WO1994006906A1 - DNA ENCODING MURINE PRECURSOR INTERLEUKIN 1β CONVERTING ENZYME - Google Patents

Abstract

Complementary DNA (cDNA) encoding the murine precursor interleukin-1β (pre-IL-1β) converting enzyme (ICE) is isolated from a murine cDNA library. A cDNA, encoding the full-length open reading frame (ORF) for the nascent ICE as well as the 20 kDa subunit and the 10 kDa subunit of ICE are identified and sequenced. ICE is useful in the conversion of pre-IL-1β into mature IL-1β. The murine ICE cDNA is useful in obtaining the genomic sequence. The recombinantly produced murine ICE is useful in the identification of inhibitors of ICE activity.

Description

TITLE OF THE INVENTION

DNA ENCODING MURINE PRECURSOR INTERLEUKIN IB

CONVERTING ENZYME

BACKGROUND OF THE INVENTION

Mammalian interleukin-1 (IL-1) is an immunoregulatory protein secreted by certain cell types as part of the general inflammatory response. The primary cell type responsible for IL-1 production is the peripheral blood monocyte. Other non-transformed cell types have, however, been described as releasing or containing IL-1 or IL-1-like molecules. These include epithelial cells (Luger et al., J. Immunol. 127: 1493-1498 [1981], Le et al, J. Immunol.138: 2520- 2526 [1987] and Lovett and Larsen, J. Clin. Invest. 82: 115-122 [1988], connective tissue cells (Ollivierre et al., Biochem. Biophys. Res. Comm. 141: 904-911 [1986], Le et al, J. Immunol. 138: 2520-2526 [1987], cells of neuronal origin (Giulian et aL, J. Exp. Med. 164: 594-604 [1986] and leukocytes (Pistoia et al, J. Immunol. 136: 1688-1692 [1986], Acres et al, Mol. Immuno. 24: 479-485 [1987], Acres et l, J. Immunol. 138: 2132-2136 [1987] and Lindemann et aL, J. Immunol. 140: 837-839 [1988]. Transformed cell lines have also been shown to produce IL-1. These include monocytic leukemia lines P388D, J774, THP.l, U-937 (Krakauer and Oppenheimer, Cell. Immunol. 80: 223- 229 [1983] and Matsushima et aL, Biochem. 25: 3242-3429 [1986], EBV-transformed human B lymphoblastoid lines (Acres, et aL, J. Immunol. 138: 2132-2136 [1987]) and transformed murine keratinocytes (Luger et aL, J. Immunol. 125: 2147-2152 [1982]).

Biologically active IL-1 exists in two distinct forms, IL-lcc with an isoelectric point of about 5.2 and IL-lβ with an isoelectric point of about 7.0 with both forms having a molecular mass of about 17,500 (Bayne et al., J. Exp. Med.163: 1267-1280 [1986] and Schmidt, J. Exp. Med. 160: 772 [1984]). The polypeptides appear evolutionarily conserved, showing about 27-33% homology at the amino acid level (Clark et aL, Nucleic Acids Res. 14: 7897-7914 [1986]). Mammalian IL-lβ is synthesized as a cell associated precursor polypeptide of about 31.5 kDa (Limjuco et al., Proc. Natl. Acad. Sci. USA 83: 3972-3976 [1986]). Precursor IL-lβ is unable to bind to IL-1 receptors and is biologically inactive (Mosley et aL, J. Biol. Chem. 262: 2941-2944 [1987]). Biological activity appears dependent upon some form of proteolytic processing which results in the conversion of the precursor 31.5 kDa form to the mature 17.5 kDa form.

Recent studies suggest that the processing and secretion of IL-lβ is specific to monocytes and monocytic cell lines (Matsushima et aL, J- Immunol. 135:1132 [1985]), For example, fibroblasts and keratinocytes synthesize the IL-lβ precursor, but have not been shown to actively process the precursor or secrete mature IL-lβ (Young et aL, J. Cell Biol. 107:447 (1988) and Corbo et aL, Eur. J. Biochem. 169:669 [1987]).

Several observations support the hypothesis that the processing and secretion of IL-lβ occurs by a unique pathway distinct from that used by classical secretory proteins. IL-lβ molecules from five different species do not contain hydrophobic signal sequences (Lomedico et aL, Nature 312:458 [1984], Auron et al., Proc. Natl. Acad. Sci. USA 81; 7907 [1984], Gray et aL, J. Immunol. 137:3644 [1986], Maliszewski et al., Mol. Immunol. 25:429 [1988], Mori et al., Biochem. Biophys. Res. Commun. 150:1237 [1988], and Furutani et al., Nucleic Acid Res. 13:5869 [1985]. Furthermore, neither the human nor the murine IL-lβ precursors, when synthesized in vitro, translocate across competent microsomal membranes, and despite the presence of N-linked carbohydrate addition sites, do not contain N-linked carbohydrate. Finally, light and electron microscopy studies immunolocalize IL-lβ to the cytoplasm and fail to demostrate IL-lβ in organelles that are involved in classical secretion (Bayne et al., J. Exp. Med. 163: 1267-1280 [1986] and Singer et aL, J. Exp. Med. 167: 389 [1988]).

In activated human monocytes, pulse-chase experiments suggest that IL-lβ secretion may be linked to processing. These experiments show that the intracellular pool of unprocessed precursor is chased to extracellular mature IL-lβ (Hazuda et aL, J. Biol. Chem. 263: 8473 [1989]. IL-lβ precursor is occasionally found extracellularly but does not appear to contribute to the formation of 17 kDa IL-lβ unless incubated at high concentrations in the presence of excess trypsin, chymotrypsin or collagenase (Hazuda et aL, J. Biol. Chem. 264: 1689 [1989], Black et aL, J. Biol. Chem. 263: 9437 [1988] and Hazuda et aL, J. Biol. Chem. 265: 6318 [1990]). However, none of these proteinases appear capable of generating mature IL-lβ terminating with Ala^.

Enzyme (ICE), that is capable of cleaving the IL-lβ precursor at AspH6-Alall^'7, _{as we}y _{as at a} h_omoι₀g_OUS site at Asp^-Gly^, and generating mature IL-lβ with the appropriate amino terminus at Ala* ^ has now been identified in human monocytes. The Asp at position 116 has been found to be essential for cleavage, since substitution of Ala (Kostura et L, Proc. Natl. Acad. Sci 86: 5227-5231 [1989] or other amino acids (Howard et aL, J. Immunol., 147. 2964-9, 1991) for Asp inhibits this cleavage event.

The substrate specificity of human ICE has been defined with the use of peptides that span the cleavage site of the enzyme. Two features of peptide substrates are essential for catalytic recognition by the enzyme. First, there is a strong preference for aspartic acid adjacent to the cleavage site, in that any substitution of this residue in the IL-lβ precursor and peptide substrates leads to a substantial reduction in the rate of catalysis (Kostura et aL, Proc. Natl. Acad. Sci. 86: 5227-5321 [1989]; Sleath et aL, J. Biol. Chem. 265, pp. 14526- 14528 [1990]; Howard et aL, J. Immunol., 147, pp. 2964-2969 [1991]). There is an equally stringent requirement for four amino acids to the left of the cleavage site, whereas methyiamine is sufficient to the right. The minimal substrate for the enzyme, Ac-Tyr-Val-Ala-Asp-NH3-CH3, is the best peptide substrate with a relative Vmax/Km similar to that of the IL-lβ precursor itself (Thornberry, et aL, Nature 356, 768-774, [1992]).

ICE is a cysteinyl proteinase by the following criteria: (1) The diazomethylketone Ac-Tyr-Val-Ala-Asp-COCHN2 is a potent, competitve, irreversible inhibitor of the enzyme, (2) inactivation of the enzyme by iodoacetate is competitive with substrate, and (3) the catalytically active Cys reacts selectively with [^C] iodoacetate more than 10 times faster than do other cysteines or dithiothreitol (Thornberry et aL, Nature 356, pp. 768-774, [1992]).

Human ICE has been purified from THP.l monocytic cells by conventional chromatography through a DEAE-5PW HPLC column, followed by a SP-5PW HPLC column. Two proteins, 20 kDa and 10 kDa in relative mass (Mr) track with enzyme activity. Purification of these proteins by RP-HPLC, followed by tryptic peptide mapping and sequence analysis, yielded primary amino acid sequence information with which to clone the enzyme. The cloning of human ICE has revealed that the enzyme is a unique heterodimeric protease consisting of 19.8 (p20) and 10.2 (plO) kDa subunits which are derived from a single 45 kDa (p45) proenzyme. The p20 subunit of human ICE contains the catalytic thiol (Cys^^) t,_ut the plO subunit is clearly required for enzymatic function since, upon dilution, the subunits dissociate and enzyme activity is lost. Saturating levels of substrate or inhibitor prevent this dissociation, suggesting that both subunits participate in binding or catalytic interactions at the active site. In the proenzyme, potential ICE-like cleavage sites flank both subunits, as well as a p22 amino-terminally extended form of the p20, suggesting that autoproteolysis may be involved in generating the heterodimeric (ρ20:pl0) form of the enzyme. Support for this hypothesis comes from the fact that the p45 protein can serve as substrate for the p20:pl0 form of the enzyme. The p45 protein is cleaved to yield multiple products, all of which are congruent with the purified forms of ICE or intermediates predicted to result from single cleavages of the proenzyme (Thornberry et al., supra). A tetrapeptide aldehyde transition-state analogue (Ac-Tyr- Val-Ala-Asp-CHO) was shown to be a reversible, selective and potent inhibitor of ICE with a Ki of 0.76 μM. This inhibitor was used to make an affinity ligand (Ac-Tyr-Val-Lys-Asp-CHO) with which to purify active ICE. Due, in part, to the enzyme's unusual substrate specificity, the affinity column can be used to purify ICE in a single step from a crude cellular lysate. This is the method of choice for purifying catalytically active enzyme (Thornberry, et aL, Nature 356, pp. 768- 774, [1992]).

The tetrapeptide aldehyde inhibitor can prevent the processing of the human IL-lβ precursor in blood monocytes stimulated with heat-killed S. aureus. The tetrapeptide aldehyde causes a dose dependent inhibition of release of mature II- lβ into plasma with an IC50 of 4 μM. (Thornberry, et aL, Nature 356, pp. 768-774, [1992]). This demonstrates that ICE is necessary and sufficient for the generation of mature IL-lβ from activated human monocytes.

ICE activity has not yet been demonstrated in species other than man. Two recent reports have investigated the ability of murine macrophages to process murine pIL-lβ. Peritoneal macrophages activated with LPS released a 20 kDa a form of IL-lβ whose amino terminus was not characterized [Beuscheer et al., (1990), J. Immunol., 144. pp. 2179-2183]. However a more recent study has provided strong evidence for a murine form of ICE in that treatment of the murine macrophage line J774 with ATP and LPS resulted in apoptosis and cleavage of the murine IL-lβ precusor at Asp 17_y_aιl 18 yielding a 17.5 kDa product [Hogquist et aL, (1991), Proc. Natl. Acad. Sci. U.S.A., 88, pp. 8485-8489].

OBJECTS OF THE INVENTION

It is, accordingly, an object of the present invention to provide a cDNA encoding murine ICE, the recombinantly produced murine ICE being capable of converting pre-IL-lβ to biologically active mature IL-lβ with Val^° as the amino-terminal amino acid. An additional object of the present invention is to provide expression vectors containing cDNA encoding full length murine ICE, or the individual 20 kDa and 10 kDa subunits of the enzyme. A further object of the present invention is to provide recombinant host cells containing cDNA encoding full length pre-IL-lβ, ICE and/or the individual 20 kDa and 10 kDa subunits of the enzyme. An additional object is to provide a method for the coexpression of murine ICE and IL-lβ in a recombinant host cell to produce biologically active IL-lβ. A further object of the present invention is to provide isolated 20 kDa murine ICE subunit, and isolated 10 kDa murine ICE subunit. An additional object of the present invention is to provide full length murine ICE. Another object is to provide monospecific antibodies which bind to either the murine ICE 20 kDa or the 10 kDa subunit, and the use of these antibodies as diagnostic reagents.

SUMMARY OF THE INVENTION

Complementary DNAs (cDNAs) are identified from a mouse cell line cDNA library, which encode the full length form, from which the individual 20 kDa and 10 kDa subunits of murine ICE are derived. The cDNAs are fully sequenced and cloned into expression vectors for expression in a recombinant host. The cDNAs are useful to produce recombinant full length murine ICE, as well as the individual 20 kDa and 10 kDa subunits of the murine enzyme.

BRIEF DESCRIPTION OF THE DRAWING

Fig. 1. Cleavage of murine and human IL-lβ precursors following incubation with cytosolic extracts from pentoneal exudate cells (PECS), IC21, and J774 cells, as well as affinity-purified human ICE as a positive control.

Fig. 2. Substrate specificity of murine and humen ICE; time course of cleavage of the murine and human IL-lβ precursors by each enzyme. Fig. 3. Determination of the K_j of Ac-YVAD-CHO for murine ICE.

Fig. 4. Active-site labeling of native murine ICE with an irreversable, active site inhibitor to delineate the structure of active murine ICE.

Fig 5. Addition of Ac-YVAD-CHO inhibiting the release of mature, 17.5 kDa IL-lβ from PECs cultured ex vivo and activated by LPS.

A. Dose-dependent in the level of secreted IL-lβ with increasing concentrations of Ac-YVAD-CHO.

B. Immunoblot analysis of intracelluar IL-lβ levels in the presence of and absence of 100 μM inhibitor.

C. Immunoblot analysis of secreted IL-lβ in the presence and absence of 100 μM inhibitor.

Fig. 6. Comparison of primary amino acid sequence of murine and human ICE. The p20 and plO subunits are boxed and the Asp-X cleavage are underlined. Indentical amino acids are boxed; related amino acids are shaded.

Fig. 7. Northern blot analysis of murine and human ICE mRNA.

Fig. 8. Recombinant murine ICE is active, as demonstrated by the incubation of radiolabeled murine IL-lβ precursor with E. coli extracts in the presence and absence of 200 nM Ac-YVAD-CHO.

Fig. 9. Purification of active recombinant p20:pl0 murine

ICE from E. coli extracts by affinity chromatography following immunoblot analysis to identify the p32, p20 and plO ICE proteins.

Fig. 10. Characterization of the structure and junction of active recombinant murine ICE.

A. Silver-strain gel of affinity purified murine ICE.

B. Cleavage of recombinant murine IL-lβ precursor by recombinant murine ICE.

C. D. & E. Molecular mass determination of the p20 (C) and plO (D) subunits of recombinant murine ICE and the 17.5 kDa cleavage prodct of murine IL-lβ (E).

Fig. 11. Recombinant murine ICE (Met - His^{1 2}) expressed in E. coli is active as demonstrated by the incubation radiolabelled murine IL-lβ precursor with an E. coli extract.

DETAILED DESCRIPTION

The present invention relates to cDNA encoding murine pre-IL-lβ converting enzyme (ICE) which is isolated from IL-1 producing mouse cells. Murine ICE, as used herein, refers to an enzyme which can specifically cleave the peptide bond between the aspartic acid at position 117 (Asp 11 ) ^d th_e valine at position 118 (Val " °) of murine precursor IL-lβ, and the peptide bond between Asp at position 27 (Asp²⁷) and Gly at position 28 (Gly²⁸). Human ICE refers to an enzyme that can cleave human precursor IL-lβ at the Asp²⁷-Gly²° and Asp ^ "-Ala ^ ' peptide bonds, which are analogous to the cleavages described for the murine II- lβ precursor.

