkyl.

According to an embodiment, the inhibitor is the compound X-VRPR-Y, wherein X is selected from H, Ac, Z, biotyl, a fluorescent substituent and from other substituents providing a desired functionality to the compound, and Y is selected, independently of X, from fmk, cmk and -CHO.

In general, inhibitors of the present invention may be functionalised as desired, preferably at the N-terminal end of the peptide fragment, in order to adapt it to a specific purpose, which increases its utility as a research tool.

An inhibitor that showed inhibition of Maltl -specific cleaving is z-VRPR-ftnk. This inhibitor may be used as such, for example in research and/or in vitro testing, such as in vitro enzymatic assays of this invention. This inhibitor may also be useful for in vivo applications, for example as a medicament and in the treatment and/or prophylaxis of the conditions mentioned below. According to preferred embodiment, an inhibitor having improved in vivo characteristics, such as fewer side effects and comparatively low toxicity and overall improved pharmacological properties is designed on the basis of the indicated z-VRPR-fink by peptidomimetics. Such an inhibitor may be synthesized according to the methodology disclosed in the following publications: Bioorganic & Medicinal Chemistry, Volume 5, Issue

5, May 1997, Pages 797-807. E. Ponnusamy, U. Fotadar, A. Spisni and D. Fiat, A novel method for the rapid, nonaqueous tert-butoxycarbonylation of some O-17-labeled amino-acids and 0-17-NMR parameters of the products, Synthesis (Stuttgart) 1 (1986), pp. 4849.

Accordingly, the present invention encompasses peptidomimetics of the target sequence peptides of SEQ ID NO 1-47, in particular 4-47, in particular RSR, RTR and RPR, optionally comprising further amino acids. The peptidomimetics may comprise the same chemical modifications as indicated for the peptide substrate sequences SEQ ID NO 1-47 above (pegylation, glycosylation, etc.) The present invention also provides a method for obtaining an inhibitor capable of inhibiting Malt 1 -specific cleaving and/or a compound for preventing and/or treating the conditions indicated below. The method comprises the steps of designing and testing peptidomimetic compounds for their Malt 1 -specific cleaving inhibitory activity. Drug design according to peptidomimetics has been discussed in the art. For example, Walensky et al., Science 2004; Sep 3;305(5689): 1411-3, describe hydrocarbon stapling for obtaining peptidomimetics having improved pharmacological properties. Li L. et al, Science 2004 Sep 3. describe a small molecule mimic of Smac, a pro-apoptotic protein. Rubin-Carrez C. published on the internet a report entitled "des peptidomimetiques peptidiques aux mimetiques non peptidiques: sur Ia voie du medicament.", which sets out some of the methodology currently use for developing peptidomimetics: http://ist.iriserm.fr/BASIS/elgis/fqmat/atelier/DDD/1235.doc

For example, the substitution of classic peptide bonds by isosteric amides resistant to peptidases is mentioned in this report, amongst many other ways. The above references are entirely incorporated herein by reference. The present invention thus encompasses peptidomimetic compounds of the peptide-based inhibitors reported herein.

The method of screening of the present invention may be a method for screening for principles having at least one of the effects selected from: (1) immunomodulation, (2) modulation of inflammatory processes, (3) suppressing an immune reaction, (4) reducing or avoiding an immune reaction against a transplant, (5) reducing and/or increasing blood pressure, (6) reducing cellular proliferation. Medicaments with these effects may be prepared from active principles selected by the screening method of the present invention.

The screening method of the present invention is susceptible of identifying inhibitors and/or principles that are useful as a medicament. For example, the principle may be used for the prevention or treatment of (1) lymphomas and/or leukaemia, (2) an immunological disorder, (3) multiple sclerosis, (4) an autoimmune disease, (5) diabetes, (6) allergies, (7) hemic disease, in particular hemic tumours, (8) tumours in general, (9) inflammatory disorders, for example colitis, arthritis and/or artheriosclerosis, or combinations of two or more of the aforementioned.

The present invention also provides inhibitors identified by the screening method, the inhibitors potentially having the characteristics indicated above. Accordingly, inhibitors may be used as medicaments.

The present invention will now be illustrated by way of examples. These examples do not limit the scope of this invention which is defined by the appended claims. Examples

Example 1 : Analysis of Human T cell Lines and Observation of Faster Migrating BcIlO Species

