CA2136981A1 - Dna encoding precursor interleukin 1.beta. converting enzyme - Google Patents

Abstract

Complementary DNA (cDNA) encoding the precursor interleukin-1.beta. (pre-IL-1.beta.) converting enzyme (ICE) is isolated from a cDNA library. A cDNA, encoding the full-length open reading frame (ORF) for the nascent ICE, as well as individual cDNAs encoding the 20 kDa subunit and the 10 kDa subunit of ICE are identified and sequenced. ICE is useful in the conversion of pre-IL-1.beta. into mature IL-1.beta.. The recombinantly produced ICE, including the individual 20 kDa and 10 kDa subunits is useful in the diagnosis of inflammatory diseases, in the production of recombinant IL-1.beta., and to identify inhibitors of ICE activity.

Description

94/~01~4 2 1 3 6 9 8 1 PCT/USg3/05687 .~,.. . ~. . .

TITLE OF THE lNVENTlON
DNA ENCODING PRECURSOR I~TERLEUKIN 1 CONVERT~G ENZYME

5 RELATED APPLICATIO~S
This application is a continuation-in-part o-f application serial number 746,454 filed Augu~t 16, 1991.

BACKGROUND OF THE INVENTION
Mammalian interleukin- 1 (IL- 1) is an immuno-regulatory protein secreted by certain cell types as par~ of the general inflammatory response. The primary cell type responslble for IL-l production is the peripheral blood monocyte. Other non-transformed cell types have, however, been described as releasing or 5 containing IL-l or IL-l-like molecu!es. These include epithelial cells (Luger et al., J. lmmunol. 127: 1493-1498 [1981], Le et ah, J.
Immunol. 13~s: 2520-2526 11987] and Lovett and Larsen, J. Clin.
Invest. ~2: 115-122 [1988], connective tissue cells (Ollivierre et al., Biochem. Biophys. Res. Comm. 141: 904-911 [1986~, Le et al., J .
20 lrnmunol. 138: 2~20-2526 [1987], cells of neuronal origin (Giulian et al., J. Exp. Med. 164: ~94-604 [1986] and leukocytes (Pistoia et ah, J. Immunol. 136: 1688-1692 [1986], Acres et al., Mol. Immuno.
24: 479~85 [1987~, Acres et al., J. lmmunol. 138: 2132-2136 [1987]
and Lindemann et al.7 J. lrnmunol. 140: 837-839 [1988].
25 Transf~rmed cell lines have also been shown tb produce IL-l. The~se include monocytic leukemia lines P3~s8D, J774, THP.l, U-937 (Krakauer and Oppenheimer, Cell. Immunol. R0: 223-229 [1983] and M..tsushima et ah, Biochem. 25: 3242-3429 [1986], EBV-tr~nsformed human B Iymphoblastoid lines (Acres, et al., J.
30 Immunol. 13~: 2132-2136 [1987]) and trans~ormed murine keratinocytes (Luger et al., J. Immunol. 125: 2147-2152 [1982]).

W O 94/00154 2l3698l~; PCI/US93~05687 ~ ~

Biologically active IL-l exists in two distinct forms, IL- .
la with an isoelectlic point of about 5.2 and IL-l,B 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, showin~ about 27-33% homology at the amino acid level (Clark et al., Nucleic Acid~s Res. 14: 7~97-7914 [1986]).
Marr~nalian L-l,B is ~synthesized as a cell associated precursor polypeptide of about 31.5 kOa (Limjuco et ah, Proc. Natl.
Acad. Sci. USA 83: 3972-3976 [1986]). Precursor IL-l,B i~s unable to bind to IL-l receptors and is kiologically 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 fo~n.
Recent studies suggest that the processing and s~cretion of IL- 1 ~ is specific to monocytes and monocytic cell lîne~s (Matsushima et al., J. lmmunoh 135:1132 [lQ85]) For example, fibroblasts and keratinocytes synthesize the IL-l~ precur,~or~ but have not been shown to actively process the precursor or secrete mature IL-l~ (Young et al., J. Cell Biol. 107:447 (1988) and Corbo et ah, Eur. J. Biochem. 169:669 [1987]).
Several obse~vations support the hypothesis tha~ the processing and secretion of IL-l ~ occurs by a unique pathway distinct from that used by clas.sical secretory proteins. IL- 1 ~
molecules from five different species do not contain hydrophobic signal sequences (Lomedico et ah, Nature 312:45Qs [1984], Auron et ah, Proc. Natl. Acad. Sci. USA 81; 7907 [1984], Gray et ah, J.
Immunol. 137:3644 [1986], Maliszewski et a!., Mol. lrnmunol.
25:4~9 [i988], Mori et ah, Biochem. Biophy~. Res. Commun.
150:1237 [19~8], and Furutani et al., Nucleic Acid Re~s. 13:5869 94/001~4 213 ~-9 8~ PCI`/US93/05687 [19~s5]. Furthermore, neither the human nor the murine IL-l,B
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.
5 Finally, light and electron microscopy studies immunolocalize IL-l~
to the cytoplasm and fail to demostrate IL-l,B in organelles th~t are involved in classical secretion (Bayne et al., J. Exp. Med. 163: 1267-128sO [1986] and Singer et ah, J. Exp. Med. 167: 389 [198~$]).
In activated monocytes, pulse-chase e~periments suggest that IL-l,B secretion may be linked to proces.sing. These experiment~
show that the intracellular pool of unprocessed precursor is chased to extracellular mature IL-l~ (Hazuda et al., J. Biol. Chem. 263: 8473 [ 1 98s9]. IL-l ,~ precursor is occasionally found extracellularly but does not appear to contribute to the folmation of 17 kDa IL-I ~
5 unless incubated at high concentrations in the presence of excess trypsin, chymotrypsin or colla~enase (Hazuda et al., J. Biol. Chem.
264: 1689 ~1989], Black et al., J. Biol. Chem. 263: 9437 ~19~$8] and Hazuda et al., J. Biol. Chem. 265: 6318 ~1990]). However, none of these proteinases appear capable of generating mature IL-l~
20 terminating with Alal 17 Proteolytic maturation of precursor L-l~ to mature, 17 kDa IL-l,B apparently results from cleavage between Aspl 16 and Alal 17. An endoproteinase, termed Interleukin-l Coverting En~yme (ICE), that is capable of cleaving the IL- 1~ precursor at 25 Aspl 16-Alal 1, as well as at a homologous site at Asp27-Gly2~, and generating mature IL-l,B with the appropriate amino terminus at Alal 17 has now been identified. The Asp at position 1 16 has been found to be essential for cleavage, since substitution of Ala (Kostura et al., Proc. Natl. Acad. Sci 86: 5227-5231 ~1989] or other amino 30 acids (Howard et al., J. Irnmunol., 147, 2964-9, 1991) for Asp - inhibits this cleavage event.

WO 94/00154 I rj~ PCr/U593/05687 ICE activity has been obtainable only from cells ~
naturally producing the enzyme. Crude cell Iysates with ICE activity are available from these cells (Black, R.A. et al., 1989, FEBS Lett., 247, pp 3~6-90; and Kostura, M.J. et ah, 19~9, P.N.A.S. USA, ~s6, pp 52~7-317 Howard, A. et ah, 1991, J.Immunol., in press).
However, purification of ICE from natural sources does not yield the quantities required for extensive study or practical application of the enzyme.

OBJECTS OF THE ~VENTlON
It is, accordingly, an object of the present invention to provide a cDNA encoding ICE, the recombinantly produced ICE
being capable of converting pre-IL-l,B to biologically active mature IL-1 ,B with Alal 17 as the amino-telmirlal amino acid. An additional object of the present invention is to provide expression vectors containing cDNA encoding full length ICE, or the individual 20 kl:)a and 10 kDa subunits of the enzyme. A further object of the present invention is to provide recombinant ho.st cells containing cDNA
encoding full length pre-IL-l,B, 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 ICE and IL-l ~ in a recombinant hos~ cell to produce biologically active IL- 1~. A
fur~her object of the present invention is to provide isolated 20 kDa ICE subunit, and isolated l0 kDa ICE subunit. An additional object of the present invention is to provide full length ICE. Another object is to provide monospecific antibodies which bind to either the ICE 20 kDa or the 10 kDa subunit, and the use of these antibodies as diagnostic reagents.

Complimentary DNAs (cDNAs) are identified from a monocytic cell line cDNA library, which encode the full length --~ g4/001~4 2 1 3 6'9 8` 1V ~ PCI ~US93/05687 form, frorn which the individual 20 kDa and 10 kDa subunîts of ICE
are derived. The cDNAs are fully sequenced and cloned into expression vectors for expression in a recombinant ho~t. The cDNAs are useful to produce recombinant full length ICE, as well as the individual 20 kDa and 10 kDa subunits of the enzyme.

BRIEF DESCRIPTION OF THE DRAWING
Figure 1 A and 1 B. Sodium dodecyl sulfate polyacrylamide gel electrophoregrams of purified ICE (panel A) and associated pre-IL- l ~ cleavage (enzymatic) activity (panel B). A.S. =
ammonium sulfate; DE-F.T. = DEAE flow through; S.P. =
sulfopropyl cation exchange; HIC = propyl hydrophobic interaction chromatography; TSK = TSK-125 size exclu~ion chromatography;
HAP = hydroxyapatite column chromatography.
Figure 2A and 2B. ICE activity which had been purified through an alternate purification scheme (A.S., DEAE, SP, HAP, TSK) was applied to a Propyl hydrophobic interaction column.
Protein was eluted with a reverse salt gradient. Eluted proteins were dialysed and analyzed by SDS-PAGE and silver staining (panel A) a~s well as converting enzyme activity (panel B). Note the correlation between appearance in the eluate of the 22 and 10 kDa protein.s (arrows3 and ICE activity.
Figure 3A and 3B. A. Illustrates the pH optimum of THP-l S-300 Interleukin-1~ conve3ting enzyme activity. B.
Illu~rates a salt titration of lnterleukin-l,B converting enzyme activity.
Figure 4. Active site labeling of ICE by 14-C-iodoacetate. ~
Figure ~. Isoelectric point of native ICE.
Figure 6A and 6B. Interleukin-l~ converting enzyme equilibrium characteristics.

WO 94/Ool~c4 2 1 3 0 ~ PCI`/US93/05687 j j;~

Figu~e 7. Interleukin-l~ converting enzyme ss-glu~aLhione reactivation.
Figure ~. lnterleukin-l,B converting enzyme s~s-glutathione stability.
Figure 9. Rate constant for ICE association.
Figure 10. Molecular weight estimation of ICE: Size exclusion chromatography of GSSG treated or cystamine treated enzyme.
Figure 1 1 A and 1 1 B. A. C-4 reverse phase HPLC
chromatogram of the 22 kDa and 10 kDa ICE subunits. B. SDS-PAGE fractionation of the 22 kDa and 10 kDa ICE subunit~s following reverse phase HPLC.
Figure 12. Capillary LC electrospray ionization mass spectrum of the 20 kDa ICE subunit.
lS Figure 13. Capillary LC electrospray ionization mass `
spectrum of the 10 kDa ICE subunit.
Figure 14A and 14B. Comparative tryptic and Asp.N
maps of the 20 kDa ICE subunit.
Figure l5A and l5B. Comparati~e tryptic and Asp.N
maps of the lO kDa ICE subunit.
Figure 16. Comparison of amino terminal sequence~
from the 20 kDa ICE subunit and the 24 kDa protein.
Figure 17. Design of degenerate oligonucleotides ~or PCR of DNA fragments to the 20 kDa ICE subunit.
2s Figure 18. ~:)esign of degenerate oligonucleotides for PCR of DNA fragments to the 10 kDa ICE subunit.
Figure 19. Sequence of the PCR product for the 20 kDa IC~ subunit.
Figure 20. Sequence of the PCR product for the 10 kDa 30 ICE subunit.
Figure 21. Structural organization of the human ICE
cDNA.