As used herein, all amino acid three letter and single letter designations conform to those designations which are standard in the art, and are listed as follows:

The amino acid sequence of murine IL-lβ is known (Gray et aL, J. Immunol. 137: pp. 3644-3648 [1986]). Murine cells capable of producing DL-lβ include, but are not limited to, keratinocytes, endothelial cells, mesangial cells, thymic epithelial cells, dermal fibroblasts, chondrocytes, astrocytes, glioma cells, mononuclear phagocytes, granulocytes, T and B lymphocytes and NK cells. Transformed murine cell lines which produce D -lβ include, but are not limited to, monocytic leukemia lines such as J774, IC21 and WEHI-3, Al, PUS-1.8, RAW 309 Cr.l, RAW 264.7, WR19M.1. and transformed murine keratinocytes. The preferred cells for isolating murine ICE- encoding DNA of the present invention include mouse macrophages and WEHI-3 cells with the most preferred cells being WEHI-3 cells.

Other cells and cell lines may also be suitable for use in isolating ICE cDNA. Selection of suitable cells may be done by screening for ICE activity in cell extracts or medium. Methods for detecting ICE activity are well known in the art (Kostura, MJ. et aL, 1989, P.N.A.S. USA, 86, pp.5227-5231) and measure the conversion of precursor IL-lβ to mature EL-lβ. Cells which possess ICE activity in this assay may be suitable for the isolation of ICE cDNA.

Interleukin-lβ producing cells such as human THP-1 cells (American Type Culture Collection, ATCC TIB) described by Tsuchiya et aL, Int. J. Cancer 26: 171-176 (1980) and murine WEHI-3 cells (American Type Culture Collection ATCC TIB 68) are grown in suspension at about 37°C in, for example, Dulbecco's modified minimal essential medium (Hazelton Research Products) with about 10% fetal calf serum (HyClone; defined sera with no detectable endotoxin) or Iscove's Modified Dulbecco's Medium (JRH Biosciences) with about 9% horse serum.

Cell-free extracts are prepared from murine peritoneal exudate cells or murine monocyte lines and by disruption of the cells by nitrogen cavitation, hypotonic lysis or the like. The cells are collected by centrifugation and may be washed in an isotonic buffer solution such as phosphate buffered saline, pH about 7.4. Hypotonic lysis is accomplished by washing the cells in about 10 volumes of hypotonic buffer (about 10 mM KC1, about 20 mM HEPES, about pH 7.4, about 1.5 mM MgC12, about 0.1 mM EDTA) or (about 25 mM HEPES, about pH 7.5, about 5 mM MgC12, and 1 mM EGTA) and collected by centrifugation. The lysis buffer may also contain a reducing agent such as dithiothreitol (DTT). The hypotonic buffer will generally contain protease inhibitors such as PMSF, leupeptin and pepstatin. The cells are resuspended in about 3 volumes of hypotonic buffer, to a denisty of 10'-10°/ml, placed on ice for about 20 minutes, and lysed by about 20 strokes in a Dounce homogenizer. Disruption of about 90 to about 95% of the cells is obtained in this mannner. Nitrogen pressure disruption also takes place in a hypotonic buffer. Resuspended cells are placed in a nitrogen pressure cell at 400 psi of nitrogen for about 30 min at about 4°C with agitation. Disruption is accomplished by releasing the pressure and evacuating the cells from the pressure cell. The cell lysate is clarified by successive centrifugation steps; at about 400 to about 1000 x g (supernatant SI), at about 30,000 x g (supernatant S2) and at about 300,000 x g (supernatant S3). The cell lysate may also be clarified by the following procedure. Unbroken cells and nuclei are removed by centrifugation at about 3000 rpm, for about 10 minutes, at about 5°C in a Beckman GPR centrifuge. The post nuclear supernatant fluid is centrifuged for about 20 minutes at about 16,000 rpm in a Sorvall centrifuge with a SS34 rotor. The supematant fluid is further clarified by centrifugation for about 60 minutes at about 50,000 rpm in a Beckman centrifuge (50.2Ti rotor) or 45,000 rpm (45Ti rotor). The resultant supernatant fluid is stored at about -80°C following the addition of about 2 mM DTT and 0.1% CHAPS.

ICE activity is monitored by an in vitro cleavage assay utilizing radiolabeled pre-IL-lβ as a substrate. Both murine and and human pre-Il-lβ can serve as substrate. An approximately 1.5 kilobase (kb) cDNA clone containing the entire coding sequence of murine pre- IL-lβ is inserted into EcoRI-PstI cleaved pGEM-3 plasmid DNA (Promega-Biotec) and propagated in E. coli according to standard methods (Maniatis et aL, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor, NY [1982]). Purified plasmid is linearized with PstI and then transcribed using a T7 RNA polymerase in vitro transcription system (Promega-Biotec) and then the mRNA processed according to the manufacturers' instructions. Translations are performed by programming micrococcal nuclease- treated rabbit reticulocyte extracts (Promega Biotec) with the in vitro synthesized mRNA in the presence of 25 μCi of -"S-methionine (Amersham) according to the manufacturers instructions. This yields labeled pre-IL-lβ which migrates as a doublet on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular mass of about 34 and about 31 kilodaltons (kDa). The cleavage of pre-IL-lβ is performed by incubating 1 μl of rabbit reticulocyte extract containing radiolabeled precursor with about 10 to about 20 μl of the sample containing IL-lβ converting enzyme. Cleavage of pre-IL-lβ to yield 17.5 kDa mature Il-lβ is assayed by SDS-PAGE according to the method of Laemmli (Nature 227: 680-685 [1970]), followed by fluorography using procedures known in the art. Specificity of enzymatic cleavage and characterization of cleavage products are also determined with mutant human pre-IL-lβ. Construction of a mutant pre-IL-lβ is performed by site directed oligonucleotide mutagenesis which is well known in the art. A synthetic double-stranded 27 nucleotide (27-mer) long oligodeoxyribonucleotide, corresponding to amino acids 115-126, with ApaLI-Hpaϋ ends is synthesized on an Applied Biosy stems DNA 380A synthesizer according to established manufacturer's protocols. The 27-mer encodes an Asp 116 --> Alall6 amino acid substitution at the -1 position adjacent to the processing site of pre-IL-lβ. The oligonucleotide is ligated by procedures well known in the art to EcoRI-ApaLI and Hpaϋ-Pstl fragments obtained from cleavage of full length pre-IL-lβ cDNA. The human nucleotide and predicted amino acid sequence of the pre-IL-lβ translation product is disclosed by March et aL, Nature 315: 641-647 (1985). The ligated fragments are added to a ligation reaction containing EcoRI-PstI cleaved pGEM-3. Clones containing the pGEM/IL-lβ mutant are identified by hybridization with the mutant oligonucleotide sequence. Clones are mapped by restriction endonuclease cleavage and the DNA sequenced to verify the authenticity of the mutation. Transcription of the vector bearing the mutant or native constructs produced a 1.5 kilobase (Kb) mRNA and translation results in a doublet of 34 and 31 kDa proteins.

When homogenat containing human pre-IL-lβ converting enzyme are incubated with human pre-IL-lβ, cleavage to 28 and 17.5 kDa products is observed. When IL-lβ converting enzyme is incubated with the mutated Aspl 16 to Alal 16 pre-IL-lβ, little or no cleavage of the mutant precursor was observed. The normal cleavage product from the interaction of pre-IL-lβ with pre-IL-lβ converting enzyme is a 17.5 kD polypeptide with the N-terminal amino acid sequence of mature IL- lβ. The Aspl 1" residue of pre-IL-lβ is therefore important to the processing of mature IL-lβ by IL-β converting enzyme.

Cytosolic extracts prepared from P. acnes elicited peritoneal exudate cells were assayed for IL-lβ cleavage activity in a gel-based assay using in vitro synthesized, radiolabeled murine pIL-lβ as the substrate (Fig. 1). Incubation of these extracts with the 31 kDa murine IL-lβ precursor yielded a 17.5 kDa product that was indistinguishable in size from that generated by cleavage with affinity- purified human ICE (Fig. IA control, lanes 1 and 7). A 28 kDa protein was generated when extracts were incubated for shorter times. Both cleavage products were also generated by incubation of PEC extracts with the human IL-lβ precursor (Fig. IB, lanes 1 and 7). Incubation of murine ICE with the mutant Alal 16 IL-lβ precursor yielded the 28 kDa but not the 17.5 kDa product, indicating that murine ICE has the same requirement as human ICE for an Asp in the PI position (Fig. 1C, lanes 1 and 5). Taken together, these studies indicate that the murine enzyme recognizes the same subse of Asp-X bonds in these macromolecular substrates as human ICE.

The peptide Ac-YVAD-AMC was determined to be a substrate for murine ICE. The specificity of this substrate is derived from the peptide sequence Tyr-Val-His-Asp-Ala that defines the minimal substrate recognition sequence for ICE cleavage at site 2 of the human EL-lβ precursor. To define further the substrate specificity of murine ICE, a tetrapeptide aldehyde inhibitor Ac-YVAD-CHO (L- 709,049) that is a potent (Ki=0.76 nM), selective, and reversible inhibitor of human ICE was tested for its ability to inhibit the murine enzyme [Thornberry et aL, supra]. Incubation of 1 μM tetrapeptide aldehyde with PEC extracts resulted in complete inhibition of cleavage of both the murine and human IL-lβ precursors (lane 2 in Fig 1. A, B, and C). The tetrapeptide aldehyde inhibits murine ICE with a Ki<3 nM as determined from the progress curve (Fig. 2) of the enzyme assayed against the YVAD-AMC substrate in the presence of 20 nM inhibitor. This is comparable to the potency observed against the human enzyme (Ki=0.76 nM), suggesting that the active sites of both convertases are similar.

To obtain a convenient source of murine ICE, several murine monocyte-macrophage cell lines were surveyed for ICE activity. Cleavage activity against the murine IL-lβ precursor was highest in cytosolic extracts of IC21 cells (Fig. IA, lane 3), but was also detectable in J774 cells (Fig. IA, lane 5) and WEHI-3 cells (data not shown). The cleavage activity in these cells was identical to the activity in murine peritoneal exudate cells in that the murine and human IL-lβ precursors were both cleaved to products of 28 kDa and 17.5 kDa and the human mutant Alal 1 IL-lβ precursor was cleaved to a 28 kDa product. In all cases the activity was inhibited by the tetrapeptide aldehyde (Fig. 1). Human ICE, like murine ICE, is capable of cleaving both processing sites in the murine and the human E -lβ precursors (Howard, et aL, J. Immunol. 147, pp. 2964-2969 [1991]). We compared the relative ablity of murine ICE and human ICE to cleave each of the precursors by analyzing the time course for the generation of the 28 kDa and 17.5 kDa proteins and found that incubation of either enzyme and the same species precursor resulted in a higher percentage of 17.5 kDa protein generated relative to 28 kDa protein than when the precursor from the heterologous species was used as the substrate (Fig. 3). We conclude that murine ICE does not cleave site 2 in the two precursors with equal efficiency. This is consistent with substrate with substrate specificity studies on human ICE which indicate a preference for Ala over Val in the PI' position and a preference for a hydrophobic residue at the P4 position (Thornberry et aL, supra; Sleath et aL, J. Biol. Chem, 265, pp. 14526-14528 [1990]).

The enzymatically active form of human ICE is a p20:pl0 complex and it has been demonstrated that the active-site cysteine residue is on the p20 subunit (Cys²^) (Thornberry, et aL, Nature 356, pp. 768-774 [1992]). This can be accomplished by (1) labeling the active-site thiol in ICE with

idodacetic acid, as this thiol is approximately 10-fold more reactive than other thiols on the protein, (2) We identified the catalytic murine ICE subunit by labeling the enzyme in PEC extracts an active site label with in the presence or absence of a competing, reversible tetrapeptide aldehyde inhibitor (Ac- Tyr-Val-Ala-Asp-CHO). Following fractionation of the cell extract by SDS-PAGE and addition of

streptavidin, a protein with a molecular weight corresponding to the p20 subunit of human ICE can be visualized (Fig. 4). Thus, as is true for human ICE, most, if not all, of the catalytically-active enzyme in cell extracts is associated with the p20 subunit, which by analogy with human ICE, is complexed with a plO subunit.

To evaluate the role of murine ICE in IL-lβ production in vivo, we examined peritoneal macrophages for their ability to synthesize and secrete mature II- lβ upon activation [Chensue, 1989]. PECs from P. acnes treated mice were isolated and cultured for 18 hours in the presence of 100 ng/ml LPS to measure Il-lβ release. Under these conditions, macrophages secrete significant levels (1-6 ng/ml) of IL-lβ (Figure 5A).

In whole human blood, the tetrapeptide aldehyde inhibitor of ICE (Ac-Tyr-Val-Ala-Asp-CHO) prevents the production and release of mature IL-lβ from activated monocytes [Thornberry et aL, Nature 356, pp. 768-774 [1992]). We, therefore, tested the ability of this inhibitor, which has a similar Ki against murine ICE, to prevent the release of IL-lβ from LPS -stimulated peritoneal macrophages. The amount of IL-lβ released was measured by ELISA from adherent cells cultured in the presence of 100 ng/ml LPS and increasing concentrations of the tetrapeptide aldehyde. The results showed that IL-lβ levels in the media decreased in proportion to the level of inhibitor present (Fig. 5A). The IC50 for this inhibitor in this system about 20 μM.

Consistent with results obtained from activated monocytes in human blood, the intracellular synthesis of the 31 kDa IL-lβ precursor, as determined by Western blot analysis, was unaffected by the inhibitor (Fig. 5B). Immunoblot analysis indicated that the majority of IL-lβ being secreted is 17.5 kDa in size and that the inhibitor effectively prevents the generation of all processed, mature IL-lβ (Fig. 5C). Because treatment with the ICE inhibitor decreased the amount of 17.5 kDa IL-lβ secreted in a dose-dependent fashion, we conclude that processing of the IL-lβ precursor to the mature form occurs at the Asp 11 '- Val 11° ICE cleavage site and, as in human monocytes , ICE is required for processing of IL-lβ in murine peritoneal exudate macrophages.

Any of a variety of procedures may be used to molecularly clone murine ICE cDNA. These methods include, but are not limited to, direct functional expression of the ICE gene following the construction of an ICE-containing cDNA library in an appropriate expression vector system. Another method is to screen an ICE- containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labelled oligonucleotide probe designed from the amino acid sequence of the ICE subunits. The preferred method consists of screening an ICE-containing cDNA library from murine WEHI cells constructed in a bacteriophage or plasmid shuttle vector with a cDNA probe encoding human ICE.

It is readily apparent to those skilled in the art that other types of libraries, as well as libraries constructed from other cells or cell types, may be useful for isolating murine ICE-encoding DNA. Other types of libraries include, but are not limited to, cDNA libraries derived from other cells or cell lines other than WEHI-3 cells, and genomic DNA libraries.