Cells Used for the Experiments

Blood samples were obtained from two HLA-A*0201 positive healthy volunteers containing readily detectable frequencies of EBV- and CMV-specifϊc CD8⁺ T-cells in ex-vivo analyses. From these blood samples, peripheral blood mononuclear cells (PBMCs) were obtained by density centrifugation using Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) and cryopreserved in RPMI 1640 supplemented with 40% FCS and 10% DMSO (lxl0⁷-2xl0⁷ cells per vial) until further use. Phycoerythrin-labeled HLA-A*0201/peptide multimers were prepared with NLVPMVATV peptide (CMV/pp65₄95_503) and were kindly provided by P. Guillaume and I. Luescher (Ludwig Institute for Cancer Research, Epalinges, Switzerland). Multimer-positive CD8⁺ T-cells were sorted by a FACSVantage^® SE using CellQuest software (Becton Dickinson), cloned by limiting dilution, and expanded in RPMI 1640 medium supplemented with 8% human serum (HS), 150 U/ml of recombinant human IL-2 (rIL-2; a gift from GlaxoSmithKline), 1 μg/ml phytohemagglutinin (PHA; Sodiag, Losone, Switzerland) and lxlO⁶/ml irradiated allogeneic PBMC (3000 rad) as feeder cells. Positive EBV- and CMV-specifϊc T-cell clones (cytotoxic T lymphocytes, CTL) were periodically (every 15 days) re-stimulated in 24-well plates with PHA, irradiated feeder cells, and rIL-2.

The human cell lines Jurkat (J77 clone 20), Hut78 and Raji cells (all are gifts of Oreste Acuto, Pasteur Institute, Paris) were grown at 37°C in RPMI 1640 supplemented with 10% FCS and antibiotics.

Stimulation of Cells

For short term stimulations, cells were re-suspended at a density of 5x10⁷ cells/ml in RPMI with 0.5% serum and pre-warmed for 10 min at 37°C before addition of the stimulating agent.

T-cell stimulation was initiated by addition of PMA (10 ng/ml, Alexis) and ionomycin (lμM, Calbiochem) (P/I) or a combination of anti-human CD3ε (10 μg/ml of OKT3, Apotech) and anti-CD28 (10 μg/ml of CD28.2, Immunotech) antibodies (CD3/CD28), immediately followed by addition of 5 μg/ml of cross-linking goat anti-mouse antibody (Southern Biotech). Pooled human CMV-specific CTL clones were stimulated by 10 μg/ml MHC -pep tetramers (HLA-A* 0201 /peptide multimers with NLVPMVATV peptide corresponding to CMV/pp65₄95_503, kindly provided by P. Guillaume and I. Luescher, Ludwig Institute, Epalinges, Switzerland) together with 10 μg/ml of anti-CD28 (CD28.2, Immunotech).

Raji B cells were stimulated using P/I as described above. Stimulations were conducted for various time intervals, depending on the cell type, as indicated in the figures.

Lvsis of Stimulated Cells and SDS-PAGE

Stimulations were stopped by addition of ice-cold Tris-NaCl buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl), and pelleted cells were lysed in Tris-NaCl lysis buffer containing 1 % NP40, proteinase inhibitors (Complete, Roche) and phosphatase inhibitors (cocktails I and II, Sigma).

Postnuclear cell lysates were boiled with reducing SDS-sample buffer and analyzed on 16x20 cm gels by 15% SDS-PAGE according to Anderson, N. L. & Anderson, N. G. Analytical techniques for cell fractions. XXII. Two-dimensional analysis of serum and tissue proteins: multiple gradient-slab gel electrophoresis. Anal Biochem 85, 341-54 (1978).

Western Blots of Cell-Lysates

Primary antibodies used are monoclonal mouse anti-phospho ERK (Sigma), rabbit anti- phospho-lKBα (5A5, Cell Signalling), rabbit anti-IκBα (Sigma), and rabbit anti-BcllO (H- 197, Santa Cruz). Western blots were revealed using HRP-coupled goat anti-mouse or anti- rabbit antibodies (Jackson Immunoresearch).

Results

The results are shown in Figure 1 a-c. A faster migrating BcIlO species, that starts to appear after 15 min of stimulation (Fig. 1, a and b) was observed.

In all Figures, black arrowheads indicate the position of unmodified BcIlO, while white arrowheads indicate a faster migrating form of BcIlO.

The appearance of this BcIlO species was delayed with respect to IKB phosphorylation (Fig. Ia), and persisted for several hours after TCR engagement (not shown). Generation of the faster migrating BcIlO species could also be observed in human CTL clones stimulated with anti-CD3 and anti-CD28, PMA and ionomycin or specific MHCp tetramers (Fig. Ib). Stimulation of human Raji B cells with PMA and ionomycin led to similar results (Fig. Ic).

Example 2: Relevance of Signalling Components Up-stream BcIlO in the NF-KB pathway

To test the relevance of signalling components acting upstream and downstream of BcIlO in the NF-κB pathway, and in particular the association of BcIlO with its upstream binding partner Carmal through a caspase recruitment domain (CARD), a number of further experiments were conducted.

Experiments

In a first approach, Jurkat cells were pre-incubated with either the pan-PKC inhibitor bisindolyl-maleimide VIII (BIM) or with G56976, an inhibitor of classical (Ca2+- and DAG- dependent) PKC isoforms, before stimulation with CD3/CD28 as indicated in Example 1. For pre-incubation, the cells were incubated with 5 mM G56976 (Calbiochem) or 500 nM Bisindoleylmaleimide VIII acetate (Alexis), or solvent control for 30 min at 37° in RPMI with 0.5 % serum directly prior to stimulation.