~ ~ 94/001~4 2 1 3 6 9 8 1 P~JIJS93/05687 Figure 22. Structural organization of the human~ICE
precursor protein.
Figure 23. ln vitro tran,slation of ICE cDNA in rabbit reticulocyte Iysates.
Figure 24. Location of the 24 kDa protein and the 20 kDa and 10 kDa ICE subunits in the ICE precursor.
Figure 25. Functional expression of ICE cDNA in transfected COS-7 cells.

DETAILED DESCRIPTION
The present invention relates to cDNA encoding pre-IL-1~ converting enzyme (ICE) which is isolated from IL-l producing cells. ICE, as used herein, refers to an enzyme which can specifically cleave the peptide bond between the aspartic acid at 15 posihon 116 (Aspl 16) and the alanine at position 117 (Alal 17) of precu~sor Il-l~, and the peptide bond Asp at position 27 ~Asp27) and Gly at position 2~ (Gly28). -The amino acid sequence of human L- 1 ,B i~s known, (Marchetal.,Nature315:641-647 [1985]). Mammaliancells 20 capable~of producing IL-l~ include, but are not limited to, keratinocytes, endothelial cells, mesangial cells, thymic epithelial cells, derrnal fibroblasts, chondrocytes, astrocytes, glioma cells, monomuclear phagocytes, granulocytes, T and B lymphocytes and NK cells. Transformed mammalian cell lines which produce JL-l 25 include, but are not limited to, monocytic leukemia lines such as P3~s8Dl, J774, THP.l, Mono Mac 6 and U-937; EBV-transformed human B lymphoblastoid lines and transformed mur~ne keratinocytes. The prefelTed cells for the present invention include normal human peripheral blood monocytes and T~P.1 cells and the 30 most preferred cells are THP.1 cells.
Other cells and cell lines may also be suitable for use to isolate ICE cDNA. Selection of suitable cells may be done by ~ .
.

WO 94/001~4 '` ~2`13 B 9`81 Pcr/US93/056~7 ,'~

screerling for ICE activity in cell extracts or conditioned medi lm.
Methods for detecting ICE activity are well known in the art (Kostura, M.J. et ah, 1989, P.N.A.S. USA, ~6, pp.5227-523 1) and measure the conver~sion of precursor IL-l,B to mature IL-l~. Cells 5 which possess ICE activity in this assay may be suitable ~or the isola~ion of ICE cDNA.
Human peripheral blood monocytes are obtained from healthy donors by leukophoresis and purified by sedimentation through Lymphocyte Separation Media (Organon Teknika) followed by elutriation on a Beckman counterflow centrifuge as described by Wicker, et al., Cell. Immunol. 106: 31~-329 (19~7). Monocytes are identified by labeling with anti-MACl antibody followed by FACS
analysis using standard procedures known in the art.
The present invention relates to a unique pre-IL-l,B
5 converting en7yme (ICE), also described as pre-IL-l,B convertase, which is isolated from IL-l producing cells. Pre-IL-1~ converting enzyme or covertase~ as used herein, refers to an enzyme which ca specifically cleave the peptide bond between aspartic acid at position 116 (Aspl 16) and alanine at position 117 (Alal 17) of the pre-IL-l,B
20 molecule. The amino acid sequence of hurnan IL-1~ is known, - (March et ah, Nature 315: 641-647 (1985). Mammalian cells capable of producing IL-l,B include, but are not limited to, keratinocytes, endothelial cells, mesangial cells, thymic epithelial cells, dermal fibroblasts, chondrocytes, astrocytes, glioma cells, 25 mononuclear phagocytes, granulocytes, T and B lymphocytes alld NK cells. Transfo~ned mammalian cell lines which produce IL-l include, but are not limited to, monocytic leukemia lines such as P3g~Dl, J774, THP-l, U-937 and Mono Mac 5; EBV-transformed human B lymphoblastoid lines and transfolmed murine 30 keratinocytes. The preferred cells for the present invention include normal human peripheral blood monocytes, MonoMac 6 cells and THP-l cells and the most prefe~Ted cells are THP-l cells.

~uo 94/001~4 PCI/US93/05~87 . . . : `2`.113 6 9'8 1 .

Interle~in- l ,B producing cells such as human THP- l cells (American Type Culture Collection, ATCC TIB 202) described by Tsuchiya et al., lnt. J. Cancer 26: 171-176 (19~0) are grown in suspension at about 37C 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. The cells are grown in roller bottles, Wheaton turbolift 46 liter suspension flasks (Wheaton), or 75, 200, or 300 liter fermenters with weekly harvests of about 1-2 x 106 celis/ml (3-4 doublings/week). Media for use in suspension flasks or fermenters may contain about 0~1-0.3 % F6 pluronic to reduce shear force on the cells. Cells are typically grown for no more than 3-4 months following initial culturing.
~ell-free extracts are prepared ~rom human peripheral blood monocytes or THP. 1 cells by disruption of the cells by `
nitrogen cavitation, hypotonic Iysis 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 20 Iy.sis is accomplished by washing the cells in about 10 volumes of ~- hypotonic buffer (about 10 mM KCl, about 20 mM HEPES, about pH 7.4, about 15 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 25 reducing agent such as dithiothreitol (Dl~). 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, placed on ice for about 20 min and Iysed by about 20 strokes in a Dounce homogenizer. Disruption of about 90 to 30 about 95 % of the cells is obtained in a 100 or 300 ml tight filling Dounce homogenizer using about 25 or about 15 strokes respectively. Nitrogen pressure disruption also takes place in a WO 94/00~ ~4 2 ~ 3 6 9 8 1 PCI /US93/05687 ,~

hypotonic buffer. Resuspended celLs are placed in a nitrogen ~
pres~cure cell at 400 psi of nitrogen for about 30 min at about 4C
with agitation. Disruption is accomplished by releasing the pressure and evacuating the cells from the pr~ssure cell. The cell Iysate is 5 clarified by successive centrifugation step~s; at about 400 to about 1000 x g (supernatant Sl), at about 30,000 x g (supernatant S2) and at about 300,000 x g ~supernatant S3). The cell Iysate may also be clarified by the following procedure. Unbroken cells and nuclei are removed by centrifugation at about 3000 rpm, ~r about 10 minutes, at about 5C in a Beckman GPR centrifuge. The post nuclear supernatant fluid is centrifuged for about 20 minutes at about 16,000 rpm in a Sorval centrifuge with a SS34 rotor. The supernatant fluid is further clarified by centrifugation for about 60 minute.s at about 50,000 rprn in a Beckrnan centrifuge (50.2Ti rotor) or 45,000 rpm 5 (45Ti rotor). The resultant supernatant fluid is stored at about -~0 C following the addition of about 2 mM DTT and 0.1% CHAPS.
Purification of ICE is monitored by an in vitro ~leavage assay utilizing radiolabeled pre-IL-l,B as a substrate. An approxirnately 1.5 kilobase (kb) cDNA clone .;ontairling the entire 20 coding sequence of pre-L-l,B is inserted into EcoRI-PstI cleaved pGEM-3 plasmid DNA (Promega-Biotec) and propagated in E. coli according to standard methods (Maniatis et ah, Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor, NY
[1982]). Purified plasmid is linearized with PstI and then transcribed 25 using a 1"7 RNA polymerase in vitro transcription system (Promega-Biotec) and then the mRNA processed according to the manufacturers' instructions. Translations are preformed by !
pro~,raming micrococcal nuclease-treated rabbit reticulocyte extracts (Promega Biotec) with the in vitro synthesized mRNA in the 30 presence of 25 ,uCi of 35S-Methionine (Amersham) according to the manufacnurers instructions. This yielded labeled pre-L-l ,B which migrated as a doublet on sodium dodecyl sulfate polyacrylamide gel `~0 94/OOlÇ4 - PCI`/IJS93/05~87 ~:

- 11'- ' electrophoresis (SDS-PAGE) with an apparent molecular mas~s~ of about 34 and about 31kilodalton (kDa). The cleavage of pre-IL-l ~
is preformed by incubating 1 111 of rabbit reticulocyte extract ;:
containing radiolabeled precursor with about 10 to about 20 ~Ll of the 5 sample containing IL-l~ converting enzyme. Cleavage of pre-IL-l,B
to yield 17.5 kDa mature Il-l~ is assayed by SDS-PAGE according to the method of Laernmli (Nature 227: 6~0-6~5 [197Q])? followed by fluorography using procedures known in the art. Specificity of enzymatic cleavage and characterization of cleavage products are deterrnined with native pre-IL-l,B and mutant pre-L~
Construction of a mutant pre-IL-l ~ is preformed 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, 5 with ApaLI-HpaII ends is synthesized on an Applied Biosystems DNA 3~s0A synthesizer according to established manufacturer's protocols. The 27-mer encodes an Asp 1 16 > Alal 16 amino acid sub~stitution at the -1 position adjacent to the processing site of pre-IL-l ,B. The oligonucleotide is ligated by procedures well known in 20 the art to EcoRI-ApaLI and HpaII-Pstl fragments obtained from cleavage of full length pre-IL- 1 ,B cDNA. The human nucleo~ide and predîcted amino acid sequence of the pre-IL-l,B translation product is disclosed by March et ah, Nature 315: 641-647 (19~5). The ligated fragments are added to a ligation reaction containing EcoR1-~5 PstI cleaved pGEM-3. Clones containing the pGEM/IL- 1~ muta~nt 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 30 produced a 1.5 kilobase (Kb) mRNA and translation results in a doublet of 34 and 31 kDa proteins.

WO 94/001~4 2 1 ~ 6 9}~1- ;, PCI /US93/05687 f j When THP-l purified pre-IL-l,B converting enzyme is combined with the mutated Asp 1 16 to Alal 16 pre-IL-l ,B~ little or no cleavage of the mutant precursor wa~s observed. The normal cleavage product from the interaction of pre-IL-l~ with pre-IL-l~
5 conver~ing enzyme is a 17.5 kD polypeptide with the N-terminal arnino acid sequence of mature IL-l~. The Aspl 16 residue of pre-IL-l~ is therefore important to the processing of mature IL-l ~ by IL-,B converting enzyme.
The bioactivity of the cleavage products is cluantitated o by determining the amount of radiolabeled, processed IL-l ~ which binds to IL-1 membrane receptors. The assay utilizes the techniques - of Chin et al., J. Exp. Med. 165: 70-~6 (19~S7) and Tocci et al, J. lmmunol. 13~: 1109-1114 (19~7). The cleavage product generated from wild type or native pre-IL-l,B by pre-IL-l,B conver~ing en~yme 15 is biologically active as determined by its ability to bind IL-l,B
receptor in a eompetitive receptor-binding assay as disclosed above.
These purification procedures yield substantially pure, biologically active ICE.
To understand the cellular location of pre-IL- l 20 converting enzyme activity, cleavage studies are carried out using mononuclear cell fractions prepared by Percoll gradient frac~ionation. Cellular homogenate from the first clarification step is layered over prefolmed 0-100 % Percoll gradients (Pharmacia) prepared with about 0.25 M sucrose, about 10 mM HEPES, pH 7.4, 2s 10 mM KCl, 1.5 mM MgC12 and 0.1 mM EDTA. The loaded gradients are centrifuged at about 4~s,000 x g for about 25 min with 1.0 ml fractions being collected from the top. Enzymatic marker assays associated with the various subcellular compartments are carried out: cytosol, lactate dehydrogenase (Morgorstern et ah, Anal.
30 Biochem. 13: 149-161 [1965]); lysosomes, N-Acetyl ~-D-glusosaminidase (Wollen et al., Biochem. 7~ 121 [1961]);
plasma membrane, 5'-nucleotidase (Rome et ah, Cell 17: 143-153 wo 94/001~4 2 1 ~ 6 9 8 1 PCI'/IJS93~05687 [1979]) and microsome.s, sulfatase C (Canonico et al., J. Reticulo.
Soc. 24: 115-135 [1978]). The cyto~solic fraction was the only fraction capable of cleaving pre-IL-l~ into a product sirnilar in size to mature IL-l~.
Since the converting enzyme activity wa.s present in the cytosolic fraction, further purification steps were carried out on supernatant S3 to obtain a subs$antially pure IL-l~ converting enzyme. The supernatant is sequentially precipitated by the addition of granular amDnonium sulfate to achieve about 45% of saturation at about 4C. The precipitated protein is pelleted at about 30,000 x g then brought from about 75% to about ~0% of saturation with ammonium sulfate. This precipitate is pelleted, resuspended in Buffer A (about 20 mM KCI, about 25 mM HEPES, about pH 7.4, about 5.0 mM EDTA, about 2 mM DTT, about 1 mM PMSF, about 15 0.01% NP-40 and about 10% glycerol) and dialyzed for about 16 hr. The dialyzed solution is centrifuged at about 30,000 x g to remove particulate material.
A sample of the ammonium sulfate precipitated material is applied to a diethylaminoethyl (DEAE) anion exchange column 20 equilibrated with Buffer A. The flow through fraction is retained and loaded onto a sulfyl propyl (SP) cation exchange column equilibrated with the same buffer. Pre-IL-l,B conver~ing enzyme i~i eluted with a linear gradient of about 30-500 mM KCl in Buffer A.
The active fractions are dialyzed against Buffer A for about 16 hr.
25 Pre-IL-l,B converting enzyme eluted as a discreet peak with 50%
recovery of activity (Table 1).
To analyze the protein compoIlents of biologically active ICE, sodium dodecyl sulfate polyacrylamide gel electophoresis was carried out on samples from the above fractionation steps essentially 30 according to the method of Laemmli (Laemmli, Nature 227: 680-6~5 [1970J). After electrphoresis, separated proteins are visualized by staining with silvPr using a modification of the method developed by WO 94/001~4 ` /. .~ ,` P~/USg3/05687 ~