It is readily apparent to those skilled in the art that suitable cDNA libraries may be prepared from murine cells or cell lines which have ICE activity. The selection of cells or cell lines for use in preparing a cDNA library to isolate ICE cDNA may be done by first measuring cell associated ICE activity using the precursor IL-lβ cleavage assay described fully above.

Preparation of cDNA libraries can be performed by standard techniques well known in the art. Well known cDNA library construction techniques can be found for example, in Maniatis, T., Fritsch, E.F., Sambrook, J., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982).

It is also readily apparent to those skilled in the art that DNA encoding murine ICE may also be isolated from a suitable genomic DNA library.

Construction of genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techiques can be found in Maniatis, T., Fritsch, E.F., Sambrook, J. in Molecular Cloning: A Laboratory Manuel (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982).

In order to clone the murine ICE gene by the preferred method, the human ICE cDNA sequence was utilized. Southern blot analysis indicated that under reduced stringency the human probe cross-hybridized with sequences from mouse, thereby allowing the human sequences to be used as a probe to isolate murine ICE cDNA clones. A partial cDNA encoding the plO subunit and a portion of the p20 subunit was isolated by screening a mouse macrophage library under reduced stringency with the full- length human ICE cDNA. Using this partial murine ICE cDNA as a probe, six full-length clones were subsequently isolated by screening a cDNA library prepared from a WEHI cell line under standard hybridization conditions. These clones contained an open reading frame encoding a 402 amino acid protein with a predicted molecular weight of 45,640 (Fig. 6). The sequence for the full-length cDNA encoding murine ICE is shown in Table 1. The deduced amino acid sequence of ICE from the cloned cDNA is shown in Table 2. The longest polyA containing cDNA was 1,378 NT in length and had 48 and 115 NT of 5' and 3' untranslated sequence, respectively. By analogy with human ICE, the murine proenzyme consists of a 118 amino acid prodomain (pi 4) followed by the p20 and plO subunits of active ICE which are connected by a polypeptide bridge that, in human ICE, is missing from the purified form of the enzyme.

A comparison of the amino acid sequence of murine and human p45 is shown in Figure 6. The overall identity between the two proteins is 62% at the amino acid level and 72% at the nucleic acid level. The degree of identity differs markedly in functionally distinct regions of the protein. The highest level of identity is in the pi 0 region, which shares 81% identity with human plO at the amino acid level. The p20 region is also well-conserved, exhibiting 62% identity with its human counterpart. There are several regions of complete identity within both the p20 and plO subunits, the most striking of which is at the amino teminus of plO, where there is an identical stretch of 26 amino acids. The remarkable level of overall homology in the plO region suggests that it plays a critical role in the function of the enzyme. The NH2 terminal domain (pi 4), for which no function has yet been assigned, is least conserved with 53% identity between species. Human ICE is a thiol protease. The catalytic Cys²°^ is conserved and lies within a stretch of 18 amino acids which is identical to that of human ICE. Murine and human ICE have no homology to known proteins including cysteinyl proteinases. (Thornberry, et aL, Nature 356, pp. 768-774 [1992]). However, a serine adjacent to the active site Cys in human ICE aligns with the consensus sequence for the catalytic residue of serine and viral cysteinyl proteases. The murine ICE consensus sequence diverges significantly from these Ser/Cys consensus sequences and, most importantly, the Ser residue in human ICE is replaced by a Lys residue. Thus, the homology to serine proteases found in the human enzyme is not conserved in the murine enzyme.

Several proteins (p22, p20 and plO) derived from the human proenzyme have been purified from cell extracts (Thornberry, et al., Nature 356, pp. 768-774 [1992]). In the human proenzyme, four ICE-like cleavage recognition sequences are present at sites that flank these polypeptides. Previous studies have shown that human p45 can be cleaved in vitro by affinity purified enzyme (p20:pl0 form) to products consistent in size with processing at these sites. Examination of the murine p45 sequence reveals that potential Asp-X cleavage sites are present at or near all four corresponding cleavage sites in the human proenzyme. The most highly conserved site is at the amino terminus of plO, where the murine and human sequence differ only by a substitution of Gly !⁵ for Ala !⁷ in the PI' position. The Asp²⁹⁶-Ser²⁹⁷ processing site at the carboxy terminus of p20 is also well conserved; the change in P4 from Tip to Leu makes this site more like the Asp²⁷- Qjy28 cleavage site in the murine Il-lβ precursor. The Aspl^³-Serl"^ site corresponding to the amino terminus of p22 protein is also retained. With Ala 10" at P4, this site is analogous to the human processing site at the amino terminus of the p20. The sequence of murine ICE diverges markedly from human ICE at the amino-terminal processing site of p20, where AspH" is deleted in the murine protein; however, an excellent candidate site for ICE cleavage is present nearby at Aspl²³-Glyl²4. The presence of this cleavage site suggests that the amino terminus of the murine p20 subunit is Glyl²^.

Previous experiments have shown that human p45 is cleaved by purified ICE, suggesting that the activation of the proenzyme is in part autocatalytic. To determine whether the murine enzyme can be cleaved by affinity purified human ICE, cleavage reactions were conducted using ³^S-methionine labelled murine p45 synthesized in rabbit reticulocyte lysate programmed with in vitro transcribed murine p45 mRNA. Following incubation with 10 units of affinity-purified human ICE overnight at room temperature, the reactions were terminated, and the cleavage products were analyzed on an SDS polyacryamide gel. The murine p45 protein was a substrate for human p20:pl0 ICE and the cleavage products generated were similar in size to those from the human p45 protein. As observed with the human enzyme, cleavage of murine p45 in vitro appears to be intermolecular and is incomplete, suggesting that full activation of the proenzyme may require additional factors.

To determine the expression pattern of ICE mRNA in murine cells, total RNA was prepared from several monocytic cell lines, a T-cell line, and a fibroblast line and analyzed by Northern blot. A 1.6 kb low-abundance ICE mRNA corresponding to the cloned cDNA is expressed in J774, IC21, RAW 8.1, and WEHI-3 cells, but is not present in P388D1 cells or 3T3 fibroblasts (Fig. 7). The T cell line (EL4) expressed ICE mRNA at levels comparable to that of J774 and IC21 cells. In the murine monocytic lines examined the amount of ICE activity correlated with the levels of ICE mRNA present. Three species of low-abundance ICE mRNA 2.3 kb, 1.6 kb and 0.5 kb in size are coexpressed in primary human monocytes and monocytes lines. The 1.6 kb mRNA is the major form and corresponds to the cloned ICE cDNA encoding p45.

The cloned murine ICE cDNA obtained through the methods described above may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant ICE. Techniques for such manipulations are fully described in Maniatis, T, et aL, supra, and are well known in the art.

Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells and animal cells.

Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.

A variety of mammalian expression vectors may be used to express recombinant murine ICE in mammalian cells. Commercially available mammalian expression vectors which may be suitable for recombinant ICE expression, include but are not limited to, pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-l(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and gZD35 (ATCC 37565).

Murine ICE protein can be synthesized by introduction of an expression vector containing DNA encoding murine ICE into a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria, yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to drosophila derived cell lines. Cell lines derived from mammalian species which may be suitable and which are commercially available, include but are not limited to, CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).

The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation. The expression vector-containing cells are clonally propagated and individually analyzed to determine whether they produce ICE protein. Identification of ICE expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti- ICE antibodies, and the presence of host cell-associated ICE activity.

Expression of murine ICE may also be performed using in vitro produced synthetic mRNA. Synthetic mRNA can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred.

To determine the murine ICE cDNA sequence(s) that yields optimal levels of enzymatic activity and/or murine ICE protein, ICE cDNA molecules including but not limited to the following can be constructed: the full-length open reading frame of the ICE cDNA (45 kDa = base 47 base 1252) and several constructs containing portions of the cDNA encoding both the 20 kDa and 10 kDa subunits. All constructs can be designed to contain none, all or portions of the 3' untranslated region of murine ICE cDNA (base 1253-1488). ICE activity and levels of protein expression can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells. Following determination of the murine ICE cDNA cassette yielding optimal expression in transient assays, this ICE cDNA construct is transferred to a variety of expression vectors, including but not limited to mammalian cells, baculoviras-infected insect cells, E. coli. and the yeast S. cerevisiae.

Mammalian cell transfectants, bacterial transformants, and other recombinant host cells are assayed for both the levels of ICE enzymatic activity and levels of ICE protein by the following methods.

A method of using a chromophore containing a peptide of the structure Ac-Tyr-Val-Ala-Asp-AMC for determining the interleukin-lβ converting enzyme activity of a recombinant enzyme sample is as follows:

(a) adding, in aqueous solution, in any order,

(1) the chromophoric substrate and

(2) recombinant enzyme sample; and

(b) measuring the interleukin-lβ converting enzyme activity of step (a) by photometric means.

Similarly, it is preferred that the aqueous solution comprises a buffer.

The pH optimum for ICE is between 6.5 and 7.5. Consequently, suitable buffer will have a pKa between 6.5 and 7.5, such as HEPES, which we use in our studies. In general, any nonreactive buffer at a concentration that will maintain the pH of the reaction between 6 and 9 will work.

Other components may be added to the reaction that stabilize the enzyme or increase the rate of the reaction. Examples are sucrose (10%), CHAPS (0.1%), DTT (1-100 mM), BSA (0.1-10 mg/ml) all of which have been demonstrated to stabilize the enzyme. Others components which may be included are glycerol, EDTA, and a variety of standard protease inhibitors.

The concentration of ICE is highly variable and may range from 1 pM to 1 μM, depending entirely on the purpose of a particular experiment, and the kinetic parameters for the chosen substrate. The volume added to a particular reaction may be very small or comprise the entire volume of the reaction less the volume of substrate required to achieve the desired concentration.

Any state of purity of ICE is acceptable (including crude cell lysates), as long as the preparation is free of contaminating proteases that will compete with ICE for cleavage of the substrate. Even in this case it is possible to use this assay if inhibitors of the contaminating proteases are included in the reaction.

This assay is typically run between 25 and 37 degrees. The use of higher temperatures will depend upon the stability of the enzyme and running the assay at low temperatures will probably be dictated by practical considerations.

As appreciated by those of skill in the art, addition step (a) results in the cleavage of compound of formula I between the aspartic acid specifically described, and the adjacent aminomethyl coumarin group. The liberation of the chromophoric group, may be monitored by fluorometric procedures. The excitation wavelength is 380 nm and the emission wavelength is 460nm.

This assay is amenable to continuous or discontinuous sampling of the reaction. The assay is also amenable to 96-well plate format for running multiple assays simultaneously.

For example, with the fluorometric leaving group (e.g. AMC), the activity of the sample is proportional to the rate of fluorescence change, and be calculated as:

Velocity of d fluorescense 1 μM AMC d AMC

ICE Catalyzed = reaction dt fluorescence dt

As appreciated by those of skill in the art, the use described above may be quite useful for determining Michaelis-Menton kinetic parameters or other characterization information concerning the Enzyme (eg when the sample contains no putatuve inhibitor) or screening for putative ICE inhibitors or assaying purification fractions. The second method for assessing ICE enzymatic activity involves the direct introduction of the native substrate for ICE, the 31.5 K IL-lβ precursor, simultaneously with ICE. To assess the substate specificity of expressed ICE, IL-lβ precursor substrates with altered amino acids in the ICE cleavage sites are tested. In the case of mammalian cells, this involves the co-transfection of two plasmids, one containing the ICE cDNA and the other containing the prelL-lβ cDNA. In the case of oocytes, this involves the co-injection of synthetic RNAs for both ICE and the IL-lβ precursor. Following an appropriate period of time to allow for expression, cellular protein is metabolically labelled with 3~>S-methionine for 24 hours, after which cell lysates and cell culture supernatants are harvested and subjected to immunprecipitation with polyclonal antibodies directed against the IL-lβ protein. Cleavage of the wild-type precursor to the 28 K and 17 K products, and cleavage of the precursor containing an altered downstream processing site (Asp 116 to Alal 16) _t0 ^ 28 K form is assessed by an SDS-PAGE gel based assay.

The third method for detecting ICE activity involves the direct measurement of ICE activity in cellular lysates prepared from cells transfected with ICE cDNA or oocytes injected with ICE mRNA. This assay can be performed using IL-lβ precursor protein or synthetic peptides spanning the IL-lβ cleavage sites. Cleavage products of the precursor are analyzed by standard gel based assay and cleavage products of the peptides are analyzed by HPLC.

Levels of ICE protein in host cells is quantitated by immunoaffinity and or ligand affinity techniques. ICE-specific affinity beads or ICE-specific antibodies are used to isolate ³^S-methionine labelled or unlabelled ICE protein. Labelled ICE protein is analyzed by SDS-PAGE. Unlabelled ICE protein is detected by Western blotting, ELISA or RIA assays employing ICE specific antibodies.

Following expression of murine ICE in a recombinant host cell, ICE protein may be recovered to provide ICE in active form, capable of cleaving precusor IL-lβ into mature IL-lβ. Several ICE purification procedures are available and suitable for use. As described (Thornberry et aL, Nature 356; pp. 768-774 [1992]) for human ICE from natural sources, recombinant murine ICE may be purified from cell lysates and extracts, or from conditioned culture medium, by various combinations of, or individual application of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography, hydrophobic interaction chromatography, and affinity chromotgraphy with the legand Ac-Tyr- Val-Lys-Asp-CHO.

In addition, recombinant murine ICE can be separated from other cellular proteins by use of an immuno-affinity column made with monoclonal or polyclonal antibodies specific for full length nascent ICE, polypeptide fragments of ICE or ICE 20 kDa and 10 kDa subunits.

ICE antibody affinity columns are made by adding the antibodies to Affigel-10 (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1M ethanolamine HC1 (pH 8). The column is washed with water followed by 0.23 M glycine HC1 (pH 2.6) to remove any non-conjugated antibody or extraneous protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) and the cell culture supernatants or cell extracts containing ICE or ICE subunits are slowly passed through the column. The column is then washed with phosphate buffered saline until the optical density (A280) falls to background, then the protein is eluted with 0.23 M glycine-HCl (pH 2.6). The purified ICE protein is then dialyzed against phosphate buffered saline.

The full length murine ICE-encoding cDNA in plasmid Bluescript SK(-) was designated [pFClAM]. The following examples illustrate the present invention without, however, limiting the same thereto. EXAMPLE 1

In Vitro Assays For Detection Of ICE Activity

Cleavage of pre-IL-lβ by samples containing ICE activity was performed by incubating 1 μl of rabbit reticulocyte extract containing radiolabeled precursor with 10-20 μl of the specific fractions. The radiolabeled precursor IL-lβ was prepared in the following manner. A 1.5 kilobase (kb) cDNA clone containing the entire coding sequence of pre-IL-lβ was inserted into EcoRI/Pst I- cleaved pGEM-3 plasmid DNA (Promega Biotec) and propagated in Escherichia coli according to standard methods know in the art, for example, see generally, Maniatis et aL, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, NY, (1982). Purified plasmid was linearized with Pst I and then transcribed by using a T7 RNA polymerase in vitro transcription system (Promega Biotec) and then the mRNA was processed according to the manufacturer's instructions. Translations were performed by programing micrococcal nuclease-treated rabbit reticulocyte extracts (Promega Biotec) with the in vitro synthesized mRNA in the presence of 25 uCi of [³^S] methionine (1 Ci = 37 GBq; Amersham) according to the manufacturer's instructions. This yielded labeled pre-IL-lβ which migrated as a doublet on SDS-PAGE with an apparent molecular mass of 34 and 31 kDa. Both bands can be immunoprecipitated with antisera directed to the carboxyl terminus of IL-lβ. Interleukin-lβ converting enzyme activity, cleavage of radiolabeled pre-IL-lβ to yield 17.5 kDa mature IL-lβ, was monitored by SDS-PAGE.