In a further approach, Jurkat cells were lentivirally transduced with Carmal -specific or control shRNA constructs as described in Rueda, D. et al. "BcIlO controls TCR- and FcγR- induced actin polymerisation". J. Immunol. 178, 4373-4384 (2007). These cells were stimulated with CD3/CD28 or with P/I as described in Example 1.

Moreover, two types of Malt 1 -silenced Jurkat cells were exposed to a CD3/CD28 stimulation as described in Example 1 above. The first type (#1) was silenced by lentiviral silencing vector for Maltl as described by Rueda, D. et al (see above), and the second, independent Maltl silencing vector (#2) was obtained from OpenBiosystems (Clone TRCN0000073826).

Furthermore, Jurkat cells expressing constructs for VSV-tagged wt or CARD-mutated, dominant negative Carmal (Carmal -DN) were prepared by retroviral transduction of Jurkat cells, as disclosed in Gaide, O. et al. "CARMAl is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation" Nat. Immunol. 3, 836-843 (2002)). This construct has a non- functional CARD motif that does not allow binding to BcIlO which is required for the formation of a Carmal -BcI 10-Maltl complex (reviewed in Thome 2004, cited above). These cells were stimulated with CD3/CD28 as described in Example 1. In another experiment, Jurkat cells were lentivirally transduced with a VSV-tagged expression construct for the Carmal coiled coil domain (or empty vector), see also A Tanner, M. J., Hanel, W., Gaffen, S. L. & Lin, X. CARMAl Coiled-coil domain is involved in the oligomerization and subcellular localization of CARMAl, and is required for T cell receptor- induced NF-kappa B activation. J Biol Chem (2007). This construct lacks the N-terminal CARD domain of Carma 1 as well as part of the C-terminus and prevents oligomerization of endogenous Carmal in the cell by interfering with the oligomerization process.

Cell lysis and western blots was done as in Example 1. The primary antibody for the experiment with VSV-tagged wt or VSV-tagged Carmal-DN and Carmal -coiled-coil construct is monoclonal mouse anti-VSV (P5D4, Sigma). For Carmal, a rabbit anti-Carmal (AL220, Alexis) antibody was used.

Results & Conclusions

The outcome of these studies can be seen in Figures 2 a-d.

Consistent with a described role for PKCtheta (a Ca²⁺-independent, so-called novel PKC isoform) upstream of the Carmal -BcI 10-Maltl complex in T cells, BIM but not G06976 prevented the generation of the faster migrating BcIlO species (Fig. 2a). Moreover, its generation was prevented by shRNA-mediated silencing of Carmal (Fig. 2b) and strongly reduced by the expression of a CARD-mutated, dominant negative mutant of Carmal that is unable to bind BcIlO (Fig 2e). Expression of a Carmal coiled coil construct, which prevents Carmal oligomerization, also had a clear inhibitory effect (Fig. 2f). Together, these findings suggest that formation of the faster migrating BcIlO species depends on its recruitment by Carmal and on Carmal oligomerization. In addition, silencing of Maltl expression by two independent shRNAs clearly impaired the generation of the observed BcIlO modification.

Example 3: Identifying cleavage by Maltl after Arg228 close to C- terminus of BcIlO

3.1 Two-D gel electrophoresis - detecting cleavage by Maltl close to C- terminus of BcIlO

In order to determine if proteolytic cleavage was at the origin of the faster migrating BcIlO species, Jurkat cells were stimulated for 30 min by PMA and ionomycin and cell extracts were prepared and analysed by two dimensional gel electrophoresis and anti-Bel 10 Western blot as described by Rueda et al. 2007, see above. The Western blot is shown in Figure 3 a. As in Figures 1 and 2, the black arrowhead indicates the presence of un-phosphorylated, intact BcIlO, and the white arrowhead indicates the faster migrating species, which was detected at a pi that was more acidic than the wildtype form of BcIlO. Transparent arrowheads show two previously described phosphorylation isoforms of BcIlO that are induced by TCR engagement (Rueda et al. 2007, see above).

The faster migrating species thus most likely resulted from proteolytic cleavage at the C- terminus of BcIlO, which contains a positively charged Arg residue (Arg 232) whose proteolytic removal could explain the more acidic pi value (Fig. 3b).

3.2 Identification of Ar g228 of BcIlO as site of proteolytic cleavage

To determine whether cleavage occurred after the preceding Arg residue 228, we stably expressed a BcIlO wt form or an Arg 228/Gly mutant in Jurkat cells and tested the effect of PMA/ionomycin stimulation on these constructs.

The BcIlO point mutant was generated by a standard double PCR approach, subcloned into expression vectors derived from pCR3 (Invitrogen) and verified by sequencing in both directions before further subcloning into the lentiviral vector pRDI_292 (a gift of R. Iggo, ISREC, Epalinges, Switzerland) that allows expression of constructs under the EFl promotor. The equivalent lentiviral expression vector for BcIlO wt has been described (Rueda et al. 2007). Transduction of Jurkat T cells was performed as described before (Rueda et al. 2007).