2 13 fi 9 8 1 `~- `

Oakley et al. (Analytical Biochemistry, 105 :361 -363 [ 1 9gO]). ~The electrophetic patterns from the stepwise purification of ICE (Figure 1) demonstrate the appearance of a 22 kDa and 10 kDa protein in the - final T~K and HAP steps that colTelates with the ICE activity. In 5 addition, the presence of a 24 kDa protein in colurnn fractions of highly purified ICE is sometimes observed.
The 22 kDa and 10 kDa proteins can be individually isolated using norrowbore C4 reverse phase HPLC. A 200 ml aliquot of SP purified ICE can be applied to an Applied Biosystems C4 (2.1 mm X 100 mm, 300 A pore size) reverse phase column equilibrated in 0.1% trifluroacetic acid (TFA) in deionized water.
Protein is eluted using a linear gradient of 15-90% acetonitrile in 0.1% aqueous TFA in 26 minutes. The 22 kDa and 10 kDa - components can be isolated in pure form from the column (Figure ; }5 2).
In order to obtain a highly accurate determination of the molecular mass of the 22 kDa and 10 kDa ICE ~subunit~s, five picomoles of each protein was purified to homogeneity by the above method and was subjected to capillary liquid chromatography on line ~- 20 to a Finnigan Triple-Sector ~uadropole Model 700 electropsray mass spectrometer. The standard error of mass determination was found to be 0.01-0.02~o using a standard protein, bovine cytochrome c~ to determine instrument accuracy. Following biomass ~ ~ deconvolution of the electrospray ionization prirnary spectrum, the - ~ 2s molecular weight of the isolated "10 kDa" component was found to have an average mass of 10,248 atomic mass units (Figure 4) and the "22 kDa" component was found to have an average mass of 19,~66 ato~ic mass units (Figure 3). Herein, thè "22 kDa" ICE subunit will be referred to as the 20 kDa subunit while the " 10 kDa" ICE subunit will be referred to as the 10 kDa ICE subunit. These two proteins are as.sociated with the fully active form of ICE. The "24 kDa"
protein that occasionally co-purifies with the 20 and 10 kDa ICE

94/001~4 2 1 3 6 9 8 1 - PCI/US93/05687 :, ' ` , '~

subunits will be re~elTed to as the 22 kDa protein, as it isapproximately 2000 daltons larger than the 20 kDa ICE subunit when analyzed by SDS-PAGE.
Any of a variety of procedures may b~ used to 5 molecularly clone ICE cDNA. ~hese 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 i~s to screen an ICE-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labelled oligonucleotide probe designed from the arnino acid sequence of the ICE subunits. The preferred method consists of screening an ICE-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA
encoding the ICE subunits. This partial cDNA is obtained by the 15 specific PCR amplification of ICE DNA fragments through the design of degenerate oligonucleotide primers from the amino acid sequence of the purified ICE subunits. ~`
It is readily apparent to those skilled in the art that o~er types of libraries, as well as libraries constructed from other cells or cell types, may be useful for isolating ICE-encoding DNA. Other types of libraries include, but are not limited to, cDNA libraries derived from other cells or cell lines other than THP. 1 cells, and genomic DNA lîbraries.
It is readily apparent to those skilled in the art that suitable cDNA libraries rnay be prepared from 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 firs~ measuring cell associated ICE activity using the precursor IL-1 ,B cleavage assay described fully above.
Preparation of cDNA libraries can be performed by standard techniques well known iIl the art. Well known cDNA
library construction techniques can be found for example, in WO 94~01~4 ~ PQ/US93/05687 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 Lhose skilled in the art that S DNA encoding ICE may al~so be isolated from a suitable genomic DNA library.
Construction of genomic DNA libraries can be performed by standard techniques well knvwn 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 Eaboratory, Cold Spring Harbor, New York, 1982).
In order to clone the ICE gene by the preferred method, the amino acid sequence of ICE is necessary. To accomplish this~
15 ICE protein may be purified and partial amino acid sequence determined ~y automated sequenators. It is not necessary to determine the entire amino acid sequence, but the linear sequence of two regions of 6 to 8 amino acids from both the 20 kDa and 10 kDa subunits is determined for the PCR amplification of a partial ICE
20 DNA fragment-Once suitab1e amino acid sequences have been identified, the DNA sequences capable of encoding them are synthesized.
Because ~e genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid 2s sequence can be encoded by any of a set of similar DNA
oligonucleotides. Only one member of the set will be identical to the ICE sequence but will be capable of hybridizing to ICE DNA even in ' the presence of DNA oligonucleotides with mismatches. The mismatched DNA oligonucleoddes may still sufficiently hybridize to 30 the ICE DNA to pe~it idendficadon and isolation of ICE encoding DNA.
.
, ; O 94/001~4 2 1 3 6 9 8 1 PCI /~S93/05687 Using the preferred method, cI)NA clones encoding ICE
are isolated in a two-stage approach employing polymerase chain reaction (PCR) ba~sed technology and cDNA library screening. ln the first stage, NH2-terminal and internal arnino acid sequence 5 informa$ion from the purified 20 kDa and 10 kDa ICE subunits is used to design degenerate o!igonucleotide primers for the ampli~lcation of ICE-~specific DNA ~ragments. In the second stage, these fragments are cloned to serve as probes for the isola~ion of full length cDNA from a comrmercially available lambda gtlO cDNA
library (Clontech) derived from THP.l cells (ATCC #TIB 202).
Arnino acid sequence information from the purified 20 kDa and 10 kDa ICE subunits is obtained by automated amino acid sequencing using Edman chemistry of both the intact 20 kDa and 10 kDa sublmits and the peptide fragments of the 20 kDa and 10 kDa 5 subunits generated by specific proteolytic cleavage. Enzymatic fragmentation of the 20 kDa and 10 kDa ICE subunits was performed using either trypsin (Promega) or endoproteinase Asp.N
(Boehringer Mannheim). Prior to digestion, the individual ICE
subunits were reductively alkylated at cysteine residue~s by 4-20 vinylpyridine. 40 to SO pmoles of the 20 kDa or 10 kDa ICEsubunits is dissolved in 50 ul of a solution containing 6M guanidine-HCl, 1 mM EDTA, 0.25 M Tris-HCI, pH ~.S. 2.5 ul of 71 mM ~-mercaptoethanol is added and the solution i~ then incubated for two hours under argon in the dark at room temperature. 2 ul of a 370 25 mM 4-vinylpyridine solution is then added and the resulting solution is incubated for an additional two hours under argon in the dark at room temperature. The alkylated ICE subunits were subsequently desalted by C-4 reverse phase chromotography (Applied Biosystem butyl, 7 micron column) employing a linear ~radient of 15% to 90%
30 B in 26 minutes (buffer A= 0.06% aqueous TFA; buffer B=~9.g9%
acetonitrile, 10 % H20, 0.055% TFA). Tryptic digests were conducted in SO ul of 50 mM ammoniurrl bicarbonate buffer, pH 9.0 WO94/001~4 2~3G9~ PCI/US93/05687,`--1~
for a period of 16 hours at 37C utilizing a trypsin to ICE subunit ratio (w/w) of 1:100 (10 ng trypsin to 1000 ng (50 pmol) of the 20 kDa subunit; S ng trypsin to 500 ng (50 pmol) of the 10 kDa subunit). Endoproteinase Asp.N digests were conducted in 50 ul of ~0 mM ammonium bicarbonate buffer pH 9.0 for a period of 36 hours at 25C utilizing an endoproteinase Asp.N to ICE subunit ratio (w/w) of 1:20 (40 ng Asp.N protease to ~00 ng t40 pmol) of the 20 kDa subunit, 20 ng A~sp.N protease to 400 ng (40 pmol) of the 10 kDa subunit~.
o Following incubation for the prescribed periods, digestion is terminated by the addition of 5 ul of 10% TFA and resulting peptide fragments are fractionated bv C-l~ reverse-phase HPLC on an Applied Biosystems 1 30A Separation Sys~em~ The reverse phase chromotography conditions include the use of a Vydac C-18 column (2.1 x 100 mm) operated at a flow rate of 200 ul/min and a temperature of 52C. Buffer A is 0.06% aqueous TFA while buffer B is 89.99% acetonitrile/10% H20/0.055% TFA. Peptides are eluted with a gradient of 2% B to 75% B in 60 minutes followed by 75% B to 95% B in 10 minutes, and detected at 214 nm (0.25 2 AUFS).
Automated amino acid sequencing using Edman chemistry was performed on an Applied Biosystem 477A instrument coupled to a 120A analyzer for on-line PTH-amino acid identification. For sequencing of the intact 20 kDa and 10 kDa subunits, 15 pmols of protein was routinely applied to the sequenator while for enzymatically derived peptide fragments 5-20 pmols was used.
Table 3 details the amino acid sequence obtained from the 20 kDa and 10 kDa ICE subunits, respectively, as determined by sequence analysis of the intact ICE subunits. When the intact 10 kDa protein w;~s subjected to sequencing, a single sequence was always observed consistent with a single, free amino terminu~s of a , .