EXAMPLE 2

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis And

Staining of Interleukin-lβ Converting Enzvme

Sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified ICE was carried out essentially according to the method of Laemmli, Nature 227: 680-685 (1970). A 0.15 cm x 10 cm x 10cm Bio-Rad mini-gel was used to cast a 15% acrylamide 0.4% Bis- acrylamide SDS-PAGE resolving gel. A second discontinuous stacking layer consisting of 5% acrylamide - 0.14% bis-acrylamide was then cast on top of the resolving gel. After polymerization, the gels were loaded with no more than 50 μg of protein dissolved in Laemmli buffer containing bromphenol blue as a tracking dye. The gels were then subjected to electrophoresis at 50 V for 1/2 to 1 hour then at 150 V until the bromphenol blue dye begins to elute from the gel. At this point electrophoresis was stopped and the gel was processed for silver staining.

After electrophoresis, separated proteins were visualized by staining with silver using a modification of the method developed by Oakley et aL, (Analytical Biochemistry, 105: 361-363. 1980) and now sold in kit form by Daiichi. Briefly, the gel was first soaked 30 minutes in 200 ml of a 50% methanoLwater mixture followed by three washes of 10 minutes each in 200 ml of deionized water. After the initial washes the gel was then soaked in a solution of 40% methanol; 10% ethanol; 0.5% glutaraldehyde; 49.5% water for 15 minutes then washed thoroughly with 4 changes of 200 ml aliquots of deionized water each lasting 10 minutes. Staining and visualization of proteins was accomplished using the protocol of Oakley.

EXAMPLE 3

Characterization of murine ICE as a thiol protease

Characterization of murine ICE in terms of protease class was ultimately accomplished by the synthesis of a mechanism based thiol protease inhibitor, a peptide aldehyde, synthesized as described in [Thornberry, et aL, supra]. This peptide aldehyde has been previously shown to reversibly inhibit human ICE reversibly inhibits murine ICE and also in a dose dependant fashion and in a manner competitive with substrate. EXAMPLE 4

Isolation of ICE cDNA clones from lambda cDNA libraries

The human ICE cDNA clone was used as a probe to screen a mouse macrophage library (Clontech) under reduced stringency. A probe containing the full length human ICE-cDNA open recording frame was generated by a PCR reaction. The probe was radiolabeled by random-priming using the Amersham multiprime kit as per the manufacturer's directions. The filters were pre-hybridized in buffer containing 30% formamide, 5X SSC, 5X Denhardt's, 0.1% SDS, and 100 μg/ml salmon sperm DNA for 1 hour at 42°C. The filters were hybridized in buffer containing 30% formamide, 5X SSC, 5X Denhardt's, 0.1% SDS, 100 mg/ml salmon sperm DNA, 10% dextran sulfate and 0.3 x lθ6 cpm/ml of probe overnight at 42°C. The filters were washed in 2X SSC, 0.1% SDS at 42°C and exposed to x-ray film for 4 days. A partial murine ICE cDNA clone isolated in this manner was used as a probe to re-screen a WEHI library (Stratagene) under standard conditions. All positive inserts were rescued as plasmids from the λ ZAP vector and sequenced on both strands by the chain termination method.

Isolation of RNA. RNA was extracted from 10° cells by a guanidine thiocyanate procedure. PolyA+mRNA was isolated using the Promega PolyA Tract System. RNA was fractionated on 1% agarose containing 2.2 M formaldehyde and blotted to a Duralon membrane. Blots were probed with a full-length murine or human ICE cDNA radiolabeled by random-priming (Amersham) with ³²P-dCTP to a specific activity of 5 x 10° cpm/μg. Blots were prehybridized at 42°C for 4 hours in 5X SSC, 5X Denhardt's solution, 250 μg/ml salmon sperm DNA, 1.0% glycine, 0.075% SDS, 50 mM NaP04, pH 6 and 50% formamide. Hybridizations were carried out at 42°C for 20 hours in 5X SSC, IX Denhardt's solution, 50 μg/ml salmon sperm DNA, 0.1% SDS, 50 mM NaPθ4, and 50% formamide containing lθ6 cpm/ml of probe. RNA blots were washed in 2X SSC, 0.2% SDS at 42°C and autoradiographed at -70°C. EXAMPLE 5

Subcloning and sequencing of ICE cDNA clones

Several cDNA clones 1.6 kb in length and containing a single open reading frame of 402 amino acids were subcloned into pGEM vectors (Promega) and bi-directionally sequenced in their entirety by the method of Sanger. The sequence for the full-length cDNA encoding murine ICE is shown in Table 5. The full length clone was designated clone pFCl AM. The deduced amino acid sequence of full length murine ICE from the cloned cDNA is shown in Table 6.

TABLE 1

GGCACGAGTT CAGTTTCAGT AGCTCTGCGT GTAGAAAAGA AACGCCATGG CTGACAAGAT CCTGAGGGCA AAGAGGAAGC AATTTATCAA CTCAGTGAGT ATAGGGACAA TAAATGGATT GTTGGATGAA CTTTTAGAGA AGAGAGTGCT GAATCAGGAA GAAATGGATA AAATAAAACT TGCAAACATT ACTGCTATGG ACA AGGCACG

GGACCTATGT GATCATGTCT CTAAAAAAGG GCCCCAGGCA AGCCAAATCT TTATCACTTA CATTTGTAAT GAAGACTGCT ACCTGGCAGG AATTCTGGAG CTTCAATCAG CTCCATCAGC TGAAAC ATTT GTTGCTACAG AAGATTCTAA

AGGAGGACAT CCTTCATCCT CAGAAACAAA GGAAGAACAG AACAAAGAAG ATGGCACATT TCCAGGACTG ACTGGGACCC TCAAGTTTTG CCCTTTAGAA AAAGCCCAGA AGTTATGGAA AGAAAATCCT TCAGAGATTT ATCCAATAAT GAATACAACC ACTCGTACAC GTCTTGCCCT CATTATCTGC AACACAGAGT TTCAACATCT TTCTCCGAGG GTTGGAGCTC AAGTTGACCT CAGAGAAATG AAGTTGCTGC TGGAGGATCT GGGGTATACC GTGAAAGTGA AAGAAAATCT CACAGCTCTG GAGATGGTGA AAGAGGTGAA AGAATTTGCT GCCTGCCCAG AGCACAAGAC TTCTGACAGT ACTTTCCTTG TATTCATGTC TCATGGTATC CAGGAGGGAA TATGTGGGAC

CACATACTCT AATGAAGTTT CAGATATTTT AAAGGTTGAC ACAATCTTTC AGATGATGAA CACTTTGAAG TGCCCAAGCT TGAAAGACAA GCCCAAGGTG ATCATTATTC AGGCATGCCG TGGAGAGAAA CAAGGAGTGG TGTTGTTAAA

AGATTCAGTA AGAGACTCTG AAGAGGATTT CTTAACGGAT GCAATTTTTG AAGATGATGG CATTAAGAAG GCCCATATAG AGAAAGATTT TATTGCTTTC TGCTCTTCAA CACCAGATAA TGTGTCTTGG AGACATCCTG TCAGGGGCTC ACTTTTCATT GAGTCACTCA TCAAACACAT GAAAGAATAT GCCTGGTCTT GTGACTTGGA GGACATTTTC AGAAAGGTTC GATTTTCATT TGAACAACCA GAATTTAGGC TACAGATGCC CACTGCTGAT AGGGTGACCC TGACAAAACG TTTCTACCTC TTCCCGGGAC ATTAAACGAA GAATCCAGTT CATTCTTATG TACCTATGCT GAGAATCGTG CCAATAAGAA GCCAATACTT CCTTAGATGA TGCAATAAAT ATTAAAATAA AACAAAACAG AAA AAAAAAA AAAAA AAA

TABLE 2

MADKILRAKR KQFINSVSIG TINGLLDELL EKRVLNQEEM DKIKLANITA MDKARDLCDH VSKKGPQASQ IFITYICNED CYLAGILELQ SAPSAETFVA TEDSKGGHPS SSETKEEQNK EDGTFPGLTG TLKFCPLEKA QKLWKENPSE

IYPIMNTTTR TRLALΠCNT EFQHLSPRVG AQVDLREMKL LLEDLGYTVK VKENLTALEM VKEVKEFAAC PEHKTSDSTF LVFMSHGIQE GICGTTYSNE VSDILKVDΗ FQMMNTLKCP SLKDKPKVπ IQACRGEKQG VVLLKDSVRD SEEDFLTDAI FEDDGIKKAH IEKDFIAFCS STPDNVSWRH PVRGSLFIES LIKHMKEYAW SCDLEDIFRK VRFSFEQPEF RLQMPTADRV TLTKRFYLFP GH 402

EXAMPLE 6

Cloning of the ICE cDNA into E. coli Expression Vectors

A construct for the expression of recombinant enzyme was generated by PCR with primers

(5'GGGCCCCATATGAACAAAGAAGATGGCACATTTC3', 5' GGGCCCCATATGTTAATGTCCCGGGAAGAGGTAGAAAC3') that contained Ndel sites and placed an initiator methionine in frame with a truncated form of the murine ICE proenzyme

lacking the prodomain. The PCR fragment was cloned into the Ndel site of the pETl la expession vector (Novagen) and placed under the expression of the inducible T7 polymerase promoter. This construct was then transformed into E. coli strain BL21(DE3)pLYS, which contains a chromosomal copy of the T7 RNA polymerase gene driven by the inducible lac promoter. Expression was induced by addition of the lac substrate (IPTG) to a log phase growth of cells. Following 3 hours of induction, the cells were harvested by resuspension in spheroplast buffer (50 mM Tris, pH 8, 0.5 mM EDTA, 0.5 μM sucrose, 1 mM PMSF, 5 μg/ml leupeptin) and homogenization in a Menton- Gaulin press. The crude cytosolic fraction was analyzed on polyacrylamide gels to visualize recombinant ICE protein expression (Fig. 9). Immunoblot analysis with polyclonal sera specific for human p20:pl0 ICE indicates that the p32 protein is synthesized intact but it is partially proteolyzed to p20 and plO cleavage products. The p20 and plO cleavage products are overexpressed at high levels and are detectable by Coomassie Blue stain. The cytosolic extract has ICE enzyme activity (Fig. 8). The recombinant activity is indistiguishable from native enzyme activity in a gel cleavage assay using murine IL-lβ precursor as substrate. The substrate is generated by incubation of in vitro transcribed murine IL-lβ precursor mRNA with rabbit reticulocyte lysate in the presence of -"S-methionine. Incubation of either the native or recombinant enzyme with murine IL-lβ precursor generates the expected 28 kDa and 17.5 kDa products that result from cleavage at the Asp²⁷-Gly²° and Aspl 1⁷-Vall 1 ° sites. Cleavage activity is completely inhibited by a potent, specific, reversible tetrapeptide aldehyde inhibitor of human ICE (L-709,049). Further fractionation of the cell lysates indicates that the majority of this ICE activity is in the soluble cytosol and only a small fraction is in the pellet. This is in contrast to the behavior of human ICE p45 protein which partitions into inclusion bodies when expressed in E. coli under identical conditions. In a continuous fluorometric assay using the tetrapeptide aldehyde- AMC substrate, the activity in the cytosolic extract is 50-100 units/ml. Active, recombinant ICE can be purified from this extract following concentration (40 X) and affinity chromatography using the tetrapeptide aldehyde as the ligand (see Fig. 10). A complex consisting exclusively of p20 and plO subunits is isolated in this matter. Liquid chromatography-electrospray ionization-mass spectroscopy (LC-ESI- MS) analysis of this material as described (Thornberry et aL, Nature 356, pp.768-774 (1992); Griffin et aL, Int. J. Mas. Spect. Ion Phys., Il l, pp. 131-149 (1991)) indicated that the p20 subunit had a molecular mass of 20171.4 Da, in excellent agreement with the predicted mass of 20171.0 Da for a protein extending from Asnl *" to Asp ⁹6. The major subunit had a molecular mass of 10478.8 Da designated plO, in excellent agreement with the predicted mass of 10479.0 Da for a protein extending from Gly³15 to His^ . Another form of the subunit was identified which had a molecular mass of 11170 Da, designated pi 1 which is the predicted mass (11169.9 Da) for a protein extending from

autocatalytic processing of a precursor protein (Asnl

lacking the prodomain. Cleavage occured at two of three Asp-X bonds (Asp²⁹⁶-Ser²⁹⁷, Asp³⁰⁸-Ala³⁰⁹, Asp³l⁴-Gly³¹⁵) to yield an active enzyme complex consisting of a p20 subunit and two different forms of the lower molecular weight subunit [pi 1 or plO].

A construct for the expression of recombinant enzyme from the full-length 402 aa p45 proenzyme was generaged by PCR with the following primers:

5' GGGCCCCATATGGCTGACAAGATCCTGAGGGCAAAG 3^* 5' GGGCCCCATATGTTAATGTCCCGGGAAGAGGTAGAAAC 3' which contained Ndel sites that flunk the entire open reading frame from Metl to

This construct was cloned into the Ndel site of the pETlla as described above and transformed into the E. coli strain BL21(DE3)pLYS for expression. The p45 ICE protein expressed in E. coli was enzymatically active in a gel cleavage assay with radiolabeled murine IL-lβ precursor as substrate (Fig. 12). By immunoblot analysis, the great majority of ICE protein is sequestered in inclusion bodies, as is in the case for the human p45 ICE protein when expressed in E. coli.

EXAMPLE 7

In Vitro Translation of ICE mRNA by Xenopus Oocyte Microinjection Vector and Expression in Mammalian Cells

ICE cDNA constructs are ligated into in vitro transcription vectors (the pGEM series, Promega) for the production of synthetic mRNAs.

Synthetic mRNA is produced in sufficient quantity in vitro by cloning double stranded DNA into a plasmid vector containing a bacteriophage promoter, linearizing the plasmid vector containing the cloned ICE-encoding DNA, and transcribing the cloned DNA in vitro using a DNA-dependent RNA polymerase from a bacteriophage that specifically recognizes the bacteriophage promoter on the plasmid vector. Various plasmid vectors are available containing a bacteriophage promoter recognized by a bacteriophage DNA-dependent RNA polymerase, including but not limited to plasmids pSP64, pSP65, pSP70, pSP71, pSP72, pSP73, pGEM-3Z, pGEM-4Z, pGEM-3Zf, pGEM-5Zf, pGEM-7Zf, pGEM-9Zf, and pGEM-HZf, the entire series of plasmids is commercially available from Promega.

The double stranded ICE-encoding DNA is cloned into the bacteriophage promoter containing vector in the proper orientation using one or more of the available restriction endonuclease cloning sites on the vector which are convenient and appropriate for cloning ICE DNA. The vector with the ligated ICE DNA is used to transform bacteria, and clonal isolates are analyzed for the presence of the vector with the ICE DNA in the proper orientation.