Stimulation and Western blots were done as described in Example 1 above, using monoclonal mouse anti-FLAG (M2, Sigma).

The result is shown in Figure 3c. It can be seen that stimulation induced the formation of a faster migrating species for the FLAG-tagged wildtype form, but not the Arg 228/Gly mutant of BcIlO, supporting the idea of a proteinase-dependent cleavage of BcIlO after Arg 228.

3.3 Testing of various Maltl- and Bell 0 mutants

Human embryonic kidney cells (293T cells) were co-transfected with combinations of VSV- tagged Maltl and FLAG-tagged BcIlO mutant constructs that were prepared with the same method (double PCR approach, etc) as detailed above. Transfection of 293T cells was done as in Rueda et al. 2007. Transfected samples included: cells transfected with mock plasmid (devoid of functional Maltl), with VSV-MaIt 1 (functional wt Maltl), with VSV-Maltl point mutated at Cys 464 (putative active site) of Maltl (= "C464/A"), and cells with VSV-Maltl with a deletion of the caspase-like domain (aa 336 to aa 566 of Maltl) (= "Δcasp") all with FLAG-tagged wt BcIlO. Further cells were prepared by transfection with mock plasmid and VSV- wt Maltl, both with FLAG-tagged, point mutated BcIlO (R228/G).

The outcome of these experiments is shown in Figure 3d. BcIlO cleavage was observed when wt BcIlO, but not the Arg 228/Gly mutant of BcIlO was co-expressed with Maltl in 293T cells, while point mutation of the neighboring residues Thr 229 and VaI 230 of BcIlO did not affect cleavage (data not shown). In this setting, BcIlO cleavage was dependent on the presence of the intact Maltl caspase-like domain, since deletion of this domain (deletion of aa 336 to 566) or mutation of the putative active site Cys 464 of Maltl abolished BcIlO cleavage.

3.4 Analysis of C-terminal GIu-C peptide of FLA G-tagged Bell 0

In order to analyse the C-terminal nature of the fast and slow migrating forms of BcIlO, FLAG-tagged BcIlO was immunoprecipitated from 293T cells co-transfected with FLAG- BcIlO and Maltl .

In particular, pooled lysates from ten 10 cm dishes of FLAG-BcIlO- or FLAG-BcIlO- and VSV-MaIt 1 -transfected 293T cells were used for anti-FLAG immunoprecipitation on 120 μl of anti-FLAG-agarose (Sigma). Samples were processed on 15% Anderson SDS-PAGE (Anderson et al. 1978, see above), and Coomassie-stained bands corresponding to uncleaved and cleaved BcIlO were cut out and analyzed by in-gel GIu-C digest and mass spectrometric (MALDI-MS) analysis (TOPLAB, Munich, Germany).

The results are shown in Figure 4. Uncleaved BcIlO contains the full length C-terminal GIu-C peptide of 1590.89 D (aa 220-233), while the sample with the Maltl-induced cleaved form of BcIlO contains a C-terminal GIu-C peptide that stops after Arg 228, with a molecular weight of 1019.65 D (aa 220-228). This experiment confirms Arg 228 of BcIlO as the cleavage site.

In conclusion of the experiments conducted under Example 3 Maltl is identified as an Arg- specific protease cleaving BcIlO after Arg 228.

3.5 Testing of effect of other proteases on BcIlO cleavage

For testing if the observed proteolytic activity is an indirect effect of Maltl on other proteases, Jurkat cells were pretreated with the lysosomal or proteasomal inhibitors (NH₄ ⁺ / leupeptin or MG132) or solvent control before stimulation with PMA and ionomycin, and postnuclear lysates were analyzed by Western blot as indicated. As is shown in Figure 5 a, inhibition of lysosomal proteases through pre-incubation of cells with NH₄CI and leupeptin prevented BcIlO degradation, but did not affect generation of the faster migrating BcIlO species corresponding to BcIlO cleavage. Inhibition of the proteasome by MG 132 also did not affect BcIlO, but efficiently stabilized P-I B (Figure 5b).

In an analogue experiment, Jurkat cells were pretreated with the pan-caspase inhibitor zVAD- fmk or solvent control, and stimulated with anti-CD3 and anti-CD28 antibodies or hexameric recombinant FasL for the indicated times. Postnuclear lysates were analyzed by Western blot with the indicated primary antibodies (Figure 5c). A non-specific band (n.s.) of the anti-Bell 0 blot served as a loading control. It can be seen from Figure 5c that pretreatment of the cells with zVAD-fmk had no effect on anti-CD3/CD28-induced reduction in the apparent molecular weight of BcIlO, while it potently inhibited FasL-induced caspase-8 activation.

In conclusion, it is unlikely that BcIlO cleavage is mediated by an indirect effect of Maltl on other proteases, since inhibition of lysosomal, proteasomal or caspase-type proteolytic activities did not affect BcIlO cleavage.

Example 4: In vitro protease activity assay

In this example, the capacity of Maltl to cleave a BcllO-derived fluorogenic substrate upon T-cell activation was assessed.