.

u/o 94/001~4 2 1 3 6 9 8` 1` PCT/US93/05687 homogeneous protein. For the most part, a single, free amino~ ;
terminu~s wa.s also observed when the intact 20 kDa protein was sequenced. However, on occa~sion, a secondary sequence was detected in highly purified preparations contain~ng the intact 20 kDa 5 protein. The observation of the secondary sequence was coincident with the co-purification of a 24 kDa protein with the 20 kDa ICE
subunit (Figure 1 1). The protein from which the secondary sequence was obtained was less prevalent in the mixture than the 20 kDa protein from which the primary sequence was obtained, allowing for the assignment of the secondary sequence to the 22 lcDa protein (Table 3). A comparison of the amino terminal se4uence~s of the 22 kDa protein and the 20 XDa ICE subunit reveals that the 22 kDa protein contains amino acid sequence corresponding directly to the NH2 terminus of the 20 kDa ICE subunit with an NH2 terminal 5 extention of 16 amino acids (Figure 16). The only difference in the overlapping sequence of the 24 kDa and 20 kDa protein~s is the substitution of an Asn for Asp at position +1 in the 24 kDa protein.
Degenerate oligonucleotides were designed based on the amino acid sequence from the amino terrninus and internal regions 20 of both the 20 kDa and 10 kDa proteins (Example 23). For the 20 kDa protein, the arnino terminal primer, ~AYCCNGCNATGCCNAC (SEQ.ID.NO.:3), was 128 fold degenerate while the internal primer, 3'-ATRGGNTADTACCTRTT-5' (SEQ.ID.NO.:4), was 4g fold 2s degenerate (Figure 17). Forthe 10 kDa protein, the amino telminal primer, GCNATHAARAARGCNCA (SEQ.ID.NO.:5), was 192 fold degenerate while ~e internal primer, GTYTACGGNTGNTGNCT
(SE~Q.ID.NO.:6), was 128 fold degenerate (Figure 18).
Single-stranded THP. 1 cDNA was synthesized from 30 THP. 1 celllllar poly A+ mRNA and used as a PCR ternplate. PCR
was conducted essentially as described by a modification of the MOPAC procedure (Lee, et ah, Science 239, 128~S (198g)). For WO 94/001~4 2 1 3 6 9~8~1 ii P(~JUS93/05687 ~

each PCR, 10 pmol of each primer was added to that cluantity~of cDNA ~synthesized for 0.4~1g of poly A+ mRNA in a reaction buffer consisting of ~0 mM KCI, 10 mM TRIS-HCI (pH ~.3), 1.5 mM
MgC12, 0.01% w/v gelatin, and 20 uM of each dNTP in a final 5 volume of 10~1. The PCR program consi.sted of one cycle of denaturation for 100C or 10 minutes, the addition of 2 units of Ta4 polymerase, ~ollowed by 30 cycles of the following steps: 9~C for 30 seconds, 4~C for 30 seconds, and 70C for 1 minute~s. For the - 20 kDa protein, a PCR product of 1 16 bp was synthesized and its identity was verifled by hybridization with an irlternal inosine-subs$ituted oligonucleotide [ATIGGRTAIATYTCIGCR]
(SE(~?.ID.NO.:7), while ~or the 10 kDa protein, a PCR product of 221 bp was synthesized and verified with a similar type of probe [ATIGARAARGAYTTYATIGC~(SEQ.ID.NO.:~). Bo~h PCR
15 prodllcts were subcloned into Blue~script vectors (Stratagene), and sequenced by the chain termination method (Sanger, et al., PNAS 74, 5463 ( 1 977)).
The deduced nucleic acid sequence of the PCR product~s derived from the 20 kDa and 10 kDa ICE subunits (Figure 19 and 20 20 respectively, Example 23) reveals complete identity with several of the previously sequenced tryptic peptides (Figures 19 and 20) not utilized in the design of prirners or probes for PCR amplification.
These PCR derived products from Example 23 were used as hybridization probes for scr~,ening a lambda gtlO cDNA
25 library derived from THP.l cells (Clontech). Plating and plaque lifts of the library were per~ormed by standard methods (T.
Maniatis, E.F. Fritsch~ J. Sarnbrook, Molecular Cloning: A
LaboratoryiManual (Cold Spring Harbor La~oratory, Cold Spring Harbor, New York, 1982). The probes were random-primed 30 labelled with 32P-dCTP to high ~specific activity and a separate screening of the library (600,000 pla4ues per screen) was conducted with each probe. The probes were added to hybridization bu~fer w0~)4/001';4 213698'11 PCI/U593~0S687 (50% formamide, 5X Denhardts, 6X SSC (lX SSC = 0.15 M~ 0.5%
SDS, 100 ,uglml salmon sperm DNA) at 1 x 106 cpm/ml.
Eleven positively hybridi~ing phage were detected using ~he 10 kDa specific probe while seven positively hybridiziIlg phage S were observed using the 20 kDa probe.
Several cDNA clones ranging in size from 1.0 kb to 1.6 kb in length and containing a single open reading frame of 404 amino acids were subcloned into pGEM vectors (Promega) and bi-directionally sequenced in their en~irety by ~he chain termination method (Sanger et al., P.N.A.S. USA. 74, pp ~S463~ 1977~
The sequence for the full-length cDNA encoding ICE i~s shown in Table 5, and was designated clone OCP9. The deduced amino acid sequence of ICE from the cloned cDNA is shown in Table 6. Inspection of the deduced amino acid sequence reveals the 5 presence of a single, large open reading frame of 404 amino acids.
By comparison with arnino acid sequences derived from the purified native ICE 20 kDa and 10 kDa .subunits, an additional in frame coding sequence of 119 amino acids is loca~ed amino terminal to the 20 kDa subunit. In addition to the sequence of the 20 kDa and 10 20 kDa ICE subunits, the entire NH2-terminus of the 24 kDa protein is encoded in this open reading frame.
A precise correlation in molecular mas~ exists for both the 20 kDa and 10 kDa ICE subunits when a comparison is made between the molecular mass determined by mass spectrometry and 25 that computed by the deduced amino acid sequence. For the 10 kDa subunit, the molecular mass determined from the deduced sequence is 10,242 (amino acid 317 to 404) which perfectly agrees with the m~ss spec~rometry determination. For the 20 kDa subunit, a di~ference of no more than 20 amu is observed between the deduced 30 sequence (19,844, amino acid 120 to 297) and the mass spectrometry determination.

WO 94/U01~;4 2 13 6 9 8 1 PCI`/US93/05687 Complete sequence identity is ob~served between the amino acid sequence determined by direct Edman sequencing of the individual ICE subunits and the deduced amino acid se4uence from the cDNA, except for one amino acid found at position 120 of the 5 full length nascent ICE protein. According to the cDNA sequence, Asn is encoded at position 120 of the full-length protein. This amino acid position corresponds to the NH2-terminus of the 20 kDa subunit. The amino acid sequence derived from the purified native 20 kDa subunit determined the NH2-terminal amino acid to be Asp.
This dlfference between the deduced amino acid sequence and the known~ amino acid sequence, the only difference found, may be attributable to deamidation of the NH2-terminal Asn of the 20 kDa .
subunit to form Asp.
Whether Asn or Asp is found at the NH2-terminus of s~ the~20 kDa~sùbunit,~or whether Asn or Asp is found within the ICE
polypéptide~at~position 120 of the full length protein~, or in a polypeptide~ fragment, -is not expected to affect ICE act~vity si:,gnificantly, if at all.
Further ~inspection of the ICE deduced amino acid 20 seyuence reveals several ICE-like cleavage sites between amino acid positions 103-104, 1 19-120, 297-29~, and 316-317. Evidence for IsuGh~a ~mechanism is ~given by ~the finding that when radiolabeled, in translated~`p45 is~incùbated in ~e presence of affinity purified I CE,;Iabeled cl~àvage products arè generated which are congruent in 25 ~ ~ ~size with the~ known~ purified ICE forms or predicted interrnediates which could resu~lt from single cleavages of p45. Processing was not due to a contaminating protease since a tetrapeptide aldehyde ICE
inhibitor speci~1caliy blocked the cleavage of p45. Mutation of the Asp to an Ala at each of these sites prevents forrnation of the ~ n `' cleavage product predicted from processing at that particular site.
Each of ~hese potential cleavage sites could serve to generate the 20 kDa ~and 10 kDa subunits from the full length ICE protein, and may ~... .
.,~. ,.:~:

,, ~ . .
~"
~ -94/001~4 13 6 9 8 i PCl /US93/05687 be involved in an autocatalytic mechanism for the activation a~d processing of ICE.
Puri~ied biologically active ICE may have several different physical foIms. ICE may exist a~s a full-length nascent or unprocessed polypeptide, or as partially processed polypeptides or combinations of processed polypeptides, as evidenced by the synthesis of a 45 kDa polypeptide by programming of a cell-free in vitro translation system with in vitro transcribed mRNA
corresponding to the full length cDNA (Figure 23). The observed ICE translation product is coincident with the predicted size of the 404 amino acid-encoding ORF of the ICE cDNA. The full-length nascent ICE polypeptide is postranslationally modified by specific proteolytic cleavage events which result in the formation of fragments of the full length nascent polypeptide. A fragment, or physical association of fragments rnay have the full biological activity associated with ICE (cleavage of precursor IL~ into mature IL-I~) however, the degree of ICE activity may vary between individual ICE fragments and physically associated ICE
polypep~ide fragments.
lCE in substantially pure forrn derived from natural sources according to the purification processes described herein, i~i found to be an association of two ICE polypeptide fragments encoded by a single mRNA. The two ICE polypeptide fragments are found to have an apparent molecular weight of 20 kDa and 10 kDa.
2s The cloned 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 30 ICE. Techniques for such manipulations are fully described in Maniatis, T, et al., supra, and are well known in the art.

WO 94/001~4 2 1 3 6 g 8 1 . PCl~US93/05687 i~``

Expression vectors are defined herein as DNA sec~uences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropliate host. Such vectors can be used to express eukaIyotic gencs in a variety of ho~sts such as 5 bacteria, bluegreen algae~ plant cells, insect cells and animal cell~s.
Specifically designed vectors allow the shuttling of DNA
between hosts such as bacteria-yeast or bactelia-al~imal cells. An appropriately constructed expression vector shvuld contain: an origin of replication for autonomous replication in host cells, selectable o markers, a limited nurnber of useful restriction enzyme ~sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sec~uence that directs RNA polymerase to bind to DNA and imtiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vector.
15 may include, but are not limited to, cloning vector~s, modified cloning vectors, specifically designed plasmids or viruses.
A variety of mammalian expression vectors may be used to expre'ss recombinant ICE in mammalian cells. Commercially available mammalian expression vectors which may be suitable for 20 recombinant ICE expression~ include but are not 11mited to, pMClneo (Strata~ene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV~ S-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 3719~$), pSV2-dhfr (ATCC 37146), pUCTag 25 (ATCC 37460), and ~ZD35 (ATCC 37565).
DNA encoding ICE may also be cloned into an expression vector for expression in a recombinant host cell.
Recombinant host cells may be prokaryotic or eukaryotic, including bu~ not limited to bacteria, yeast, mammalian cells including but not 30 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 denved from mammalian species which .~, 0 94/001 i4 2 1 3 ~ 9 ~ 1: ` PCI /lJS93/05687 may be suitable and which are commercially available, include but are not limited to, CV-l (ATCC CCI~ 70), COS-I (ATCC CRL
1650), COS-7 ~ATCC CRL 1651), CHO-Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 165~), HeLa (ATCC CCL
2), C127I (ATCC CRL 1616), BS-C-l (ATCC CCL, 26) and MRC-5 (ATCC CCL 171).
The expression vector may be introduced into host cell~s via any one of a number of techinques 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 imm~Lnological reactivity with anti-ICE antibodies, and the presence of host cell-associated ICE activity.
Expression of ICE DNA may also be performed using in vitro produced ~synthetic mRNA. Synthetic rnRNA can be efficiently translated in various cell-free systems, including but not limited to wheat germ extra~ts and reticulocyte extracts, as well as 2Q ef~lciently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred.
To determine the ICE cDNA sequence(s) that yields optimal levels of enzymatic activity and/or ICE protein, ICE cDNA
molecules including but not limited to the following can be constructed: the full-length open reading frarne of the ICE cDNA
(45 kDa = base 4- base 1215) and several constructs containing pcrtions of ~é 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 ICE cDNA (base 1216-148~). ICE activity and levels of protein expression can be determined following the introduction, both singly and in WO94/001`4 213698 1 PCli/US93/05687 combination, of the~se con.structs into appropriate host cells.
Following dete~nination of the ICE cDNA ca~ssette yielding optimal expression in transient assays, this ICE cDNA construct is transferred to a variety of expression vectors, including but not 5 limited to marnmalian cells, baculovirus-infected insect cells, E. Coli, and the yeast S. cerevi~siae.
Mammalian cell transfectants and microinjected oocytes are assayed for both the levels of ICE enzymatic activity and levels of ICE protein by the following methods. The first method for asse~ssing ICE enzymatic activity involves the direct introduction of the native substrate for ICE, the 31.5 K IL-l~ precursor, simultaneously with ICE. To assess the substate specificity of expressed ICE, IL-l~ precursor substrates with altered amino acids in the ICE cleavage sites will be tested. In the case of mammalian cells, this involves the co-transfection of two plasmids, one containing the ICE cDNA and the other contairlirlg the preIL-l cDNA. ln the case of oocytes, this involves the co-injection of synthetic RNAs for both ICE and the IL- 1 ,B precursor. Following an appropriate period of time to allow for expression, cellular protein 20 iS me$abolically l~belled with 35S-methionine for 24 hours~ after which cell Iysates and cell culture supernatants is harvested and `
subjected to immunprecipitation with polyclonal antibodies directed against the IL-I ,B protein. Cleavage of the wild-type precursor to the 28 K and 17 K forms, and cleavage of the precursor containing 25 an altered downstream processing site (Aspl 16 to Alal 16) to the 2 K form is assessed by an SDS-PAGE gel based assay.
The second method for detecting ICE activity involves the direct measurement of ICE activity in cellular Iy~sates prepared from mammalian cells transfected with ICE cDNA or oocytes 30 injected with ICE mRNA. This as~say can be performed using IL-l ,B
precursor protein or synthetic peptides spanning the IL- l ~ cleavage sites. Cleavage products of the precursor is analyzed by standard gel "'? 94/001~4 ` . . PCr/~JS93/05687 `.-; .