Once a vector containing the ICE-encoding DNA in the proper orientation is identified and isolated, it is linearized by cleavage with a restriction endonuclease at a site downstream from, and without disrupting, the ICE transcription unit. The linearized plasmid is isolated and purified, and used as a template for in vitro transcription of ICE mRNA.

The template DNA is then mixed with bacteriophage- specific DNA-dependent RNA polymerase in a reaction mixture which allows transcription of the DNA template forming ICE mRNA. Several bacteriophage-specific DNA-dependent RNA polymerases are available, including but not limited to T3, T7, and SP6 RNA polymerase. The synthetic ICE mRNA is then isolated and purified.

It may be advantageous to synthesize mRNA containing a 5' terminal cap structure and a 3' poly A tail to improve mRNA stability. A cap structure, or 7-methylguanosine, may be incorporated at the 5'terminus of the mRNA by simply adding 7-methylguanosine to the reaction mixture with the DNA template. The DNA-dependent RNA polymerase incorporates the cap structure at the 5' terminus as it synthesizes the mRNA. The poly A tail is found naturally occurring in many cDNA's but can be added to the 3' terminus of the mRNA by simply inserting a poly A tail-encoding DNA sequence at the 3' end of the DNA template.

The isolated and purified ICE mRNA is translated using either a cell-free system, including but not limited to rabbit reticulocyte lysate and wheat germ extracts (both commercially available from Promega and New England Nuclear) or in a cell based system, including but not limited to microinjection into Xenopus oocytes, with microinjection into Xenpus oocytes being preferred.

Xenopus oocytes are microinjected with a sufficient amount of synthetic ICE mRNA to produce ICE protein. The microinjected oocytes are incubated to allow translation of the ICE mRNA, forming ICE protein.

These synthetic mRNAs will be injected into Xenopus oocytes (stage 5 - 6) by standard procedures [Gurdon, J.B. and Wickens, M.D. Methods in Enzymol. 101: 370-386, (1983)]. Oocytes will be harvested and analyzed for ICE expression as described below.

EXAMPLE 8

Cloning of ICE cDNA into a Mammalian Expression Vector

ICE cDNA expression cassettes are ligated at appropriate restriction endonuclease sites to the following vectors containing strong, universal mammalian promoters: pBC12BI [Cullen, B.R. Methods in Enzymol. 152: 684-704 1988], and pEE12 (CellTech EP O 338,841) and its derivatives pSZ9016-l and p9019. p9019 represents the construction of a mammalian expression vector containing the hCMVTE prm, polylinker and SV40 polyA element with a selectable marker/amplification system comprised of a mutant gene for dehydrofolate reductase (mDHFR) (Simonsen, C.C. and Levinson, A. D. Proc. Natl. Acad. Sci USA 80: 2495-2499 [1983]) driven by the SV40 early promoter. An SV40 polyadenylation sequence was generated by a PCR reaction defined by primers 13978-120 and 139778-121 using pD5 (Berker and Sharp, Nucl. Acid Res. 13: 841-857 [1985]) as template. The resulting 0.25 Kb PCR product was digested with Clal and Spel and ligated into the 6.7 Kb fragment of pEE12 which had been likewise digested. The resultant plasmid was designated p9018. p9018 was digested with Bgiπ and Sfil to liberate the 3' portion of the SV40 early promoter and the GScDNA from the vector. A 0.73 Kb Sfil-Xhoϋ fragment isolated from plasmid pFR400 (Simonsen, C.C. and Levinson, A. D. Proc. Natl. Acad. Sci USA 80: 2495-2499 [1983]) was ligated to the 5.6 Kb vector described above, reconstituting the SV40 early promoter, and inserting the mdHFR gene. This plasmid is designated p9019. pSZ9016-l is identical to p9019 except for the substitution of the HIV LTR for the huCMVIE promoter. This vector was constructed by digesting p9019 with Xbal and Mlul to remove the huCMVIE promoter. The HTV LTR promoter, from residue -117 to +80 (as found in the vector pCD23 containing the portion of the HTV-1 LTR (Cullen, Cell 46:973 [1986]) was PCR amplified from the plasmid pCD23 using oligonucleotide primers which appended to the ends of the product the Mlul and Spel restriction sites on the 5' side while Hind in and Xba I sites were appended on the 3' side. Following the digestion of the resulting 0.2 kb PCR product with the enzymes Mlul and Xba I the fragment was agarose gel purified and ligated into the 4.3 Kb promoterless DNA fragment to generate the vector pSZ9016-l.

Cassettes containing the murine ICE cDNA in the positive orientation with respect to the promoter are ligated into appropriate restriction sites 3' of the promoter and identified by restriction site mapping and/or sequencing. These cDNA expression vectors are introduced into various fibroblastic host cells: [COS -7 (ATCC# CRL1651), CV-1 tat [Sackevitz et aL, Science 238: 1575 (1987)], 293, L (ATCC#CRL6362)] by standard methods including but not limited to electroporation,or chemical procedures (cationic liposomes, DEAE dextran, calcium phosphate). Transfected cells and cell culture supernatants can be harvested and analyzed for ICE expression as described above.

All of the vectors used for mammalian transient expression can be used to establish stable cell lines expressing murine ICE. Unaltered murine ICE cDNA constructs cloned into expression vectors will be expected to program host cells to make intracellular murine ICE protein. In addition, murine ICE is expressed extracellularly as a secreted protein by ligating ICE cDNA constructs to DNA encoding the signal sequence of secreted protein such as the human growth hormone or human lysozyme. The transfection host cells include, but are not limited to, CV-l-P [Sackevitz et aL, Science 238: 1575 (1987)], tk-L [Wigler, et al. Cell 11: 223 (1977)], NS/0, and dHFr- CHO [Kaufman and Sharp, J. Mol. Biol. 159: 601, (1982)].

Co-transfection of any vector containing murine ICE cDNA with a drug selection plasmid (included, but not limited to G418, aminoglycoside phosphotransferase, pLNCX [Miller, A.D. and Rosman G. J. Biotech News 7: 980-990 (1989)]; hygromycin, hygromycin-B phosphotransferase, pLG90 [Gritz. L. and Davies, J., GENE 25: 179 (1983)] ; APRT, xanthine-guanine phosphoribosyltransferase, pMAM (Clontech) [Murray, et aL, Gene 31: 233 (1984)] will allow for the selection of stably transfected clones. Levels of ICE are quantitated by the assays described above.

ICE cDNA constructs are ligated into vectors containing amplifiable drug-resistance markers for the production of mammalian cell clones synthesizing the highest possible levels of ICE. Following introduction of these constructs into cells, clones containing the plasmid are selected with the appropriate agent, and isolation of an overexpressing clone with a high copy number of the plasmid is accomplished by selection in increasing doses of the agent. The following systems are utilized: the 9016 or the 9019 plasmid containing the mutant DHFR gene [Simonson, C. and Levinson, A., Proc. Natl. Acad. Sci. USA 80: 2495 (1983)], transfected into dHFR- CHO cells and selected in methotrexate; the pEE12 plasmid containing the glutamine synthetase gene, transfected into NS/O cells and selected in methionine sulfoximine (CellTech International Patent Application 2089/10404); and 9016 or other CMV prm vectors, co-transfected with pDLAT-3 containing the thymidine kinase gene [Colbere and Garopin, F., Proc. Natl. Acad. Sci. 76: 3755 (1979)] in APRT and TK deficient L cells, selected in APRT (0.05 mM azaserine, 0.1 mM adenine, 4 ug/ml adenosine) and amplified with HAT (100 uM hypoxanthine, 0.4 uM aminopterin, 16 uM thymidine).

The expression of recombinant murine ICE is achieved by transfection of full-length ICE cDNA, including the complete ORF of the 45 kDa ICE preprotein (Fig. 23), into a mammalian host cell. The 1.6 kb EcoRI fragment containing the full length ICE cDNA is cloned into vector pSZ-9016. 6mg of this DNA along with 0.6 ug of pX8TAT, a mammalian expression vector which places the trans-activating protein (TAT) of HIV under the control of the SV40 early promoter, is transfected into COS-7 cells by the cationic-liposome method. Cells are harvested 48 hours later and lysed in detergent buffer (25 mM HEPES pH 7, 1% Triton-X-100, ImM EDTA, 2mM DTT, 10 ug/ml aprotinin, 10 ug/ml leupeptin, 10 ug/ml pepstatin, and 2 mM PMSF). Cell lysates are incubated with radiolabeled IL-lβ precursor to measure ICE activity. Cleavage products of IL-lβ are analyzed by immunoprecipita- tion with IL-lβ antibody and fractionation on SDS polyacrylamide gels. The IL-lβ precursor is cleaved to the mature, 17 kDa form by cells transfected with ICE cDNA, but not by cells transfected with the expression plasmid alone. The cleavage product comigrate with mature IL-lβ produced by incubation of substrate with affinity purified ICE and is completely inhibited by the specific ICE inhibitor (L-709,049). The substrate specificity of the expressed ICE activity is verified by incubating lysates with an IL-lβ precursor cleavage site mutant (Alal 1 ) _which cannot be cleaved by ICE. As with native ICE, the activity from the transfectants will cleave the mutant protein to a 28 kDa product but not to the 17 kDa form.

EXAMPLE 9

Cloning of murine ICE cDNA into a Baculovims Expression Vector for

Expression in Insect Cells

Baculovims vectors, which are derived from the genome of the AcNPV vims, are designed to provide high level expression of cDNA in the Sf9 line of insect cells (ATCC CRL#1711). Recombinant baculoviruses expressing ICE cDNA is produced by the following standard methods (InVitrogen Maxbac Manual): the ICE cDNA constmcts are ligated into the polyhedrin gene in a variety of baculovims transfer vectors, including the pAC360 and the BlueBac vector (InVitrogen). Recombinant baculoviruses are generated by homologous recombination following co-transfection of the baculovims transfer vector and linearized AcNPV genomic DNA [Kitts, P.A., Nuc. Acid. Res. 18: 5667 (1990)] into Sf9 cells. Recombinant pAC360 vimses are identified by the absence of inclusion bodies in infected cells and recombinant pBlueBac vimses are identified on the basis of B- galactosidase expression (Summers, M. D. and Smith, G. E., Texas Agriculture Exp. Station Bulletin No. 1555)). Following plaque purification, ICE expression is measured by the assays described above.

The cDNA encoding the entire open reading frame for p45 is inserted into the BamHI site of pBlueBacϋ. Constmcts in the positive orientation are identified by sequence analysis and used to transfect Sf9 cells in the presence of linear AcNPV wild type DNA.

Authentic, enzymatically-active ICE is found in the cytoplasm of infected cells. Active ICE is extracted from infected cells under native conditions by hypotonic or detergent lysis.

EXAMPLE 10

Cloning of murine ICE cDNA into a veast expression vector

Recombinant murine ICE is produced in the yeast S. cerevisiae following the insertion of the optimal ICE cDNA cistron into expression vectors designed to direct the intracellular or extracellular expression of heterologous proteins. In the case of intracellular expression, vectors such as EmBLyex4 or the like are ligated to the ICE cistron [Rinas, U. et aL, Biotechnology 8: 543-545 (1990); Horowitz B. et aL, J. Biol. Chem. 265: 4189-4192 (1989)]. For extracellular expression, the ICE cistron is ligated into yeast expression vectors which fuse a secretion signal (a yeast or mammalian peptide) to the NH2 terminus of the ICE protein [Jacobson, M. A., Gene 85: 511-516 (1989); Riett L. and Bellon N. Biochem. 28: 2941-2949 (1989)].

These vectors include, but are not limited to pAVEl>6, which fuses the human serum albumin signal to the expressed cDNA [Steep O. Biotechnology 8: 42-46 (1990)], and the vector pL8PL which fuses the human lysozyme signal to the expressed cDNA [Yamamoto, Y., Biochem. 28: 2728-2732)]. In addition, ICE is expressed in yeast as a fusion protein conjugated to ubiquitin utilizing the vector pVEP [Ecker, D. J., J. Biol. Chem. 264: 7715-7719 (1989), Sabin, E. A., Biotechnology 7: 705-709 (1989), McDonnell D. P., Mol. Cell Biol. 9: 5517-5523 (1989)]. The levels of expressed ICE are determined by the assays described above.

EXAMPLE 11

Murine IL-l β antibodies

Polyclonal antiserum for an ELISA assay nd immunoblot analysis was generated by intrapopliteal lymph node injection of female New Zealand rabbits with 50 μg of recombinant murine IL-lβ (R & D Sytems) in CFA followed by two additional intranodal injections (at two and four weeks) and one s.c. injection (at eight weeks) of 50 μg of IL- lβ in IFA. To generate immunoprecipitating antisera, NZW rabbits were injected with recombinant murine IL-lβ adsorbed to alum (13).

EXAMPLE 12

ELISA assay for murine IL-l β

Plates (96 well, Costar) were coated with 5 mg/ml of a monoclonal anti-mlL-lβ antibody [Hogquist et aL, supra] in PBS reference, washed in PBS, and blocked by the addition of BSA solution for 30 minutes. After three washes, 100 μl aliquots of sample were added to the plate and incubated for 2 hours at 25°C. The plates were then washed four times and incubated for 1 hour at 25 °C with a 1:500 dilution of the rabbit polyclonal semm. Plates were washed three times and incubated with a 1:10,000 dilution of peroxidase conjugated F(ab')2 fragment goat anti-rabbit IgG (Accurate Scientific) for 1 hour at 25°C. The peroxidase assay (TMB substrate, Kierkegaard and Perry) was terminated after 1 minute by the addition of 1 M phosphoric acid and color development was quantitated by measurement of OD450 on a plate spectrophotometer (Molecular Devices).

EXAMPLE 13

Isolation And Characterization of ICE in Murine PEC

Female CD-I mice (6-8 weeks) were injected intraperitoneally with 1 mg of heat-killed Propionibacterium acnes. Cells were harvested 6 days following injection by lavage with 5 ml PBS containing 10 U/ml heparin (Upjohn). Cells (2 x lθ6/ml) were plated for 2 hours in RPMI, 10% FCS (Hazelton) at 37°C and then washed to remove non-adherent cells. Adherent cells were then cultured in fresh medium containing 100 ng/ml LPS for 18 hours prior to harvest. Cell free extracts were prepared from peritoneal exudate cells and tissue culture cells by hypotonic lysis as described previously [Kostra, supra]. Briefly, cells were washed in PBS without MgCl2 and CaCl2, pelleted and resuspended in buffer containing 10 mM KC1, 20 mM Hepes, pH 7.4, 1.5 mM MgCl2, 0.1 mM EDTA at a concentration of 10° cells/ml. Following incubation for 20 minutes on ice, cells were lysed by 20 strokes in a tight-fitting Dounce homogenizer. Cellular homogenates were first clarified by sequential centrifugation at 1000 x g for 20 minutes, 30,000 x for 10 minutes, and 230,000 x g for 60 minutes (4°C) and then dialyzed overnight against 100 mM Hepes, 10% sucrose, 0.1% CHAPS, 2 mM DTT, pH 7.5 (4°C). Extracts were stored at -80°C. EXAMPLE 14

Characterization of IL-lβ in stimulated murine macrophages

For detection of intracellular IL-lβ, adherent murine peritoneal exudate cells (2 x lθ6) were lysed in 100 μl of 2X Laemmli buffer, electrophoresed on a 12.5% polyacrylamide gel and analyzed by immunoblot with polyclonal serum against murine IL-lβ. Culture supernatants from peritoneal exudate cells were first immunoprecipitated and then analyzed by immunoblot. Culture supernatants (2 ml) were incubated with 200 μl of a 25% slurry of IgG coated protein beads overnight at 4°C. The immune complexes were washed three times in RIPA buffer (25 mM Tris, pH 7.5, 10 mM NaCl, ImM EDTA, 1% Triton-X-100, 0.5% sodium deoxycholate and 0.2% SDS) and resuspended in 20 μl of 2X Laemmli buffer. Electrophoresis and Western blotting were carried out as described [Limjuco et aL, Proc. Natl. Acad. Sci., USA pp. 3972-3976 [1986] except that the blots were incubated in SuperBlock (Pierce Biochemicals) for 1 hour at 25°C prior to addition of antibody.