Unstimulated or PMA/ionomycin-stimulated Jurkat T cells were mechanically lysed in cleavage assay buffer (50 mM Tris-HCl pH 7.4, 60 mM NaCl, 100 mM CaCl₂ and 10 mM

DTT) using a dounce homogenizer. Maltl protease activity was determined upon addition of

50 μM Ac-LRSPv-amc (amc = 7-amino-4-methylcoumarin) (Peptides International, Inc.) and incubation at 30⁰C for 4 h, using a Synergy microplate reader (BioTek). The experiment was repeated for various time-intervals of stimulation (0, 10, 30, 45 and 60 min). In another setting, stimulation was for 20 minutes and the cleavage of Ac-LRS R-amc (Assay time) was followed during indicated times.

The results are shown in Figure 6a and 6b. As can be seen, stimulation of Jurkat cells using PMA and ionomycin led to a specific increase in proteolytic activity (Fig. 6a) that was impaired when Maltl expression was silenced (the values in Fig. 6b are values of those samples activated for 20 min normalized to unstimulated controls). Thus, T-cell activation leads to activation of the Maltl proteolytic activity and to a Maltl -dependent cleavage of BcIlO after Arg 228. Example 5: Functional consequence of Maltl -dependent BcIlO cleavage on IKB phosphorylation

In order to assess if Maltl -mediated cleavage of BcIlO had an influence on IKB phosphorylation, and, consequently, on NF-kB activation, the following experiment was conducted.

Jurkat cells stably transduced with wildtype BcIlO or its Arg 228/Gly mutant (see Example 3 and Figure 3d) were stimulated with anti-CD3 and anti-CD28 for different times (0, 5, 10, 15, 30 and 60 minutes), and cell extracts prepared as in Example 1 and analyzed by Western blot for BcIlO, P-IKB, P-ERK and P-JNK as indicated. Antibodies are as in Example 1. For P- JNK, a rabbit anti-phospho-JNK primary antibody (Biosource) was used.

It was found that intensities and kinetics of IκBα phosphorylation and degradation are similar in Jurkat cells stably expressing the wt or Arg 228/Gly form of BcIlO, indicating that the classical IκBα-dependent pathway of NF-κB activation was unaffected by BcIlO cleavage.

Example 6: Functional consequence of Maltl -dependent BcIlO cleavage on relative NF-KB activity

In another experiment designed to assess the influence of Maltl -mediated cleavage of BcIlO on NF-KB response, the following experiment was conducted.

For the experiments, cells with inactive Malt 1 as described in Example 3 above were used: VSV-Maltl (wt with functional Maltl), Δcasp, C464/A, and also another mutant, H415/A, which is mutated by a point mutation at the active site, obtained by the standard double PCR approach as described with respect to C464/A above (Example 3).

These cells were co-transfected with a renilla-luciferase and an NF-κB-firefly luciferase construct and mock plasmid to normalize transfection efficiency as disclosed by Rueda et al. (2007). Relative NF-KB activity was determined 24 h after transfection by dual luciferase assay (Rueda et al. 2007).

The result can be seen in Figure 7. It can be seen that inactivation of the Maltl catalytic activity by deletion of the caspase-like domain (Δcasp) or mutation of the active site residues Cys 464 or His 415 resulted in total NF-KB activation levels of only 50-65 % of the maximum level achieved with wt Maltl. Malt 1 dependent cleavage is, therefore, necessary for an optimal NF-KB response.

Example 7: Effect of BcIlO cleavage on T-cell adhesion

7. / Adhesion assays:

Central areas of tissue culture dishes (35-mm x 10-mm) were coated for 90 min at 25 ⁰C with 50 μl of fϊbronectin solution (40 μg/ml; Roche). Coated dishes were washed three times with PBS (without Ca2+ and Mg2+) and were blocked for 2 h with 1% (wt/vol) BSA in PBS. Cells were washed with Hank's balanced-salt solution (Gibco) and were left unstimulated or were stimulated at 37 ⁰C for 30 min with MnCl₂ (1 mM) or PMA (100 ng/ml), or for 5 min with anti-human CD3 and anti-CD28 (see example 1). Subsequently, cell suspensions were added to fibronectin-coated tissue culture dishes followed by incubation for additional 30 min at 37 ⁰C. Non-adherent cells were removed by washing of the dishes three to five times with Hank's balanced-salt solution. Adherent cells were counted in six separate fields with a microscope with an ocular counting reticule.

7.2 Results

After activation with either anti-CD3 and anti-CD28 or PMA, cells expressing the R228G mutant had less adhesion to fibronectin than did cells expressing wild-type BcI- 10 (Fig. 8). In contrast, manganese-induced adhesion was similar in cells expressing wild-type BcI- 10 or the R228G mutant (Fig. 8).