- ~7 -based assay and cleavage products of the pep~ides are analyzed by HPLC.
Levels of ICE protein in host cells is quanti$ated by immunoaffinity and/or ligand affinity techniques. ICE-specific 5 affinity beads or ICE-specific antibodies are used to isolate 35S-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 ICE in a recombinant host cell, ICE protein may be recovered to provide ICE in active form, capable of cleaving precusor IL-l~ into mature IL-l~. Several ICE
purification procedures are available and suitable for use. As described above for purification of ICE from natural sources, 5 recombinant ICE may be purified from cell Iysates and extracts, or from conditioned culture medium, by valious combinations of, or individual application of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction 20 chromatography.
In addition, recombinant ICE can be separate~ 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 I Q
25 kDa subunits-Monospecific antibodies to ICE are purified frommammalian antisera containing antibodies reactive against ICE or are prepared as monoclonal antibodies reactive with ICE using the technique of Kohler and Milstein, Nature 256: 495-497 (1975).
30 Monospeci~l& antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for ICE. HQmogenous binding as used herein refers ~, r,,: ,., ,. ,, ., ,. . . ,,,, ;. . , WO 94~001~4 2 13 6 9 8 I PCI /VS93fO~687 ~

' ' . : , , ~;

- 2~ -to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the ICE, as described above.
Enzyme specific antibodies are raised by immunizing animals such a~
mice, rats, guinea pigs, rabbits, goats, horses and the like, with 5 rabbits being preferred, with an appropriate concentration of ICE
either with or without an immune adjuvant.
Preimmune serum is collected prior to the first immunization. Each animal receives between about 0.1 ~g and about `
1000 ~g of ICE associated with an acceptable immune adjuvant.
o Such acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corvnebacterium parvum and tRNA. The initial immunization consisted of the en~yme in, preferably, Freund's complete adjuvant at multiple sites either subcutaneously (SC), intraperitoneally (IP~ or both. Each animal is bled at regular intervals, preferably weekly, to determine antibody titer. The ~nimals may or may not receive booster injections following ~e initial immunizaiton. Those anirnals receiving booster injections are generally given an equal amount of the enzyme in Freund's incomplete adjuvant by the same route. Boo~ster injections are given at about three week intervals until maximal titers are obtained. At about 7 days after each booster immunization or about weekly after a single immunization, the animals are bled, the serum collected, and aliquots are stored at abollt -20C.
Monoclonal antibodies (mAb) reactive with ICE are prepared by immunizing inbred mice, prefeMbly Balb/c, with ICE.
The mice are immunized by the IP or SC route with about 0.1 ~lg to about 10 ~g, preferably about 1 ~lg, of lCE in about O.S ml buffer or saline incorporated in an equal volume of an acceptable adjuvant, a~s 30 discussed above. Freund's complete adjuvant is preferred. The mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster `"0 ~4/001~4 ' .' ' ' PCr/US93/05687 r .....
2 1~ 6 9 8 1 immuni~ations of about 0.1 to about 10 ,ug of ICE in a buf~er~
solution such as phosphate bu~fered saline by the intravenous (IV) route. Lymphocytes, from antibody positive mice, preferably splenic IymphGcytes, are obtained by removing spleens from 5 immunized mice by standard procedures known in the art.
Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion par~er, preferably myeloma cell~st under conditions which will allow the formation of stable hybridomas.
Fusion par~ers may include, but are not limited to: mou~se myelomas P3/NSl/Ag 4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0 being preferred. The antibody producing cells and myeloma cells are fused in polyethylene glycol, about 1000 mol. wt., at concentrations from about 30% to about 50%. Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin 15 supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art. Supernatant fluids are collected form growth positive wells on about days 14, 18, and 21 and are screened for antibody produciton by an immunoas~say such as solid phase immunoradioassay (SPIRA) using ICE as the antigen. The culture 20 fluids are also tested in the Ouchterlony preGipitation assay to determine the isotype of the mAb. Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPh~rson, Soft Agar Techniques, in Tissue Culture Methods and Applications? Kruse and Paterson, Eds., Academic 2s Press, 1973.
Monoclonal antibodies are produced in vivo by injection of pristane primed Balb/c mice, approximately 0.S ml per mouse, with about 2 x 106 to about 6 x lo6 hybridoma cells about 4 days after priming. Ascites fluid is collected at approximately ~-12 days 30 after cell transfer and the monoclonal antibodies are purified by techniques known in the art.

WO94tl)01~4 1 3691~ PC~/US93/()~687 .

In vitro production of anti-pre-IL-l~ mAb is calTied out by growing the hydridoma in DMEM containing about 2% fetal calf serum to obtain sufficient quantities of the specific mAb. The mAb are purified by techniques known in ~he art.
Antibody titers of ascites or hybridoma culture fluids are dete~nined by various serological or immunological assays which include, but are not limited to, precipi~ation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of ICE in body fluid.s or tissue and cell extracts.
It is readily apparent to those skilled in the art that the above described method~s for producing monospecific antibodies may be utilized to produce antibodies specific for ICE polypeptide fragments, or full-length nascent ICE polypeptide, or the individual 20 kDa and lO kDa subunits. Specifically, it is readi;y apparent to those skilled in the art that monospecific antibodies may be g~nerated which are specific for only the 20 kDa ICE subunit or only the 10 kDa ICE subunit, or only the full-length nascent ICE molecule.
ICE antibody affinity columns are made by adding t'ne antibodies to Affigel-lO (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies forrn covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the 2s 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 HCl (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 supernat~nts or cell extracts containing ICE or ICE subunits are sl~wly passed through the column. The coiurnn is then washed with phosphate buffered saline until the optical density (A2~0) falls ~) 94/001~4 ;, ~, ~ PCl/US93/05687 `` 2136g81 to background, then the protein is eluted with 0.23 M glycine-HCl (pH 2.6). The purified ICE protein i~ then dialyzed against phosphate buffered saline.
lhe full length ICE-encoding cDNA-containing in 5 plasmid pGEM-Zf(-) was designated pOCP9. A sample of pOCP9 in E. coli strain HB 101, was deposited under the terms of the Budapest Treaty, on or before August I5, l 991, in the pelmanent culture collection of the Arnelican Type Culture Collection, at 12301 Parklawn Drive, Roekville, MD., 20~52, and has been assigned the accession number ATCC 6g655.
The following examples illustrate the present invention without, however, limiting the sarne thereto.

Prep~ration of Cell-Free Extracts Human peripheral blood monocy~es were obtained from healthy donors by leukophoresis and purified by sedirnentation through Lymphocyte Separation Media (Organon Teknika) followed 20 by elutration on a Beckman counterflow centrifuge as described by Wicker et al., Cell. Lrnmunol. 106: 318-329 (19~7). Monocytes were identified by labeling with anti-MACl antibody followed by FACS
analysis. Human THP-l cells (American Type Culture Collection) were grown in su.spension at 37C in Dulbecco's Modified Minimal 25 Essential Media (Hazelton) with 10% fetal calf serum (Hyclone Laboratories, de~ined sera with no detectable endotoxin) to a concentration of 1 x 106 cells/ml. THP-l cells were also grown in ei~er 75 or 200 Liter fermenters in lscoves media with a supplement of 10% horse serum containing low endotoxin under 30 controlled conditions of pH 7.2, 20 to 50% dissolved 02, 2 to S%
dissolved CO2, 37C temperature and 90 rpm agitation speed using a marine propeller blade. Cells were harvested using a Millipore WO 94/001 '4 2 1 3 6 9 8 1 PCI /US9V056g7, -`

Prostak cross-flow membrane filtration .system run with a pressure of less than 2 psi across 50 sq ft of 0.6 uM membrane. Harvested cells were centrifuged to a pellet, washed with Dulbecco's PBS and Iy~sed with hypotonic buffer contair.ing 25 mM HEPES, pH 7.5, 5 5 mM MgC12, 1 mM EGTA, and protease inhibitors including 1 mM
PMSF, 10 ~glml leupeptin, 10 ~g/ml pepstatin A. After preparation of the S-3, Dl~ and CHAPS is added to final concentrations of 2 mm and 0.1% respectively. Media used in suspension flasks or fe~nenters also contained 0.1-0.3% F6~s pluronic to reduce shear force on the cells. Cells were typically grown for no more than 3-4 months followirlg initial reculturing from preserved cell sarnple~s.
Cell-free extracts were prepared from human peripheral blood monocytes and THP-l cells by nitrogen cavitation or by homogenization in hypotonic Iysis buffer. CeIls were collected by 15 centrifugation at 1,000 x g, (for example 15 ~ of THP-l cells) washed 3 time~s with Dulbecco's phosphate buffered saline containing no magnesium or calcium chloride and were pelleted at 1,000 x g for 10 minutes. The resulting cell pellets were resuspended in 3 volume~
of hypotonic buffer ~20 mM KCI, 25 mM HEPES, pH 7.4, 1.5 mM
20 MgC12, 0.1 mM EDTA, 1 mM DTT), and placed in an ice bath for 20 minutes. The cells were lysed in a Dounce homogenizer and homogenized with 20 strokes. For gentler Iysis, the cell pellets were resuspended m hypotonic buffer, placed in a stainless steel nitrogen pressure cell and pressurized to 400 psi with nitrogen gas and held 25 for 30 minutes at 4C with agitation. The cells were Iysed by simultaneously releasing the pressure and evacuating the cells from the container. At this point the cell membranes were effectively broken by rapid decompression and shear flow. Cellular homogenates were clarified by centrifugation at either 400 or 1,000 30 x g for 20 min, the supernatant fluid, designated S-l, being saved and stored at -80C. Freshly prepared S-l ~supernatant fluid was further centri~uged at 30,000 x g for 10 min and designated S-2.