EXAMPLE 15

Cleavage of recombinant murine IL-lβ precursor by recombinant murine ICE

A fragment containing the full-length murine pIL-lβ [Howard, et aL, J. Immun.147, 2964-2969 (1991)] flanked by Ndel sites was generated by PCR and cloned into the pET 11a expression vector as described above. Recombinant pIL-lβ was isolated from the inclusion body pellet by washing 3 times in 1% Triton-X-100, 50 mM Tris, pH 8, 1 mM PMSF, 10 μg/ml pepstatin, 10 mg/ml leupeptin followed by solubilization in 10 volume of 6 M guanidine HCl, 100 mM DTT, 50 mM Tris pH 7.5. The sample was fractionated on a C4 RP- HPLC column, diluted into ICE buffer, and purified to homogeneity by DEAE-5PW-HPLC [Thornberry, et aL, Nature 356, 768-744 (1992)]. Purified pIL-lβ (300 μg) and recombinant murine ICE (200 units) were incubated in 500 μl ICE buffer (100 mM Hepes, 10% sucrose, 0.1% CHAPS, 1 mM DTT, pH 7.5) for 5 h at 30°C. Cleavage of the 31 kDa IL-lβ precursor to both the 28 kDa and 17.5 kDa proteins is observed under these conditions. An aliquot of the cleaved material was injected onto a narrow bore C4 RP-HPLC column and the molecular mass of the 17.5 kDa pIL-lβ cleavage product was determined by LC-ESI-MS as described previously [Thornberry, et aL, Nature 356, 768-744 (1992)]. The molecular mass of this protein was determined to be 17395.8 Da, which is in excellent agreement with the predicted mass (17394.9 Da) of a cleavage product with a ValH° amino terminus.

EXAMPLE 16

Continuous fluorometric peptide substrate assay for ICE activity

The peptide Ac-Tyr-Val-Ala-Asp-NH-CH3 defines the minimal substrate for human ICE and was the basis for the design of a fluorogenic substrate, Ac-Tyr-Val-Ala-Asp-7-AMC. Cleavage of this substrate by human ICE shows Michaelis-Menten kinetics with a Km=14 + 3 μM. This substrate can be used to monitor the ICE activity in partially purified preparations of human ICE. This substrate was also successfully employed to measure the activity of murine ICE from S-300 cytosolic extracts of peritoneal exudate cells. The assay conditions are as follows: 50mM Ac-Tyr- Val- Ala-Asp- AMC (from a 5 mM stock in DMSO) is added to a 500 μl solution containing ICE buffer (100 mM Hepes, 10% sucrose, 0.1% CHAPS, 1 mM DTT, 1 mg/ml BSA, pH 7.5) and the reaction commences with the addition of approximately 0.25 units (5 μl) of enzyme. The reaction is incubated at 25 °C and monitored continuously in a Gilford Fluoro-IV Spctrofluorometer with an excitation wavelength of 389 nm and an emission wavelength of 460 nm. A unit is defined as the amount of enzyme required to produce 1 pmole of AMC/minute at 25°C using saturating levels (> 50 μM) of substrate. Peptides were synthesized via the Merrifield solid-phase technique using pheny lacetamidomethyl resins and tBoc amino acids. Synthesis was performed on an Applied Biosystems 430A peptide synthesizer according to the manufacturers suggested protocols. Peptides were simultaneously deprotected and cleaved from the resin with 90% anhydrous HF, 10% anisole at 0° C for 1 h and then extracted from the resin with 10% acetic acid and lyophilized. The resulting cmde peptides were purified by reversed phase HPLC on Waters C18 DeltaPak columns with a gradient of 5 to 70% acetonitrile in aqueous 0.2% trifluoroacetic acid (TFA). The structure of purified peptide was confirmed by mass spectral analysis.

A peptide of the sequence Ac-Tyr-Val-Ala-Asp-7- aminomethylcoumarin (Ac-Tyr-Val-Ala-Asp-AMC), N-(N-Acetyl- tyrosinyl-valinyl-alaninyl)-aspartic acid a-7-amino-4 methylcoumarin amide was synthesized by the following process.

Step A: N-Allyloxycarbonyl aspartic acid b-t-butyl ester a-7- amino-4-methylcoumarm amide

To a solution of N-alylloxycarbonyl aspartic acid b-t-butyl ester (3.44g, (12.6 mmol) and 7-amino-4-methylcoumarin (2.00 g, 11.42 mmol) in 15 mL of anhydrous dioxane was added ethyl dimethylaminopropyl carbodiimide (2.66 g, 13.86 mmol). After 75 min at reflux, the mixture was diluted with ethyl acetate and washed three times with 1 N hydrochloric acid and three times with saturated sodium bicarbonate. The solution was dried over sodium sulfate and concentrated in vacuo. The mixture was purified by HPLC on silica-gel (35x300 mm column, 10% ethyl acetate in dichloromethane as eluent) to give the title compound as a colorless foam: 1H NMR (200 MHz, CD30D) d 7.77 (d, 1H, J = 2.39 Hz), 7.68 (d, IH, J = 9.06 Hz), 7.49 (dd, IH, J = 2.36, 9.10 Hz), 6.21 (q, IH, J = 1.30 Hz), 5.95 (m, IH), 5.4-5.15 (m, 2H), 4.72- 4.58 (m, 3H), 2.85 (dd, IH, J = 6.17, 15.73 Hz), 2.65 (dd, IH, J = 7.62, 16.37 Hz), 2.43 (d, 3H, J = 1.44 Hz), 1.43 (s, 9H).

Step B: Aspartic acid b-t-butyl ester a-7-amino-4-methyl- coumarin amide

To a solution of N-Allyloxycarbonyl aspartic acid b-t-butyl ester a-7- amino-4-methylcoumarin amide (435 mg, 1.01 mmol) and Dimedone (1.13 g, 8.08 mmol) in 10 mL of anhydrous tetrahydrofuran was added tetrakis triphenylphosphine palladium (117 mg, 0.1 mmol). After 45 min, the mixture was diluted with ethyl acetate and washed five times with saturated sodium bicarbonate, dried over sodium sulfate and concentrated in vacuo. The mixture was disolved in a small amount of a solution of 1% ammonia and 10% methanol in dichloromethane and filtered through a 0.22 mm filter. The mixture was then purified by HPLC on silica-gel (22x300 mm column, eluting with a gradient of dichloromethane to 0.25% ammonia and 2.5 % methanol in dichloromethane) to give the title compound as a colorless foam: IH NMR (200 MHz, CD30D) d 7.93 (d, IH, J = 1.76 Hz), 7.82 (d, IH, J = 8.50 Hz), 7.63 (dd, IH, J = 2.40, 9.10 Hz), 6.34 (q, IH, J = 1.31 Hz), 3.89 (t, IH, J = 6.35 Hz), 2.88 (dd, IH, J = 6.03, 16.72 Hz), 2.75 (dd, IH, J = 6.77, 16.75 Hz), 2.56 (d, 3H, J = 1.37 Hz), 1.54 (s, 9H). Step C: N-(N-Acetyl-tyrosinyl-valinyl-alaninyl)-aspartic acid b-t-butyl ester a-7-amino-4-methylcoumarin

To a solution of N-(N-Acetyl-tyrosinyl-valinyl-alanine (288 mg, 0.733 mmol), aspartic acid b-t-butyl ester a-7-amino-4-methylcoumarin (242 mg, 0.698 mmol) and hydroxybenzotriazole (149 mg, 1.10 mmol) in 2 mL of dimethyl formamide at 0°C was added dicyclohexylcarbodiimide (151 mg, 0.733). After 16 h at ambient temperature, the mixture was filtered and purified by Sephadex" LH-20 chromatography (1M x 50 mm column, methanol eluent). The resulting product was triturated with methanol to give the title compound as a colorless solid: IH NMR (200 MHz, DMF-d7) d 8.3-7.5 (m, 7H), 7.09 (br d, 2H, J = 8.61 Hz), 6.72 (br d, 2H, J = 8.64 Hz), 6.27 (q, IH, J = 1.31 Hz), 4.84 (m, IH), 4.62 (m, IH), 4.44-4.14 (m, 2H), 3.15-2.7 (m, 4H), 2.45 (d, 3H, J = 1.37 Hz), 2.13 (m, IH), 1.87 (s, 3H), 1.41 (s, 9H), 1.37 (d, 3H. J = 7.38 Hz), 0.94 (d, 3H, J = 7.12 Hz), 0.93 (d, 3H, J = 7.12 Hz).

Step D: N-(N-Acetyl-tyrosinyl-valinyl-alaninyl)-aspartic acid a-7-amino-4-methylcoumarin amide

N-(N-Acetyl-tyrosinyl-valinyl-aIaninyl)-aspartic acid b-t-butyl ester a- 7-a__nino-4-methylcoumarin amide was disolved in trifluoroacetic acid. After 15 min the mixture was concentrated in vacuo to give the title compound as a colorless solid: IH NMR (200 MHz, DMF-d7) d 8.3-7.5 (m, 7H), 7.09 (br d, 2H, J = 8.61 Hz), 6.72 (br d, 2H, J = 8.64 Hz), 6.27 (q, IH, J = 1.31 Hz), 4.84 (m, IH), 4.62 (m, IH), 4.44-4.14 (m, 2H), 3.15-2.7 (m, 4H), 2.45 (d, 3H, J = 1.37 Hz), 2.13 (m, IH), 1.87 (s, 3H), 1.41 (s, 9H), 1.37 (d, 3H. J = 7.38 Hz), 0.94 (d, 3H, J = 7.12 Hz), 0.93 (d, 3H, J = 7.12 Hz). Microanalysis calculated for C33H39N5O1071.65 H20: C, 57.00, H, 6.13, N, 10.07; found: C, 56.97, H, 5.84, N 10.16.

EXAMPLE 17

Affinity Chromatography of Interleukin-IB Converting Enzvme Preparation of a chromatographic matrix from Compound A

An affinity column for interleukin-1 converting enzyme was prepared from the potent peptide aldehyde inhibitor Acetyl-Tyr- Val-Lys-Asp-CHO (Compound A), coupled via a 12-atom bis-oxirane spacer to SEPHAROSE CL-4B through the lysine residue.

Synthesis Of Affinity Matrix Step A: Epoxy-activated SEPHAROSE CL-4B

Epoxy-activated SEPHAROSE CL-4B was prepared as described in the literature (Sundberg, L., and Porath, J. (1974) J. Chromatogr. 90, 87-98). Specifically, a slurry consisting of 100 gm suction-dried SEPHAROSE CL-4B, 100 ml of 1,4-butanediol diglycidyl ether (a nominal 70% solution), and 100 ml 0.6M NaOH containing 2 mg/ml NaBH4 was mixed with an overhead stirrer for 16 hours at ambient temperature. The resulting epoxy-activated SEPHAROSE CL-4B was washed exhaustively on a coarse sintered glass funnel with 10 liters of water, and stored in water at 4°C.

Step B: Coupling of Peptide Aldehyde Dimethyl Acetal

A'

The blocked aspartyl-t-butyl ester, dimethyl acetal (Compound A') of the active aldehyde, Compound A, was dissolved as a 10 mM solution in methanol, and then combined with more methanol, water, and a 400 mM sodium carbonate solution adjusted to pH 11.00 with HCl, to give a 50% methanol solution containing 2 mM inhibitor and 200 mM carbonate buffer. This solution (10 ml) was mixed with the suction-dried cake (10 gm) of epoxy-activated SEPHAROSE CL-4B, and the slurry was stirred by rotation at 37°C for 3 days. The resulting affinity matrix was washed thoroughly with 1M KC1 and water, and was stored as a slurry at 4°C. The incorporation, based on results with [14-C]-lisinopril (Bull, H.G., Thornberry, N.A., and Cordes, E.H. (1985) J. Biol. Chem. 260, 2963-2972), is estimated to be 1 umol/ml packed affinity matrix.

Step C: Activation to Aldehyde

The above procedure gave the dimethyl-acetal of Acetyl- Tyr-Val-Lys-Asp-CHO coupled to the spacer arm, the t-butyl protecting group on the aspartate residue being lost during the coupling conditions. Activation of this matrix to the aldehyde was carried out in the affinity column just prior to use, by equilibrating the matrix with 0.01N HCl and letting it stand for 2 hours at 25 °C. A control matrix containing [14-C]glycine as a tracer gave no evidence (<1%) for loss of ligand under these conditions.

Synthesis of N-(N-Acetyl-tyrosinyl-valinyl-lysinyl)-3-amino-4- oxobutanoic acid dimethyl acetal b-t-butyl ester.

Step A: N-allyloxycarbonyl-3-amino-4-hyroxybutanoic acid tert-butyl ester

To a solution of N-allyloxycarbonyl (S)-aspartic acid b-tert-butyl ester (2.00 g, 7.32 mmol) in 50 mL of tetrahydrofuran (THF) at 0°C, was added N-methyl morpholine (NMM, 885 mL, 8.05 mmol) followed by isobutyl chloroformate (IBCF, 997 mL, 7.68 mmol). After 15 min, this mixture was added to a suspension of sodium borohydride (550 mg, 14.55 mmol) in 50 mL of THF and 12.5 mL of methanol at -45°C. After 30 min at -45°C, the mixture was warmed to 0 °C and held at that temperature for 30 min. The reaction was quenched with acetic acid, diluted with 1:1 ethyl acetate :hexane, and washed 3 times with dilute sodium bicarbonate. The organics were dried over sodium sulfate, filtered, and concentrated. The residue was purified by MPLC on silica-gel (35x350 mm column, 30% ethyl acetate/hexane) to give the desired product: IH NMR (200 MHz, CD30D) d 5.9 (m, IH), 5.28 (br d, IH, J = 17 Hz), 5.15 (br d, IH, J = 9 Hz), 4.52 (br d, 2H, J = 6 Hz), 3.98 (m, IH), 3.48 (ABX, 2H, J = 5, 6, 11 Hz), 2.53 (dd, IH, J = 5, 16 Hz), 2.32 (dd, IH, J = 9, 16 Hz), 1.43 (s, 9H).