Example 8: Detection of cleaved BcIlO in B-cell lymphomas

In order to know whether the cleaved form of BcIlO is detectable in B-cell lymphomas samples from biopsies were taken and analysed as follows: Lymphoma samples from lymph node, spleen and gastrointestinal biopsies from patients with mucosa-associated lymphoid tissue (MALT) lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL) or follicular cell lymphoma (FCL) were immediately frozen without additives and stored for 3-24 months before lysis. Cells were lysed in 50 mM Hepes pH 7.6, 4 mM EDTA, 150 mM NaCl und 1% Triton X-100 supplemented with the following proteinase and phosphatase inhibitors: 2 mM PMSF, 2 μg/ml aprotinin and 5 μg/ml leupeptin, 2 mM Na3VO₄, 100 mM NaF und 50 mM Na₄P₂O₇. Lysates were stored at -8O⁰C before analysis by SDS-PAGE and Western blot. Controls include lysates of non-stimulated (-) and PMA/ionomycin-stimulated (+) CD8⁺ T cell clones. The result can be seen in Figure 9. BcIlO cleavage was clearly detectable in two samples of MALT lymphomas characterized by the t (11; 18) translocation which leads to cIAP2-Maltl fusion proteins with strong NF-KB activating capacity. Moreover, BcIlO cleavage was detectable in 11 out of 12 additional lymphoma samples tested, suggesting that BcIlO cleavage might be a frequent and relevant event in the pathogenesis of B-cell lymphomas.

Example 9: Inhibition of the proteolytic activity of Maltl by small peptide inhibitors

To test the efficiency of inhibitors, Jurkat T cells stimulated or not with PMA and ionomycin for 20 min are mechanically lysed in cleavage assay buffer (see example 4) in the absence or presence of increasing concentrations of a irreversble cell permeable inhibitor peptide (z- VRPR-fmk). This inhibitor was designed based on an optimal substrate cleavage sequence reported for the plant metacaspase AtMC9 (see ref. by Vercammen et al. 2006, J. MoI. Biol., 364(4):625-36). Maltl protease activity is determined as described in example 4. To test the efficiency of the peptide inhibitor on living cells, Jurkat T cells or human primary cytolytic T cells (CTLs) are pre-incubated for Ih with indicated concentrations of z- VRPR- fink (see above), before stimulation with PMA and ionomycin for 20 min, lysis of the cells and detection of BcIlO cleavage by Western blot (see example 1). Moreover, experiments are performed to assess the effect of the inhibitor on anti-CD3/CD28-induced NF-KB activation in Jurkat T cells, on IL-2 production of Jurkat cells stimulated by incubation with antigen- presenting cells (Raji cells in combination with the superantigen SEE) and on stimulation- induced adhesion to fibronectin (see example 7)).

In the presence of the inhibitor peptide, we observe a reduction of the activation-induced LRSR-amc cleavage activity (Fig. Ha) and the activation-induced cleavage of BcIlO (Fig. 11, b and c). Moreover, the inhibitor impairs the capacity of T cells to activate NF-KB, to produce

IL-2 and to adhere to fibronectin upon T-cell activation (Fig. 11, d-f). This suggests that small peptide inhibitors with homology to the Maltl recognition sequence can be used to inhibit

Maltl activity in vitro and in living cells.

Example 10: Detection of Maltl -specific cleavage with a cleavage-specific anti-Bell 0 antibody

Antibodies that exclusively recognize the cleaved form of BcIlO were produced. To generate these antibodies, a rabbit was immunized with a short peptide comprising the 8 amino acids aminoterminal to the cleavage site (the peptide sequence is MFLPLRSR). The peptide was coupled to the carrier protein KLH and used for repeated immunization of a rabbit. Peptide synthesis, coupling to the carrier and immunization were performed by Eurogentech according to standard procedures. The affinity and/or specificity of the antibodies contained in the serum was further increased by affinity-purification over a CNBr-Sepharose column to which the peptide used for immunization was covalently bound.

In the experiment shown in Figure 12, the affinity-purified antibodies were used to assess the presence of cleaved BcIlO in lysates of human cytotoxic T lymphocytes (CTL) that were either left unstimulated or stimulated for the indicated times with PMA and ionomycin, as described above. Proteins contained in the lysates were separated electrophoretically and transferred to nitrocellulose. Samples were analysed by Western blot using a commercially available anti-BcllO antibody (left panel in Fig. 12), the affinity-purified anti-cleaved BcIlO antibodies (middle panel) or an irrelevant antibody to demonstrate equivalent protein content of samples (right panel). The anti-BcllO antibody detects BcIlO in all samples, and stimulation-induced phosphorylation isoforms of BcIlO that are present predominantly in the lysates of activated cells. In contrast, the anti-cleaved BcIlO antibodies detect only the cleaved (unphosphorylated and phosphorylated) forms of BcIlO, which are present in stimulated cells, but absent from unstimulated cells.