94~001~4 213 6 9 8 I PCI`/US93/05687 The S-2 supernatant fluid was further centrifuged at 300,000~x g for 15 hr and the supernatant fluid, designated S-3, collected.
Cell-free extracts of THP-l cell were also prepared with the following technique. Cells were washed 3 times in P13S and 5 suspended for 20 min at 0C at l 0~ cells/ml in a hypotonic buffer containing 25 mM HEPES, pH 7.5, 5 mM MgC12, and 1 mM EGTA.
Protease inhibitors were added (l mM PMSF and lO mg/ml of pepstatin and leupeptin), and the cells were broken in l O0 or 300 ml tight fitting Dounce homogenizers using 25 or 15 strokes respectively to yield 90-95% breakage. The broken cells were centrifuged at 3000 rpm for lO min at 5C in a Beckman GPR
centrifuge to remove nuclei and unbroken cells. The postnuclear supernatant was centrifuged for 20 min,at 16,000 rpm in a Sorval centrifuge with an SS34 rotor followed by a second centrifugation 5 for 60 min at 50,000 rpm in a Beckman centrifuge (50.2Ti rotor) or 45,000 rpm (45Ti rotor). After addition of 2 mM DTT and 0.1 %
CHAPS, the resultant supernatant was stored at -~0C.

Salt Fractionation ,; ~ The S-3 supernatant fluid, from Example l, was sequentially precipitated by the addition of arnrnonium sulfate in order to both concèntrate and partially purify pre-IL- l ,B converting 2s enzyme activity. Granular amrnonium sulfate was added to 50 ml of S-3 supernatant fluid to reach 45% saturation at 4C. The fluid was allowed to equilibrate on ice with stirring for 15 minutes. The ' tu'.-bid precipitate~ was clarified by centrifugation at l O,000 x g` with ' the resulting pellet being discarded. The supernatant fluid was then 30 brought to 80% saturation of ammonium sulfate using the above -~ protocol. The precipitate is then pelleted, resuspended in Buffer A
('~O mM KCl, 25 mM HEPES, pH 7.4, 5.0 mM EDTA, 2 mM DTT, WO 94/001~C4 2 1 ~ 6 9 8 1 Pcr/l~S93/05687 i```-``
`. ~ , . ;.. . . ~.. ,.. ;
;, I; ~

1 mM PMSF, 0.1% NP-40 (Nonident detergent P-40), 10% glycerol) and dialyzed overnight against the same buffer. The dialyzed precipitate was centrifuged at 30,000 x g to remove particulate material and then stored at -~0C.

EXAMP~E 3 ~ . .

Ion Exchange Chromatography Ten ml of the ammonium sulfate precipitated protein from Example 2, 110 mg total proteiIl~ wa~s applied to a Bio-Rad DEAE-5-P~v' HPLC anion exchange column equilibrated in Buffer A. Fractions containing protein were detected by absorbance at 2~0 nm. The flow through fraction was retained and then loaded onto a Bio-Rad SP-5PW sulfopropyl cation exchange column equilibrated 5 with Buffer A. A linear gradient of from 30-500 mM KCl in Buffer A was then used to elute converting enzyme activity. After elution the column ~ractions containing protein, as determined by absorbance at 2~0 nm, were dialyzed (Spectrum Laboratory products, Spectra/Por membrarle, ~,000 molecular weight cutoff) 20 against Buffer A for 16 hr. Individual fractions were assayed for converting enzyme activity using the protocol de~scribed in Example 4. ICE activity cleaves pre~ 1,B and generates a 17.S kDa product which is the biologically active Iymphokine. The converting enzyme activity, using these salt and pH conditions will not bind to DEAE
25 anion exchange resins but will bind to sulfopropyl cation exchange resins. ICE was eluted as a discrete peak with recovery of at least 50% of the starting activity.
.

`"~) 94/001~4 ` '` ~ '` ;` PCI/US93/056~7 EXAMpLE 4 In Vitro Assav~s For Detection Of ICE Activity Cleavage of pre-IL-l,B with fractions from Ex~nplex 1-5 3 and 5-7 was performed by incubating 1 ~1 of rabbit reticulocyte extract containing radiolabeled precursor with 10-20 ~1 of the speci~lc fractions. The radiolabeled precursor IL-l~ was prepared in the following manner. A 1.5 kilobase (kb) cDNA clone containing the entire coding sequence of pre-IL-l ~ was inserted into EcoR1/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 e~ al., Molecular Cloning: A Laboratory Manual ,Cold Spring Harbor Lab., Cold Spring Harbor, NY, (1982). Purified plasmid was linearized with 5 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 per~o~ned by programing micrococcal nuclea~e-treated rabbit reticulocyte extracts (Promega Biotec) wi~h the in vitro synthesized 20 mRNA in the presence of 25 uCi of [35S] methionine (1 Ci = 37 GBq; Amersham) according to the manufacturer's instructions. This yielded labeled pre-IL-l~ which migrated as a doublet on SDS-P~GE with an apparent molecular mass of 34 and 31 kDa. Both bands can be immunoprecipitated wi$h antisera directed to the 25 carboxyl telminus of IL- l ~. lnterleukin- l ~ converting en~yme activity, cleavage of radiolabeled pre-IL-l,B to yield 17.5 kDa mature Il-l ,B ,was monitored by SDS-PAGE.
Proteolytic cleavage of peptide substrates from Example 12 was carried out in the following manner. S~nples of peptides 30 were prepared in dimethyl sulfoxide at a concentration of 10 mM.
The enzyme used in these studies was purified through sulfopropyl cation exhange chromatography. Reaction mixtures contained W O 9 4 / 0 0 1 ~ 4 2 1 ~ . 6 . 9 8, 1 p c ~, u s 9 3 / 0 5 6 8 7 ~ ~
, j approximately 0.2 mg/ml enzyme, 0.2 mM peptide, 10% sucrose, 0.1% CHAPS (3-[(3-cholamidopropyl)d~nethyl-ammonio]-1-propansulfonat), 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic Acid), pH 7.5 in a 50 ul reaction volume. After 5 incubation at 25 degrees for variable leng~hs of time, reactions were quenched with the addition of 450 ul 0.1% trifluoroacetic acid. The sarnples were analyzed by reverse-pha.se high performance liquid chromatography using a Vydac C-18 column (4.6 mm x 25 cm, 5 um particle size, 300 pore size) e4uilibrated with 5% acetonitrile, 0.1%
o trifluoroacetic acid at a flow rate of 1 ml/min. Peptides were eluted using a 10 min linear gradient bf 5 to 30% acetonitrile and quantitated by monitoring the column effluent at 2~0 nm. lJnder these conditions substrate and tyrosine-containing product were separated with baseline resolution in every case. The identity of 5 cleavage products was confirmed using peptide standards.

Size Exclusion Chromatographv ICE from Example 3 was further purified by ~si~e exclusion chromatography. The protein, 0.5 ml, was loaded onto a 7.5 x 600 mm TSK G3000SW gel exclusion column which had been equilibrated in Buffer A. The column was eluted with the same buffer and the respective fractions, monitored by absorbance at 2RsO
25 nm, were assayed for converting enzyme activity. lnterleukin-l~
converting enzyme activity eluted from the column with an apparent molecular weight of 30,000. Similar experirnents using TSK G2000 col.L~nn resulted in an apparent molecular mass of 23,000. Thus the apparent molecular weight of active native ICE is between Mr 23,000 and 30,000.
The unique separation and purification processes described above have been summarized in Table 2.

) 94/001~4 2 1 3~6 9 ~ 1 PCT/US93/05687 Puri~lcation of ICE
Purificatinn Total Activity Fold Step Protein Recovered Purity (mg) (%)l Salt l lO.0 loOl l Fractionation DEAE-5-PW 52.0 l O0 2 sp 5-PW l.5 ~0 50 TSK 0.05 25 550 1 l The yield of activity of each fraction was estimated by limit dilution of that fraction.

- ~ EXAMPLE 6 2S HYdrophobic Interaction Chromato~raph~
The ICE from Example 3, 5 or 6 was further purified by hydrophobic interaction chromatography. After the addition of ' so!id ammonium sulfate to a final concentration of 1.5 M, the protein (1.5 ml) was loaded onto a silica based hydrophobic interaction 3G COlUIIlrl (Synchrom Synchropak propyl, 4.6 x 250 mm) which had been equilibrated in a buffer containing 20 mM HEPES, pH 7.4, 1~5 M (NH4)2S04, lO % glycerol, S mM EDTA, 1 mM PMSF and 2 WO 94/001~;4 . . . PCT/US93/05687 .~
2l`~`~9`i81 - 3~ -mM DDT. The column was eluted with a continuous, linear, ~
de~scending salt gradient fo~n 1.5 to 0.0 M (NH4)2S04. Column fractions were dialyzed against Buffer B and as.sayed for converting enzyme activity.
The unique separation and puri~lcation proce~sses described above have been summarized in the following table.

o Fraction Protein Total Spec. Act Total Yield _ mg/ml Protein Units/m~ Units _ %

AS 1 13~.0 20,700.0 76.~ 1,5~9,760 100.0 DEAE-FT 44.2 13,260.0 113.1 1,499,760 94.0 SP 15.2 167.0 3,289.0 549,263 34.0 HIC-3 11.3 33.9 4,424.0 149,937 9.4 TSK 0.3~ 2.~5 26,315.0 74,997 4.7 HAP 0.006 0.0361,666,666.~ 60,000 3.7 AS = ammonium sulfate fraction; DEAE-FT = DEAE anion exchange cholmatography flow through; SP - sulfopropyl cation exchange chromatography; HIC propyl hydrophobic interaction 25 chromatography; TSK = TSK size exclusion chromatography; HAP
= hydroxyapatite adsolption chromatography.

30 Hvdroxvlapatite Ad~orption Chromatographv The ICE from Example S or 6 was further ~uri~ied by hydroxylapatite adsorption chromatography on a Bio-Rad HPHT
analytical cartridge. The sample must be free of EDTA, which is WO 94/~ol ~4 2 1 3 6 9 8 1 PCr/USg3/05687 generally removed by dialysi~s. The colurnn was equilibrated in Buffer B (20 mM HEPES, pH 7.4, 5% glycerol and 2 mM Dl~).
Following application of 1.5 ml of ICE, the column was eluted with a continuous and Iinear gradiennt of 0-500 mM potassium phosphate in buffer B at room temperature. Fractions were collected, immediately placed on ice, and samples of each were dialyzed again~st Buffer B and measured for ICE activity.

EXAMPLE

Purification Process For Amino Acid Sequencing Mass Spectrophotometric Analvsi.s Of Interleukin-l~ Convertin E~yme The supernatant fluid S-3 from Example 1 were clarified by 0.22 m hoilow fiber filtration and concentrated 10-20 fold~with an Amicon YM3 spiral cartridge and dialyzed overnight (R000 molecular weight cutoff dialysis membrane) against a buf~er of 20 mM Tris, pH 7.8, 10% sucrose, 0.1% CHAPS, and 2 mM
DTT. The dialyzed supernatant (about 3-5 g total protein, - ~ corresponding to 1000 ml of cytosolic extract) was adjusted to less ~an 500 micro Siemans conductivity with water and applied to a 475 ml bed volume DEAE-5PW HPLC (Biorad) column. ICE activity - (Example 4) was eluted at about 40 mM NaCI in a gradient with the same buffer and increasing proportion~s of 0.5 M NaCI and 220 mM
Tris HCI.
2s The ICE active fractions from the DEAE column were pooled, diluted with an equal volume of 75 mM HEPES, 10% (vtv) sucrose, 0.1% (v/v) CHAPS, 2 mM Dl~ buffer, adjusted to pH 7.0, and appliéd to a 150 ml bed volume SP-5PW HPLC (Biorad) column and eluted with a KCl gradient (0-0.5 M) in the same buffer. The 30 ICE active fractions (about 75 mM salt) were chromatographed by SDS-PAGE (10-20%, 17-27%, 16%, or 18% gels) and silver stained to dete~nine the bands that tracked with activity, see Example 9.
.
~;:
~ .
:

WO 94/001:`4 2 1 ~ 6 9 8 1 PCl`~lJS93/05687 These SP-HPLC fractions were also further chromatographed on a C4-nalTowbore (ABI) HPLC eolurnn eluted with an acetonitrile gradient in 0.1% TFA. The various peaks (214 nm) were dried, chromatographed on SDS-PAGE, and silver stained to confirm the relative migration rates of the 20-22 and 10 kDa proteins as shown in Figure 1 (Example ~
The NH2-terminal amino acid se4uence for the 20 kDa subunit and the 10 kDa subunit ls shown in Table 3a and 3b.