Step B: N-allyloxycarbonyl-3-amino-4-oxobutanoic acid b-tert-butyl ester dimethyl acetal

To a solution of dimethyl sulf oxide (757 mL, 10.67 mmol) in 10 mL of dichloromethnane at _45°C was added oxalyl chloride (508 mL, 5.82 mmol). After 5 min, a solution of N-allyloxycarbonyl-3-amino-4- hyroxybutanoic acid tert-butyl ester (1.25 g, 4.85 mmol) in 10 mL of dichloromethane was added. After 15 min, triethyl amine (2.03 mL, 14.55 mmol) was added. After 30 min, the mixture was warmed to -23 °C and stirred for 30 min. The mixture was diluted with 1:1 ethyl acetate/hexane, washed with water, 1 N sodium hydrogensulfate, and twice with water. The organics were dried over sodium sulfate, filtered, and concentrated. The resultant oil was disolved in 200 mL of methanol and 20 mL of trimethyl orthoformate and 100 mg of p- toluene sulphonic acid were added. After 16 hours, the reaction was quenched with saturated sodium bicarbonate and concentrated in vacuo. The mixture was diluted with ether and washed 5 times with dilute sodium bicarbonate. The ether layer was dried over magnesium sulfate, filtered, and concentrated to afford the title compound as a colorless oil: IH NMR (200 MHz, CD30D) d 5.9 (m, IH), 5.26 (br d, IH, J = 17 Hz), 5.14 (br d, IH, J = 10 Hz), 4.51 (br d, 2H, J = 5.33 Hz), 4.25 (d, IH, J = 4.79 Hz), 4.11 (m, IH), 3.40 (s, 3H), 3.39 (s, 3H), 2.52 (dd, IH, J = 4.86, 15.27 Hz), 2.30 (dd, IH, J = 9.00, 15.28 Hz), 1.43 (s, 9H). Step C: 3-Amino-4-oxobutanoic acid b-tert-butyl ester dimethyl acetal

To a solution of N-allyloxycarbonyl-3-amino-4-oxobutanoic acid b-tert- butyl ester dimethyl acetal (312 mg, 1.03 mmol) in 10 mL of THF was added morpholine (897 mL, 10.3 mmol) and tetrakis triphenyl- phosphine palladium (100 mg). After 3 hours, the mixture was diluted with 1:1 ethyl acetate/hexane and washed 5 times with dilute sodium bicarbonate. The orgains were dried over sodium sulfate, filtered, and concentrated. The resulting oil was purified by MPLC on silica-gel (22x300 mm column, linear gradient of dichloromethane to 1% ammonia and 10% methanol in dichloromethane) to afford the title compound as a pale-yellow oil: IH NMR (200 MHz, CD30D) d 4.15 (d, IH, J = 5.67 Hz), 3.41 (s, 3H), 3.40 (s, 3H), 3.19 (m, IH), 2.47 (dd, IH, J = 4.88, 16.06 Hz), 2.22 (dd, IH, J = 7.86, 16.16 Hz), 1.45 (s, 9H).

Step D: N-(N-Acetyl-tyrosinyl-valinyl-(e-CBZ-lysinyl))-3- amino-4-oxobutanoic acid b-tert-butyl ester dimethyl acetal

To a solution of 3-Amino-4-oxobutanoic acid b-tert-butyl ester dimethyl acetal (238 mg, 1.09 mmol) in 5 mL of DMF at 0°C was added N- methyl morpholine (599 mL, 5.45 mmol) followed sequentially by N- Acetyl-tyrosinyl-valinyl-e-CBZ-lysine (735 mg, 1.09 mmol), hydroxybenzotriazole (221 mg, 1.64 mmol), and dicyclohexyl¬ carbodiimide (225 mg, 1.09 mmol). After 16 hours at ambient temperature, the mixture was filtered and purified by Sephadex" LH-20 chromatography (IM x 50 mm column, methanol eluent). The resulting product was further purified by MPLC on silica-gel (22 x 300 mm column, eluting with a linear gradient of dichloromethane to 1% ammoinia and 10% methanol in dichloromethane) to give the title compound as a colorless solid: IH NMR (200 MHz, CD30D) d 7.31 (br s, 5H), 7.04 (br d, 2H, J = 8.35 Hz), 6.67 (br d, 2H, J = 8.45 Hz), 5.04 (s, 2H), 4.61 (m, IH), 4.44-4.25 (m, 3H), 4.17 (d, IH, J = 7.27 Hz), 3.39 (s, 3H), 3.38 (s, 3H), 3.1-2.9 (m, 3H), 2.75 (dd, IH, J = 9.28, 14.12 Hz), 2.53 (dd, IH, J = 5.47, 15.58 Hz), 2.33 (dd, IH, J = 7.96, 15.53 Hz), 2.04 (m, IH), 1.88 (s, 3H), 1.8-1.2 (m, 6H), 1.41 (s, 9H), 0.94 (d, 6H, J = 6.74 Hz).

Step E: N-(N-Acetyl-tyrosinyl-valinyl-lysinyl)-3-amino-4- oxobutanoic acid dimethyl acetal b-t-butyl ester

A solution of N-(N-Acetyl-tyrosinyl-valinyl-e-CBZ-lysinyl)-3-amino-4- oxobutanoic acid b-tert-butyl ester dimethyl acetal (15.6 mg) was disolved in 2 mL of methanol and 10 mg of Pearlman's catalyst (Pd(OH)2 on Carbon) was added. After 30 min under hydrogen, the mixture was filtered and concentrated to give the title compound: IH NMR (200 MHz, CD30D) d 7.04 (br d, 2H, J = 8.44 Hz), 6.67 (br d, 2H, J = 8.54 Hz), 4.57 (dd, IH, J = 5.23, 9.04 Hz), 4.5-4.0 (m, 4H), 3.38 (s, 3H), 3.34 (s, 3H), 3.02 (dd, IH, J = 5.17, 13.81 Hz), 2.75 (dd, IH, J = 9.23, 14.06 Hz), 2.66 (t, 2H, J = 7.08 Hz), 2.53 (dd, IH, J = 5.47, 15.58 Hz), 2.34 (dd, IH, J = 7.91, 15.57 Hz), 2.03 (m, IH), 1.88 (s, 3H), 1.9-1.2 (m, 6H), 1.41 (s, 9H), 0.94 (d, 6H, J = 6.69 Hz), 0.93 (d, 3H, J = 6.64 Hz).

Affinity Chromatography Procedure

The starting enzyme preparation was purified about 100- fold from THP-1 cell lysate by anion exchange chromatography as described in Examples 2, 3, 5-8 and or 10.

Step A: Binding of ICE

The activated affinity column (5 ml, 1 cm x 6.5 cm) and a guard column of native SEPHAROSE CL-4B of equal dimensions were equilibrated with 10 column volumes of the chromatography buffer (100 mM hepes, 10% sucrose, and 0.1% 3-[(3-cholamidopropyl)- dimethylammonio]-l-propanesulfonate (CHAPS) at pH 7.50) supplemented with 1 mM dithiothreitol. The enzyme solution (15 ml, 150,000 units, 150 mg protein) was applied through the guard column and mn onto the affinity column at a flow rate of 0.022 ml/min at 4°C, and washed through with an additional 10 ml chromatography buffer at the same flow rate. During loading, 8% of the enzymatic activity was not retained, presumably due to the slow rate constant for binding. After loading, the guard column was removed and the affinity column was washed with 25 column volumes of buffer at a faster flow rate of 0.5 ml/min at 4°C. No enzymatic activity was detected in the wash fractions.

Step B: Elution of Bound ICE

To elute the enzyme, the column was then flooded with 1 column volume of buffer containing 200 mM Acetyl-Tyr-Val- Lys(CBZ)-Asp-CHO (Compound B), and left for 24 hours at room temperature to achieve dissociation of the matrix-bound enzyme. The free enzyme-inhibitor complex was then recovered from the affinity column by washing with 2 column volumes of buffer at a flow rate of 0.022 ml/min. Repeating the exchange with fresh inhibitor produced < 5% more enzyme, indicating that the first exchange had been adequate.

Step C: Reactivation of ICE

The eluted ICE was reactivated using two synergistic chemical approaches: conversion of the inhibitor to its oxime, and oxidation of the active site thiol to its mixed disulfide with glutathione by thiol-disulfide interchange.

EXAMPLE 18

Active-site labeling of native murine ICE

Purification of human ICE from cell extracts yields an active enzyme complex composed of p20 and plO subunits; however, the cloning of human ICE cDNA revealed that the enzyme was synthesized as a p45 proenzyme for which no enzymatic activity has jet been demonstrated. In cytosolic extracts, p45 is present and the active p20:pl0 enzyme appears to be generated, in part, by an autocatalytic processing mechamsm. Active-site labeling of partially purified human ICE from THP.l cells with 14C-iodoacetimide identified the active site thiol (Cys285) to be on the p20 subunit. Active-site labeling of the ICE in a cmde S-300 cytosolic extract of peritoneal exudate cells from P. acnes sensitized mice was performed with active site label by the following procedure: the samples (10 μl, 0.25 u/ul) were pre-incubated for 5 minutes at room temperature in the presence or absence of an excess (10 mM Ac-Tyr-Val-Ala-Asp-CHO of a reversible, competitive inhibitor. Then the active site label was added to a final concentration of 100 nM and the samples were incubated for 10 minutes at room temperature and fractionated by SDS-PAGE. The bound label was detected by transferring the fractionated proteins to PVDF membranes and incubating the blot with an iondinated probe in l²^I-streptavidin. As can be seen in Fig. 4, a protein of approximately 20 kDa was specifically labeled by the active site label. In the presence of excess levels of the reversible tetrapeptide aldehyde inhibitor L-709,049 (Ac- Tyr-Val-Ala-Asp-CHO), the active site was protected from reaction with the active site label. Thus, the active-site thiol of murine ICE is, like the active site for the human enzyme, localized on a p20 subunit.

The enzyme-inhibitor solution recovered from the affinity column was adjusted to contain 100 mM neutral hydroxylamine and 10 mM glutathione disulfide to effect reactivation. Under these conditions, after a short lag with a halflife of 100 sec for consumption of excess free inhibitor, the dissociation of E-I complex is entirely rate determining with a halflife of about 100 min at 25°C. After allowing 10 halflives for the exchange, the inhibitor oxime and excess reagents were removed by exhaustive desalting in an AMICON CENTRICON-10 ultrafiltration cell using the chromatography buffer at 4°C. When desired, the enzyme-glutathione conjugate was reduced with 10 mM dithiothreitol (halflife < 1 min) to give active enzyme. The purified enzyme is stable indefinitely at -80°C. The recovery of enzymatic activity by affinity chromatography was >90%, and the final purification achieved was about 75, 000-fold, as measured by SDS- polyacrylamide gel electrophoresis. The results are summarized on Table 4.

TABLE 4 AFFINITY PURMCAΉON OF ICE

vol. units/ units/

(ml) units ml mg mg/ml mg

3 DEAE sample 15 150,000 10 150 10 10

Affinity Eluate 0.2 140,000 700 0.03* 0.15 4.7 x 106

* estimated from silver staining intensity on SDS-PAGE

Recovery of ICE was 93% with a purification of 4700-fold.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Molineaux, Susan M. Rolando, Anna M. Casano, Francesca J.

(ii) TITLE OF INVENTION: DNA Encoding Murine Precursor Interleu in 1 Beta Converting Enzyme

(iii) NUMBER OF SEQUENCES: 8

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: John W. allen III

(B) STREET: P.O. Box 2000

(C) CITY: Rahway

(D) STATE: NJ

(E) COUNTRY: USA

(F) ZIP: 07065

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: Macintosh Ilci

(C) OPERATING SYSTEM: System 7.0.1

(D) SOFTWARE: Microsoftword 5.0a

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/947,330

(B) FILING DATE: 18-SEP-1992

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Wallen, John W. Ill

(B) REGISTRATION NUMBER: 35,403

(C) REFERENCE/DOCKET NUMBER: 18857

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (908) 594-3905

(B) TELEFAX: (908) 594-4720

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Tyr Val Ala Asp

1 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Tyr Val Lys Asp

1

(2) INFORMATION FOR SEQ ID NO:3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1378 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GGCACGAGTT CAGTTTCAGT AGCTCTGCGT GTAGAAAAGA AACGCCATGG CTGACAAGAT 60

CCTGAGGGCA AAGAGGAAGC AATTTATCAA CTCAGTGAGT ATAGGGACAA TAAATGGATT 120

GTTGGATGAA CTTTTAGAGA AGAGAGTGCT GAATCAGGAA GAAATGGATA AAATAAAACT 180

TGCAAACATT ACTGCTATGG ACAAGGCACG GGACCTATGT GATCATGTCT CTAAAAAAGG 240

GCCCCAGGCA AGCCAAATCT TTATCACTTA CATTTGTAAT GAAGACTGCT ACCTGGCAGG 300

AATTCTGGAG CTTCAATCAG CTCCATCAGC TGAAACATTT GTTGCTACAG AAGATTCTAA 360

AGGAGGACAT CCTTCATCCT CAGAAACAAA GGAAGAACAG AACAAAGAAG ATGGCACATT 420

TCCAGGACTG ACTGGGACCC TCAAGTTTTG CCCTTTAGAA AAAGCCCAGA AGTTATGGAA 480

AGAAAATCCT TCAGAGATTT ATCCAATAAT GAATACAACC ACTCGTACAC GTCTTGCCCT 540

CATTATCTGC AACACAGAGT TTCAACATCT TTCTCCGAGG GTTGGAGCTC AAGTTGACCT 600

CAGAGAAATG AAGTTGCTGC TGGAGGATCT GGGGTATACC GTGAAAGTGA AAGAAAATCT 660

CACAGCTCTG GAGATGGTGA AAGAGGTGAA AGAATTTGCT GCCTGCCCAG AGCACAAGAC 720

TTCTGACAGT ACTTTCCTTG TATTCATGTC TCATGGTATC CAGGAGGGAA TATGTGGGAC 780

CACATACTCT AATGAAGTTT CAGATATTTT AAAGGTTGAC ACAATCTTTC AGATGATGAA 840 CACTTTGAAG TGCCCAAGCT TGAAAGACAA GCCCAAGGTG ATCATTATTC AGGCATGCCG 900

TGGAGAGAAA CAAGGAGTGG TGTTGTTAAA AGATTCAGTA AGAGACTCTG AAGAGGATTT 960

CTTAACGGAT GCAATTTTTG AAGATGATGG CATTAAGAAG GCCCATATAG AGAAAGATTT 1020

TATTGCTTTC TGCTCTTCAA CACCAGATAA TGTGTCTTGG AGACATCCTG TCAGGGGCTC 10^'80

ACTTTTCATT GAGTCACTCA TCAAACACAT GAAAGAATAT GCCTGGTCTT GTGACTTGGA 1140

GGACATTTTC AGAAAGGTTC GATTTTCATT TGAACAACCA GAATTTAGGC TACAGATGCC 1200

CACTGCTGAT AGGGTGACCC TGACAAAACG TTTCTACCTC TTCCCGGGAC ATTAAACGAA 1260

GAATCCAGTT CATTCTTATG TACCTATGCT GAGAATCGTG CCAATAAGAA GCCAATACTT 1320

CCTTAGATGA TGCAATAAAT ATTAAAATAA AACAAAACAG AAAAAAAAAA AAAAAAAA 1378 (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 79 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: AAGTGATAAC GATCCAGAAT ATGGTGTGTG TCATTTTACT GAGTTAAACT TGGCGTTTTC 60 ACGGTAGAAC TGACTATTG 79

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GGGCCCCATA TGAACAAAGA AGATGGCACA TTTC 34

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid .

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

( i) SEQUENCE DESCRIPTION: SEQ ID NO:6: GGGCCCCATA TGTTAATGTC CCGGGAAGAG GTAGAAAC 38

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GGGCCCCATA TGGCTGACAA GATCCTGAGG GCAAAG 36

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GGGCCCCATA TGTTAATGTC CCGGGAAGAG GTAGAAAC 38

Claims

WHAT IS CLAIMED IS:

1. A DNA molecule which encodes the complete unmodified form of murine precursor interleukin 1 beta converting enzyme, having the nucleotide sequence:

GGCACGAGTT CAGTTTCAGT AGCTCTGCGT GTAGAAAAGA AACGCCATGG CTGACAAGAT CCTGAGGGCA AAGAGGAAGC AATTTATCAA CTCAGTGAGT ATAGGGACAA TAAATGGATT GTTGGATGAA CTTTTAGAGA AGAGAGTGCT GAATCAGGAA GAAATGGATA AAATAAAACT TGCAAACATT ACTGCTATGG ACAAGGCACG GGACCTATGT GATCATGTCT CTAAAAAAGG GCCCCAGGCA AGCCAAATCT TTATCACTTA CATTTGTAAT GAAGACTGCT ACCTGGCAGG AATTCTGGAG CTTCAATCAG CTCCATCAGC TGAAACATTT GTTGCTACAG AAGATTCTAA AGGAGGACAT CCTTCATCCT CAGAAACAAA GGAAGAACAG AACAAAGAAG ATGGCACATT TCCAGGACTG ACTGGGACCC TCAAGTTTTG CCCTTTAGAA AAAGCCCAGA AGTTATGGAA AGAAAATCCT TCAGAGATTT ATCCAATAAT GAATACAACC ACTCGTACAC GTCTTGCCCT CATTATCTGC AACACAGAGT TTCAACATCT TTCTCCGAGG GTTGGAGCTC AAGTTGACCT CAGAGAAATG AAGTTGCTGC TGGAGGATCT GGGGTATACC GTGAAAGTGA AAGAAAATCT CACAGCTCTG GAGATGGTGA AAGAGGTGAA AGAATTTGCT GCCTGCCCAG AGCACAAGAC TTCTGACAGT ACTTTCCTTG TATTCATGTC TCATGGTATC CAGGAGGGAA TATGTGGGAC CACATACTCT AATGAAGTTT CAGATATTTT AAAGGTTGAC ACAATCTTTC AGATGATGAA CACTTTGAAG TGCCCAAGCT TGAAAGACAA GCCCAAGGTG ATCATTATTC AGGCATGCCG TGGAGAGAAA CAAGGAGTGG TGTTGTTAAA AGATTCAGTA AGAGACTCTG AAGAGGATTT

CTTAACGGAT GCAATTΠTG AAGATGATGG

CATTAAGAAG GCCCATATAG AGAAAGATTT TATTGCTTTC TGCTCTTCAA CACCAGATAA TGTGTCTTGG AGACATCCTG TCAGGGGCTC ACTTTTCATT GAGTCACTCA TCAAACACAT GAAAGAATAT GCCTGGTCTT GTGACTTGGA GGACATTTTC AGAAAGGTTC GATTTTCATT TGAACAACCA GAATTTAGGC TACAGATGCC CACTGCTGAT AGGGTGACCC TGACAAAACG TTTCTACCTC TTCCCGGGAC ATTAAACGAA GAATCCAGTT CATTCTTATG TACCTATGCT GAGAATCGTG CCAATAAGAA GCCAATACTT CCTTAGATGA TGCAATAAAT ATTAAAATAA AACAAAACAG AAAAAAAAAA AAAAAAAA

2. An expression vector for the expression of cloned genes in a recombinant host, the expression vector containing one or more cloned genes with a nucleotide sequence selected from the group consisting of:

GGCACGAGTT CAGTTTCAGT AGCTCTGCGT GTAGAAAAGA AACGCCATGG CTGACAAGAT CCTGAGGGCA AAGAGGAAGC AATTTATCAA CTCAGTGAGT ATAGGGACAA TAAATGGATT GTTGGATGAA CTTTTAGAGA AGAGAGTGCT GAATCAGGAA GAAATGGATA AAATAAAACT TGCAAACATT ACTGCTATGG ACAAGGCACG GGACCTATGT GATCATGTCT CTAAAAAAGG GCCCCAGGCA AGCCAAATCT TTATCACTTA CATTTGTAAT GAAGACTGCT ACCTGGCAGG AATTCTGGAG CTTCAATCAG CTCCATCAGC TGAAACATTT GTTGCTACAG AAGATTCTAA AGGAGGACAT CCTTCATCCT CAGAAACAAA GGAAGAACAG AACAAAGAAG ATGGCACATT

⁵ TCCAGGACTG ACTGGGACCC TCAAGTTTTG

CCCTTTAGAA AAAGCCCAGA AGTTATGGAA AGAAAATCCT TCAGAGATTT ATCCAATAAT GAATACAACC ACTCGTACAC GTCTTGCCCT CATTATCTGC AACACAGAGT TTCAACATCT ⁱ o TTCTCCG AGG GTTGGAGCTC A AGTTGACCT

CAGAGAAATG AAGTTGCTGC TGGAGGATCT GGGGTATACC GTGAAAGTGA AAGAAAATCT CACAGCTCTG GAGATGGTGA AAGAGGTGAA AGAATTTGCT GCCTGCCCAG AGCACAAGAC

15 TTCTGACAGT ACTTTCCTTG TATTCATGTC TCATGGTATC CAGGAGGGAA TATGTGGGAC CACATACTCT AATGAAGTTT CAGATATTTT AAAGGTTGAC ACAATCTTTC AGATGATGAA CACTTTGAAG TGCCCAAGCT TGAAAGACAA

20 GCCCAAGGTG ATCATTATTC AGGCATGCCG TGGAGAGAAA CAAGGAGTGG TGTTGTTAAA AGATTCAGTA AGAGACTCTG AAGAGGATTT CTTAACGGAT GCAATTTTTG AAGATGATGG CATTAAGAAG GCCCATATAG AGAAAGATTT

25 TATTGCTTTC TGCTCTTCAA CACCAGATAA TGTGTCTTGG AGACATCCTG TCAGGGGCTC ACTTTTCATT GAGTCACTCA TCAAACACAT GAAAGAATAT GCCTGGTCTT GTGACTTGGA GGACATTTTC AGAAAGGTTC GATTTTCATT

³ ° TGAACA ACCA GAATTTAGGC TACAGATGCC

CACTGCTGAT AGGGTGACCC TGACAAAACG TTTCTACCTC TTCCCGGGAC ATTAAACGAA GAATCCAGTT CATTCTTATG TACCTATGCT GAGAATCGTG CCAATAAGAA GCCAATACTT CCTTAGATGA TGCAATAAAT ATTAAAATAA AACAAAACAG AAAAAAAAAA AAAAAAAA

3. A recombinant host cell containing one or more recombinantly cloned genes, the recombinantly cloned genes having a nucleotide sequence selected from the group consisting of:

GGCACGAGTT CAGTTTCAGT AGCTCTGCGT GTAGAAAAGA AACGCCATGG CTGACAAGAT CCTGAGGGCA AAGAGGAAGC AATTTATCAA CTCAGTGAGT ATAGGGACAA TAAATGGATT GTTGGATGAA CTTTTAGAGA AGAGAGTGCT GAATCAGGAA GAAATGGATA AAATAAAACT TGCAAACATT ACTGCTATGG ACAAGGCACG GGACCTATGT GATCATGTCT CTAAAAAAGG GCCCCAGGCA AGCCAAATCT TTATCACTTA CATTTGTAAT GAAGACTGCT ACCTGGCAGG AATTCTGGAG CTTCAATCAG CTCCATCAGC TGAAACATTT GTTGCTACAG AAGATTCTAA AGGAGGACAT CCTTCATCCT CAGAAACAAA GGAAGAACAG AACAAAGAAG ATGGCACATT TCCAGGACTG ACTGGGACCC TCAAGTTTTG CCCTTTAGAA AAAGCCCAGA AGTTATGGAA AGAAAATCCT TCAGAGATTT ATCCAATAAT GAATACAACC ACTCGTACAC GTCTTGCCCT CATTATCTGC AACACAGAGT TTCAACATCT TTCTCCGAGG GTTGGAGCTC AAGTTGACCT CAGAGAAATG AAGTTGCTGC TGGAGGATCT GGGGTATACC GTGAAAGTGA AAGAAAATCT CACAGCTCTG GAGATGGTGA AAGAGGTGAA AGAATTTGCT GCCTGCCCAG AGCACAAGAC TTCTGACAGT ACTTTCCTTG TATTCATGTC TCATGGTATC CAGGAGGGAA TATGTGGGAC CACATACTCT AATGAAGTTT CAGATATTTT AAAGGTTGAC ACAATCTTTC AGATGATGAA CACTTTGAAG TGCCCAAGCT TGAAAGACAA GCCCAAGGTG ATCATTATTC AGGCATGCCG TGGAGAGAAA CAAGGAGTGG TGTTGTTAAA AGATTCAGTA AGAGACTCTG AAGAGGATTT CTTAACGGAT GCAATTTTTG AAGATGATGG CATTAAGAAG GCCCATATAG AGAAAGATTT TATTGCTTTC TGCTCTTCAA CACCAGATAA TGTGTCTTGG AGACATCCTG TCAGGGGCTC ACTTTTCATT GAGTCACTCA TCAAACACAT GAAAGAATAT GCCTGGTCTT GTGACTTGGA GGACATTTTC AGAAAGGTTC GATTTTCATT TGAACAACCA GAATTTAGGC TACAGATGCC CACTGCTGAT AGGGTGACCC TGACAAAACG TTTCTACCTC TTCCCGGGAC ATTAAACGAA GAATCCAGTT CATTCTTATG TACCTATGCT GAGAATCGTG CCAATAAGAA GCCAATACTT CCTTAGATGA TGCAATAAAT ATTAAAATAA AACAAAACAG AAAAAAAAAA AAAAAAAA

4. A recombinant host cell expressing recombinant murine interleukin 1 beta and containing one or more recombinant genes selected from the group consisting of:

GGCACGAGTT CAGTTTCAGT AGCTCTGCGT GTAGAAAAGA AACGCCATGG CTGACAAGAT CCTGAGGGCA AAGAGGAAGC AATTTATCAA CTCAGTGAGT ATAGGGACAA TAAATGGATT GTTGGATGAA CTTTTAGAGA AGAGAGTGCT GAATCAGGAA GAAATGGATA AAATAAAACT TGCAAACATT ACTGCTATGG ACAAGGCACG GGACCTATGT GATCATGTCT CTAAAAAAGG GCCCCAGGCA AGCCAAATCT TTATCACTTA CATTTGTAAT GAAGACTGCT ACCTGGCAGG AATTCTGGAG CTTCAATCAG CTCCATCAGC TGAAACATTT GTTGCTACAG AAGATTCTAA AGGAGGACAT CCTTCATCCT CAGAAACAAA GGAAGAACAG AACAAAGAAG ATGGCACATT TCCAGGACTG ACTGGGACCC TCAAGTTTTG CCCTTTAGAA AAAGCCCAGA AGTTATGGAA AGAAAATCCT TCAGAGATTT ATCCAATAAT GAATACAACC ACTCGTACAC GTCTTGCCCT CATTATCTGC AACACAGAGT TTCAACATCT TTCTCCGAGG GTTGGAGCTC AAGTTGACCT CAGAGAAATG AAGTTGCTGC TGGAGGATCT GGGGTATACC GTGAAAGTGA AAGAAAATCT CACAGCTCTG GAGATGGTGA AAGAGGTGAA AGAATTTGCT GCCTGCCCAG AGCACAAGAC TTCTGACAGT ACTTTCCTTG TATTCATGTC TCATGGTATC CAGGAGGGAA TATGTGGGAC CACATACTCT AATGAAGTTT CAGATATTTT AAAGGTTGAC ACAATCTTTC AGATGATGAA CACTTTGAAG TGCCCAAGCT TGAAAGACAA GCCCAAGGTG ATCATTATTC AGGCATGCCG TGGAGAGAAA CAAGGAGTGG TGTTGTTAAA AGATTCAGTA AGAGACTCTG AAGAGGATTT CTTAACGGAT GCAATTTTTG AAGATGATGG CATTAAGAAG GCCCATATAG AGAAAGATTT TATTGCTTTC TGCTCTTCAA CACCAGATAA TGTGTCTTGG AGACATCCTG TCAGGGGCTC ACTTTTCATT GAGTCACTCA TCAAACACAT GAAAGAATAT GCCTGGTCTT GTGACTTGGA GGACATTTTC AGAAAGGTTC GATTTTCATT TGAACAACCA GAATTTAGGC TACAGATGCC CACTGCTGAT AGGGTGACCC TGACAAAACG TTTCTACCTC TTCCCGGGAC ATTAAACGAA GAATCCAGTT CATTCTTATG TACCTATGCT GAGAATCGTG CCAATAAGAA GCCAATACTT CCTTAGATGA TGCAATAAAT ATTAAAATAA AACAAAACAG AAAAAAAAAA AAAAAAAA

5. Murine interleukin 1 beta converting enzyme having the amino sequence:

MADKILRAKR KQFESfSVSIG TINGLLDELL EKRVLNQEEM DKKLANITA MDKARDLCDH VSKKGPQASQ IHTYICNED CYLAGILELQ SAPSAETFVA TEDSKGGHPS SSETKEEQNK EDGTFPGLTG TLKFCPLEKA QKLWKENPSE

IYPIMNTTTR TRLALΠCNT EFQHLSPRVG

AQVDLREMKL LLEDLGYTVK VKENLTALEM VKEVKEFAAC PEHKTSDSTF LVFMSHGIQE GICGTTYSNE VSDILKVDΗ FQMMNTLKCP SLKDKPKVII IQACRGEKQG VVLLKDSVRD SEEDFLTDAI FEDDGIKKAH IEKDFIAFCS STPDNVSWRH PVRGSLFIES LD HMKEYAW SCDLEDIFRK VRFSFEQPEF RLQMPTADRV TLTKRFYLFP GH

6. Murine interleukin 1 beta convertin enzyme p20 subunit having an amino acid sequence corresponding to Asnl *" - Asp²"" of the murine interleukin 1 beta converting enzyme of claim 5.

7. Murine interleukin 1 beta converting enzyme plO subunit having an amino acid sequence corresponding to Gly315 . His^² of the murine interleukin 1 beta converting enzyme of claim 5.

8. Murine interleukin 1 beta converting enzyme pll subunit having an amino acid sequence corresponding to Ala-^ . His^² of the murine interleukin 1 beta converting enzyme of claim 5. 9. A murine interlukin

subunit having an amino acid sequence Asp²"" of murine interleukin 1 beta converting enzyme of claim 5.

interleukin 1 beta converting enzyme.

11. Murine interleukin 1 comprising a first subunit correspondin

murine

a converting enzyme or Gly l - His " of murine interleukin 1 beta enzyme.

12. A monospecific antibody capable of binding to murine interleukin 1 brta having the amino acid sequence to claim 5.