Antibodies as produced in this example are useful for the specific detection of BcIlO cleavage, which serves as a readout for MALTl activity, for purposes of research, drug development and diagnosis. With such antibodies, cleaved BcIlO can for example be specifically detected in samples of lysates of lymphocytes that have been stimulated to mimic T-cell activation (see Figure 12). Furthermore, such antibodies can be used in a quantitative immunoassay to detect cleaved BCLlO in cell lysates. Such an assay, for example an ELISA assay, could be performed with lysates of cells that were pre-incubated, before stimulation, with compounds acting as Maltl inhibitors, and thus serve as a screening assay for therapeutically useful Maltl inhibitors. Another interesting application of antibodies specific for cleaved BcIlO could be their use in a immunohistochemistry-based assay to diagnose the status of Maltl activation in suitable cells or tissue samples obtained from patients suffering for example from lymphoma, auto-immune disease or inflammatory conditions.

Overall conclusion

The examples demonstrate that Maltl has arginine-directed proteolytic activity and that an increase in this activity is specifically induced by T-cell activation. A C-terminal LRSR₂₂8 motif at the C-terminus of BcIlO was identified as a Maltl target sequence. Maltl -dependent BcIlO cleavage is crucial for T-cell activation, since mutation of the BcIlO cleavage site prevents Maltl -dependent BcIlO processing and results in impaired TCR-induced T-cell adhesion. Moreover, inhibition of the proteolytic activity of Maltl using a Maltl inhibitor impairs various parameters of T-cell activation, including NF-KB activation, IL-2 production and T-cell adhesion. Importantly, processed BcIlO is also detectable in various forms of B- cell lymphoma. Thus, Maltl is essential for lymphocyte activation by cleavage of BcIlO and of additional protein substrates, and therefore represents an interesting target of immunomodulatory drug development.

Claims

Claims

1. A method for detecting the occurrence of Malt 1 -specific cleavage, the method comprising the steps of assessing the consumption of a substrate cleaved by Maltl enzymatic cleaving activity and/or the presence of a product obtained by Maltl enzymatic cleaving activity.

2. The method of claim 1 , wherein the substrate comprises a marker molecule, including marker peptides, suitable for assessing, preferably quantifying, the consumption of the substrate and/or the presence of the product.

3. The method of claim 1 or 2, wherein the substrate is a compound which, when cleaved, loses or obtains a chemical, physical or biological property, including properties selected from fluorescence, catalytic and/or other biologic activity, said property depending on the close physical proximity or on the absence of such proximity, respectively, of the two parts of the substrate that precede and follow the cleavage site and which physical proximity or absence of proximity is thus lost or obtained, respectively, by MaIt-I specific cleavage.

4. The method of claim 1, wherein the substrate is human BcIlO or a homologue or a variant thereof.

5. The method of any one of the preceding claims, which comprises the step of providing an antibody specifically binding to the product obtained by Maltl cleaving and detecting said antibody bound to said product and/or the product bound to said antibody.

6. The method of any one of the preceding claims, for detecting the occurrence of intracellular Maltl -specific cleavage, comprising the step of providing a sample of destroyed cells, in particular a cell lysate, and assessing the consumption of a substrate cleaved by Maltl -specific enzymatic cleaving activity and/or the presence of a product obtained by Maltl enzymatic cleaving activity in the sample of destroyed cells.

7. The method of any one of the preceding claims, wherein the substrate comprises a peptide comprising an amino acid sequence of Xaa-R- (SEQ ID NO: 1), wherein Xaa stands for the any one amino acid selected from the groups of S, T, V, and G, and wherein said substrate is cleaved by Maltl following the carboxyl terminus of the R.

8. The method of any one of the preceding claims, wherein the substrate comprises a peptide comprising an amino acid sequence of -S/T-R- (selected from the group of SEQ ID NO: 2 and 3), and wherein said substrate is cleaved by Maltl following the carboxyl terminus of the R.

9. The method of any one of the preceding claims, wherein the substrate comprises a peptide comprising an amino acid sequence of -S-R- (SEQ ID NO: 2), and wherein said substrate is cleaved by Maltl following the carboxyl terminus of the R.

10. The method of any one of the preceding claims, wherein the substrate comprises a peptide comprising an amino acid sequence of -R-S/T-R- (selected from the group of SEQ ID NOs.: 4 and 5), and wherein said substrate is cleaved by Maltl following the carboxyl terminus of the R at the carboxyl terminus of said amino acid sequence.

11. The method of any one of the preceding claims, wherein the substrate comprises a peptide comprising an amino acid sequence selected from any one of SEQ ID NO: 4-47, and wherein said substrate is cleaved by Maltl following the carboxyl terminus of the R at the carboxyl terminus of said amino acid sequence.

12. An assay for conducting the method of any one of claims 1-10.

13. The assay of any one of claim 12, comprising an enzyme selected from the group of human Maltl (SEQ ID NO: 48), a homologuous protein of human Maltl of another species, and from a functional variant of any of the foregoing.

14. The assay of any of any one of claims 12-13, further comprising a peptide selected from human Carmal (SEQ ID NO: 51), a non-human homologue thereof and/or a functional variant thereof.

15. The assay of any one of claims 12-14, comprising human or non-human cells expressing at least one selected of said enzyme, substrate and optional peptide.