A 20 kDa subunit NH2-termiml.s Amino Acid Sequence:
Asp Pro Ala Met Pro Thr Ser Ser Gly Ser Glu Gly 5' 10 Asn Val (SEQ.ID.NO.:l) B 10 kDa Subunit NH2-terrninu~s AminQ Acid Sequence:
Ala Ile I,ys Lys Ala His Ile Glu Lys Asp Phe Ile Ala Phe ~ys Ser (SEQ.ID.NO.:2) C 24 kDa Subunit NH2-te.rrninu~ Amino Acid Se4uence:

Ser Gln Gly Val Leu Ser Xaa Phe Pro Ala Pro Gln Ala Val Gln Asp ASI1 Pro Ala Met Pro Thr 3 0 (SEQ.ID.No.: l 9) ~!(~ 94J001~4 PCI /US93~05687 21~69~1 ~
!

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis And Staining of lnterleukin-l ,B Converting Enzvme Sodium dodecyl .sulfate polyacrylarnide gel electrophoresis of purified ICE from Examples 2, 3, 5, 6, 7,~, 10 was carried out essentially according to the method of Laemmli, Nature 227: 680-685 (1970). A 0.15 cm x 10 cm x 10 cm Bio-Rad mini-gel is used to cast a 15 % acrylarnide 0.4% Bis-acrylamide SDS-PAGE resolving gel. A second discontinuous stacking layer consisting of 5% acrylamide - 0.14% bis-acrylamide is then cast on top of the resolving gel. After polymerization, the gels are loaded with no more than 50 ~g of protein dissolved in Laemmlis buffer containing bromphenol blue as a tracking dye. The gels are 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 is stopped and the gel is proces~sed for silver staining.
;~- After electrophoresis, separated proteins can be visualized by stainLng with silver using a modification of the method developed by Oakley et ah (Analytical Biochemistry, 105: 361-363.
1980) and now sold in kit fo~n by Daiichi. Briefly, the gel is fir~st soaked 30 minutes in 200 ml of a 50% methanol:water mixture followed by three washes of 10 minutes each in 200 ml of deionized 25 water. After the initial washes the gel is then soaked in a solution of 40% methanol; 10% ethanol; 0.5% glutaraldehyde; 49.5% water for 15 minutes ~en washed thoroughly with 4 changes of 200 ml !~ ~ aliquots of deionized water each lasting 10 minutes. Staining and visualization of proteins is accomplished using the protocol of 30 Oakley.
The electrophoretic patterns from the stepwise purification of ICE are shown in Figure 1. Silver stains are depicted WO 94/0~1';4 1 3 6 9 8~1 PCr/USg3/0S687 -!

in panel A and can be compared with pre-IL-l~ cleavage acti~ity in panel B. Separation was carried out on 200 nanogram aliquots from the a~ove described converting en7yme purification protocol. Each - purification step is represented: A.S; am~rnonium sulfate fraction.
5 DE-FT; DEAE flow through; S.P.: SP cation exchange .step. HIC;
Propyl hydrophobic interaction chromatography. TSK; TSK-125 size exclusion chromatography. HAP; hydroxyapatite column chromatography. Note the appearance of a 22 and 10 KO protein in the ~inal TSK and HAP steps correlates with the appearance of ICE
activity. ICE activity which had been purified through an altemate purification scheme (A.S., DEAE, SP~ HAP, TSK) was applied to a Propyl hydrophobic interaction co!umn as described in Example 7.
Protein was eluted with a reverse salt gradient a~ described in the methods Example 7. Eluted proteins were dialysed and analyzed by 5 SDS-PAGE and silver staining (Panel A) a~s well as ICE activity (panel B). Note the correlation between the e!ution of the 22 and 10 KD proteins (arrows) and ICE activity.

EXAMPLE I I

Protea~se Inhibitor Sensitivity of ICE
The ability to inhibit ICE convertase activity by variou~
inhibitors was determined by adding the inhibitor to THP. 1 S-300 and measuring IL-~ cleavage as described in Example 4. Table 4 25 lists the inhibitors tested and whether the inhibitor had inhibitory activity.

wo 94,00lC4 2 13 6 9 8 1 P~/US93/0~687 Protease Inhibitor Sensitivit~ of ICE

lnhibitorand cla~ss Conc. Inhibition Serine PMSF 1.0 mM
DFP 10 mM
0 alPI 50 ~lg/ml Leupep~in 100 ,uM

Elastase L-659,166 1 mM
:~, 15 Serine/thiol Chloromethyl ketone TPCK 300 ~lM +
TL(: K 300 ~M +
PheCK 1 mM +
LeuCK 1 mM +
PheALaCK 1 mM +
Thiol proteases l:)ia~ome~yl ketones A-PhePheDK lmM
PheALaDK lmM
L-65 lmM -`
.

WO 94/~01~4 2 1 ~ 6 9 8`1 PCI`/US93/05687, -;

TABLE 4 Cont'd Protease Inhibitor Sensitivity of ICE

Cystarnine lmM +
N-ethyl maleimide lmM +
N-phenyl maleimide lmM +
Methyl methane-thiosulfonate l mM +

Iodoacet~nide 0.2mM +
Iodoacetic acid 0.2mM +

Metalloprotease ~DTA 50 mM
EGIA lOmM
l,lO-phenanthroline lO mM

Aspartyl Pepstatin lOO,uM

ICE activity was inhibited only by sullhydryl alkylating reagent.s.
The inhibition may or may not be mechanistically related to ICE
activity. Further testing on later purification stages in all cases 2s yielded s~nilar results. ln all cases inhibition was measured as either complete (+) or no observable inhibition (-).

3 Synthesis Of Pe~tide Substrate~s Peptides were synthesized via the MerrifIeld solid-phase technique using phenylacetamidomethyl resins and tBoc amino acids.

~) 94~001~4 2 1 3 ~ 9 ~ i PCI /US93/05687 ., Synthesis was performed on an Applied Biosystems 430A peptide synthesizer according to the manufac~urers suggested protocols.
Peptides were siinultaneously deprotected and cleaved from the resin with 90% anhydrous HF, 10% anisole at 0C for 1 h and then 5 extracted from the resin with 10% acetic acid and Iyophilized. The resulting crude peptides were purified by reversed phase HPLC on Waters Cl~ DeltaPak columns with a gradient of S to 70%
acetonitrile in aqueous 0.2% trifluoroacetic acid (TFA). The structure of purified peptides was confirmed by mass spectral analysis.
Peptides of the general sequence Asn-Glu-Ala-Tyr-Val-His-Asp-Ala-Pro-Val-Arg-Ser-Leu-Asn (hereafter referred to as P14mer) were also u~sed for ICE
characterization and were synthesized, purified and characterized as described above. Peptîdes 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-arnino-4 methylcoumarin amide were synthesized by the following process.

20 Step A: N-Allyloxycarbonyl aspartic acid ,B-t-butyl ester oc-7-amino-4-methvlcoumarin amide ~,O~N~o O
CO2t-Bu To a solution of N-alylloxycarbonyl aspartic acid ~-t-butyl ester (3.44g, (12.6 mmol) and 7-amino-4-methylcoumarin (2.00 g, 11.42 mmol~ in 15 mL of anhydrou~s dioxane was added WO 94/001~4 PCr/US93/05687 .-~
213~;9&~' . ~

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 sablrated sodium bicarbonate. Tihe solution was dried over sodium sulfate and concentrated in yacuo. The mixture was purified by HPLC on silica-gel (35x300 mmi column, 10 % ethyl acetate in dichloromethane as eluent) to give the title compound a,s a colorles~s foam: 1 H NMR (200 MHz, CD30D) ~ 7.77 (d, 1 H, J = 2.39 Hz), - 7.6~ (d7 lH, J = 9.06 Hz), 7.49 (dd, lH~ J = 2.36, 9.10 Hz), 6.21 (4, o lH, J = 1.30 Hz), 5.95 (m, lH), 5.4-5.iS (m, 2H), 4.72-4.5~ (m, 3H), 2.~5 (dd, lH, J = 6.17~ 15.73 H~), 2.65 (dd, lH, J = 7.62, 16.37 Hz), 2.43 (d, 3H, J = 1.44 Hz), 1.43 (s, 9H).

~: Aspartic acid ~-t-butyl ester a-7-amino-4-methylcoumarin amide CO~t-Bu 2s To a solution of N-Allyloxycarbonyl a~spartic acid ~-t-butyl ester a-7-amino-4-methylcoumarin amide (435 mg, 1.01 mmol3 iand Dimedone (1.13 g, 8.08 mmol) in 10 mL of anhydrous tetrahydrofuran was added tetrakis triphenylphosphine palladium (1;7 mg, 0.1 mmol). After 45 min, the mixture was diluted with ethiyl acetate and washed five time~s with saturated sodiurn bicarbonate, drled 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 .-"-'0 g4/001~4 PCl/US93/0~i6t'.7 .' ., a 0.22 mrn 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: lH NMR (200 M~z, CD30D) ~ 7.93 (d, lH, J = 1.76 Hz), 7.g2 (d, l~I, J = ~.50 Hz), 7.63 (dd, lH, J = 2.40, 9.10 Hz~, 6.34 (q, lH, J = 1.31 Hz), 3.~9 (t, lH,J
= 6.35 Hz), 2.~ (dd, lH,-J = 6.03, 16.72 Hz), 2.75 (dd, lH, J =
6.77, 16.75 Hz), 2.56 (d, 3H, J = 1.37 H7), 1.54 (s, 9H).

Step C: N-(N-Acetyl-tyrosinyl-valinyl-alaninyl)-aspartic acid ~-t-butvl ester a-?-amino-4-methylcoumarin ~OH

15 0 ,J~Nllf ~N~

H - H - H
CO2t-Bu To a solution of N-(N-Acetyl-tyrosinyl-valinyl-alanine (2~sg mg, 0.733 mmol), aspar~ic acid ~-t-butyl ester oc-7-amino-4-methylcoumarin (242 mg, 0.698 mmol) and hydroxybenzotriazole (149 mg, 1.10 mmol) in 2 mL of dimethy,l'formamide at 0C was 25 added dicyclohexylcarbodiimide (151 mg, 0.733). After 16 h at ambient temperature, the mixture was filtered and purified by Sephadex" LH-20 chromatography (lM x 50 mm column, methanol ell~ent). The resul~ing product was triturated with methanol to give the title compound as a colorless solid: lH NMR (200 MHz, DMF-30 d7) ~ 8.3-7.5 (m, 7H), 7.09 (br d, 2H, J - 8.61 Hz), 6.72 (br d, 2H, J
= ~s.64 Hz), 6.27 (q, lH, J = 1.31 Hz), 4.P~4 (m, lH), 4.62 (m, lH~, 4.44-4.14 (m, 2H), 3.15-2.7 (m, 4H), 2.45 (d, 3H, J = 1.37 Hz~, 2.13 WO 94~001~4 2 1 3 6 9 81 PCI /US91/0~687 ~

- 4~ -(m, lH), 1.~7 (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-~yrosinyl-valinyl-alanirlyl)-aspa~ic acid Ol-7-amino-4-methvlcoumarin amide N-(N-Acetyl-tyrosinyl-valinyl-alaninyl)-aspartic acid ,B-t-butyl ester ~-7-arnino-4-me~ylcoumarin amide was disolved in trifluoroacetic acid. After 15 min the mixture was concentrated in vacuo to give the title compound as a colorless solid: 1 H NMR (200 MHz, DMF-d7) ~ 8.3-7.5 (m, 7H~7 7.09 (br d, 2H, J = 8.61 Hz), 6.72 (br d, 2H, J = 8.64 Hz), 6.27 (q, lH, J = 1.31 Hz), 4.~4 (m, lH), 4.62 (m, lH), 4.~4-4.14 (m, 2H), 3.15-2.7 (m, 4H), 2.45 (d, 3H, J =
1.37 Hz), 2.13 (m, lH), 1.8~ (s, 3H), 1.41 Is, 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 calGulated for C33H~gN5010-1.65 H2O: C, 57.00, H, 6.13, N, 10.07, found: C, 56.97, H, 5.84, N 10.16.