16. A method of assessing cleaving activity on a substrate having a bond to be cleaved following an amino acid sequence according to any one of SEQ ID NO: 1-47, preferably 4- 47, said method comprising the steps of contacting the substrate with a sample for which cleaving activity is to be assessed.

17. The method of claim 15, wherein the sample comprises cells selected from human, animal, plant and/or bacterial cells, and wherein intracellular cleaving activity of these cells is assessed.

18. A screening assay comprising a proteolytic enzyme and a substrate, said substrate comprising a peptide comprising an amino acid sequence selected from any one of SEQ ID NO: 1-47, preferably 4-47, said substrate being susceptible of being cleaved by said enzyme.

19. The screening assay of claim 18, wherein the proteolytic enzyme is a recombinant protein and/or is provided in a substantially purified form.

20. A method of screening for a bioactive principle, the method comprising the steps of: - providing a system comprising an enzyme selected from: human Maltl (SEQ

ID NO:48), a non-human homologue thereof or a functional variant of any of the foregoing; exposing and/or contacting the system to a principle to be screened, measuring a proteolytic activity of the enzyme; and, optionally, selecting a particular principle on the basis on an effect the principle exerts on the proteolytic activity.

21. A compound capable of inhibiting the proteolytic cleavage catalysed by Maltl .

22. The compound of claim 21, which is capable of inhibiting cleavage of human BcIlO or a homologue thereof at a position corresponding to Arg228 of SEQ ID NO: 50.

23. A compound comprising a amino acid sequence according to any one of SEQ ID NO. 1-47, preferably 4-47, linked at the C-terminal end of the respective sequence to a group selected from chloromethylketone (cmk), fiuoromethylketone (fink), aldehyde (-CHO), and, said amino acid sequence being optionally further substituted.

24. The compound of claim 23, comprising a peptide comprising said amino acid sequence, in which the N-terminus of said peptide is substituted by a group selected from H, Ac (acetyl), Z (benzyloxycarbonyl) and biotinyl.

25. The compound of any one of claims 21-24, comprising an amino acid sequence selected from any one of SEQ ID NO: 1, 2, 3, and/or from any one of SEQ ID NO: 4-47.

26. The compound of any one of claims 21-25, comprising an amino acid sequence selected from -R-S-R- and -R-T-R-.

27. The compound of any one of claims 21-26, which is X-VRPR-Y, wherein X is selected from H, Ac, Z, biotyl, a fluorescent substituent and from other substituents providing a desired functionality to the compound, and Y is selected, independently of X, from fink, cmk and -CHO.

28. The compound of any one of claims 21-27 for use as an inhibitor for inhibiting Maltl- specific proteolytic cleavage.

29. Use of hMaltl, a homologue and/or variant thereof, as a specific protease.

30. Use of compounds comprising a peptide comprising an amino acid sequence according to any one of SEQ ID NO: 1-47, preferably 4-47, as substrates susceptible for

Malt 1 -specific proteolytic cleavage.

31. An antibody specifically binding to a product of Malt- 1 -specific cleavage.

32. The antibody of claim 31, which does not bind, under conditions conducive for the specific binding of an antibody to a binding substrate, to a substrate from which said product is obtained by Malt- 1 -specific cleavage.

33. The antibody of any one of claims 31-32, which specifically binds to cleaved BcIlO.

34. An isolated polypeptide selected from:

(a) a polypeptide having an amino acid sequence SEQ ID. NO 49;

(b) a homologue of SEQ ID. NO 49, or,

(c) a variant of SEQ ID NO: SEQ ID. NO 49, wherein the polypeptide of (a), (b) and/or (c) has a C-terminal -S-R amino acid sequence at the end.

35. Use of the polypeptide of claim 34 as target for detecting the occurrence of enzymatic activity of hMalt 1.

36. A compound which, when cleaved by hMaltl, loses or obtains fluorescence, a catalytic, chemical, physical or biologic property depending on the close physical proximity or on the absence of such proximity, respectively, of the two parts of the substrate that precede and follow the cleavage site and which physical proximity or absence of proximity is thus lost or obtained, respectively, by cleavage by Maltl .

37. The compound of any one of claims 36, comprising a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1-47, preferably 4-47.

38. A method of preparing and/or designing an inhibitor of Maltl proteolytic cleaving activity, the method comprising the steps of: (a) providing a peptide comprising an amino acid sequence according to any one of SEQ ID NO: 1-47, preferably 4-47 or any of the preferred sequences selected from SEQ ID NO: 1-47 as defined in the present specification; (b) modifying said peptide, preferably by using peptidomimetics approaches, thereby obtaining a modified peptide; (c) exposing said modified peptide to enzymatically active Maltl, a homologue and or functional variant thereof; (d) determining the presence or absence of inhibition of Maltl proteolytic cleaving activity; and, if no inhibition is found, (e) repeating the previous steps, whereby in step (b) modification of the peptide is such that a modified peptide is obtained that is structurally different from any modified peptide obtained previously.