Salt And pH Optimum Of Interleukin-l ,B Converting EnzYme The pH optimum of THP-1 S-3 (Example 1) converting enzyme activity was determined. Sarnples of S-3 extracts were dialyzed against an assor~nent of buffers at pH values ranging form '`'0 94/001~4 2 1 ~ 6 9 8 1 PC~/US93/05687 --4~ -S to 9. Aliquots were removed from the dialyzed ~samples with the remainder being redialyzed against pH 7.4 HEPES buffer. The ;:
dialyzed samples were then tested again for converting enzyme activity. Pre-IL-l~ converting en~yme has a narrow pH optimum 5 situated between pH 7.0 and ~.0 and is not tolerant to exposur~ to acidic pH. Ion re4uirements of Pre-IL-l~ converting enzyme were determined by salt titration. Pre-IL-1~ converting erlzyme (THP-l derived) wa~s incubated with increasing concentrations of KCl in the reaction. The results indicated that converting enzyme activity was optimal at low (<50 mM) concentration~s. The extent of cleavage wa~s assayed by SDS/PAGE according to Laemmli, Nature 227: 6~0-6~5 (1970), (see Example 9) followed by fluorography.
Using either crude THP- 1 S-3 or partially purified enzyme as sources of ICE activity, salt and pH titrations were 15 performed to deteImine optimum assay conditions a.s measured the by the gel based or P14mer cleavage assays, as described above. An example of both a salt and pH titration are included using crude THP-1 S-3. The data ~Figure 3A) indicate that ICE activity i~s inhibited in a dose dependant fashion by the inclusion of KCI in the 20 gel based cleavage assay. The pH optimurn for ICE is approximately 7.4 in the gel based cleavage assay. The same experiment indicates that ICE activity is labile to acid pH but is stable in a basic pH
environment. Cleavage of P14mer and Ac-YVAD-AMC exhibits a similar pH optimum and stability ~rofile. The effects of salt, 2s however, are somewhat different since the P14mer assay exhibits a sensitivity similar to the gel-based assay but the Ac-YVAD-AMC
assay is essentially insensitive to salt. The data indicate that ICE is a ne.ltral proteinase and exhibits no requirement for monovalent ions.
Figure 3A is a determination of the pH optimum of 30 THP-l S-3 converting enzyme activity. S-3 extracts were dialyzed again~t buffers at various pH values ranging from S to 9. Aliquots were removed from the dialysates and ~e remainder redialyzed WO 94/001~4 PCr/US93/05687 ~
213 6981 ` ~ ~

- 5~ -against pH 7.4 HEPES buffer. All dialysates were then te~ted~for converting enzyme activity. (-), S-300 at stated pH; (+), S-300 redialyzed against pH 7.4 HEPES buffer. The results demonstrate - that ICE has a narrow pH optimum ~situated between pH 7.0 and ~.0 5 and is no$ tolerant to exposure to acid pH.
Figure 3B is a salt titration of converting enzyme activity. THP-l S-300 extract was incubated with increasing concentrations of K(:~l in the gel-based cleavage assay. The results indicate that ICE activity is optimal at low (<50 mM) concentrations 10 of KCl.

Characterizatio Of Interleukin-1~ As A Thiol Protease 15As shown in Example 6, Table 3 ICE is only inhibited by reagents capable of modifying thiol residues. 'Fhe data in Table 3 was generated using partially purified enzyme or THP- 1 S-3 and the gel-based cleavage assay as an indicator of ICE activity. This has been verified and extended using the P14mer assay as well as Ac-20 Tyr-Val-Ala-Asp-AMC as substrates. The only exceptlon to the results of this table came when 1,10 phenanthroline (OP) was tested as an inhibitor. Originally it was shown that this compound did not inhibit ICE activity in the gel based cleavage assay. Using a discontinuous kinetic assay with P14mer as substrate (Example 12) 2s revealed that OP can inhibit ICE activity in a time and concentration dependant manner. The rate constant (8 x 10-2 s-l M-1) for inactivation is slow and it is not possible to reconstitute ICE activity by addition of ZnC12 following treatment with the chelator.
Additional tests proved that, in fact, OP was functioning as an 30 ;~nhibitor by oxidizing lCE via a phenanthroline-metal complex.
This was confi~ned by: 1~ demonstra~ing that the presence of high concentrations of the reducing agent Dl~ (10 mM) could protect ~"0 94/001'4 PC~/US93/05~87 5 ~
I(: E from the ef~ects of OPA, suggesting that oxidation of a necessary component is responsible for ICE inactivation by OP. 2) Co-treatment of ICE with two metal chelators, EDTA and OP, did - not result in an inhibition of ICE, a result that is inconsistent with the hypothesis that chelation of protein-bound metal is responsible for the observed inhibition of ICE activi~y by OP. 3) Addition of 10 mM CuSO4 to an ICE as~say contairling 1.0 mM OP results in a 17-fold increase in the rate at which ICE is inhibited by the compound, suggesting that a metal-phenanthroline complex is resporl~sible for inhibition o~ ICE by OP. These data are consistent with the hypothe~sis that ICE contains in its structure a reduced thiol residue that, once oxidized or alkylated by a variety of means, renders~the enzyme inactive. The data is not consistent with the hypothesis that ICE is a metalloprotease.
EXAMPLE lS

Identification of the 22 kD protein a~s being a component of ICE by active ~ite labelin Since thiol alkylating reagents and metal-phenthroline complexes are capable of inhibiting ICE, it is possible that a necessary thiol group is located at or near the active ~site although o~her explanations (e.g. allosteric ef~ects through a distant thiol) can be proposed to account for the effect. A necessary experiment. then, ~5 is to document that the thiol group is at a position capable of competing with ICE substrate. One method for obtaining this result is to demonstrate that the presence of saturating levels of substrate car. block the inactivation of the enzyme by a reagent capable of inhibiting ICE through a covalent modification of a thiol group. A
30 model reagent, iodoacetate, was chosen since the rate constant for inactivation of ICE (24 M-l s-l ) is much faster than carboxymethylation of simple cysteine residues. It has been WO 94/001~4 2 1 3 6 9 ~ `1 PCI iUS93/05687 ~ `

dete~nined that 100 mM iodoacetate will give a half-time of ~
inhibition of 9.13 minutes (1 * Km). Including saturating levels of the substrate Ac-Tyr-Val-Ala-Asp-AMC (200 * Km or 2.~ mM) in the reaction results in theoretical protection of the enzyme to 5 inactivation. Using 14C-iodoacetate as the alkylating reagent a similar protocol was used to identify proteins that could be labeled in the presence or absence of 200 x Krn substrate. At a concentration of 100 mM radiolabeled iodoacetate a significant number of pro~ein~s are labeled during the course of the reaction. When 2.~ mM Ac-o Tyr-Val-Ala-Asp-AMC is included in the reaction < 2% of ICE is inactivated, a value close to that which is predicted. When the number and extent of labeled proteins is assessed by SDS-PAGE and fluorography it is clear that the labeling of a single protein of 22 kDa molecular weight was significantly reduced (Figure 4). The 15 labeling of other proteins in the mixture was not affected, thus suggesting th~t the 22 kDa protein contains a thiol group capable of being alkylated with iodoacetate and that this thiol group is in a position capable of competing with the ICE substrate. As alkylation of this thiol colTelates with the presence or absence of ICE activity, 20 it can be stated that the 22 kDa protein comprises at least part of the ICE enzyme. ICE and 100 mM 14-C-iodoacetate were incubated in the presence (+S) or absence (-S) of 2.8 mM Ac-Tyr-Val-Ala-Asp-AMC and incubated for 1 hour. At 1 hour the reaction is quenched by the addition of 10 rnM unlabeled iodoacetate and 10 mM DTT.
2s Aliquots of the reaction were analyzed by SDS-PAGE followed by coomassie blue staining or autoradiography. The data indicate that no observable changes in protein composition occurred during the course of the reaction as based on coomassie blue staining.
Autoradiography, however, indicates that a 22 kDa protein is labeled 30 in the absence, but not the presence of ICE substrate.

Y'C~94/00l~4 213~9~81 PCI/US93/05~87 ;.

Characterization of ICE a~ a thiol protea~se Characterization of ICE in term.s of protease class was 5 ultimately accompli.shed by the synthesis of two types of mechanism based thiol protease inhibitors, a diazomethyl ketone and a peptide aldehyde, both constructed from the Ac-Tyr-Val-Ala-Asp peptide.
The peptidyl diazomethyl ketone (Ac-Tyr-Val-Ala-Asp-CHN2) and the peptide aldehyde (Ac-Tyr-Val-Ala-Asp-CHO~ were both synthesized as described in an accompanying patent. lt can be shown that Ac-Tyr-Val-Ala-Asp-CHN2 inhibits I(: E activity irreversibly in a time and dose dependant manner, consistent with an irreversible alkylation of an ac~ive site thiol. Addition of excess amounts of substrate effectively compete against this inactivation, again 5 indicatIng that an active site thiol is being modified.
Ac-Tyr-Val-Ala-A~sp-CHO reversibly inhibits ICE in a dose dependant fashion and in a manner competitive with substrate.
However~ the specificity of action on ICE has been determined by syn~esis of a chemically similar peptide aldehyde, Ac-Tyr(dAla)-20 Val-Ala-Asp-CHO and tested for it~s ability to inhibit ICE in a competitive and reversible manner. This compound was found to be 300-fold less potent at inhibiting ICE activity thus indicating that inhibition is dependant on recognition of a particular peptide structure represented by Ac-Tyr-Val-Ala-Asp-(~HO and is not 25 simply due to ~e presence of an aldheyde group on the C-terminus of a peptide.

WO 94/001~4 2 1 ~ 6 9 8 ~ PCI`/US93/0~687 ~ .;, .
EXAMPI,E 17 ~ -Molecular Ma~s~s Determination Of lnterleukin-l ~t Converting Enzvme _ _ Five picomoles of the 22 and 10 kD proteins purified to homogeneity by method in Example P~ were ~subjected to capillary liquid chromatography, electrospray ionization, mass spectrometry.
Mass determination was performed on a Finnigan Triple-Sector Quadropole Model 700 mass spectrometer. The standard error of o mass determination was found to be 0.01 - .02% using a standard protein, bovine cytochromec, to determine machine accuracy.
Following biomass deconvolution of the electro~spray ionization mass spectrum, the molecular weight of the isolated " 10 kDa" component was found to have an average mass of 10,248 atomic mass units and the "22 kDa'i protein was found to have an average mass of 19,~66 - ~ atomic mass units.

-I~oe!ectric Focu~sing Of lnterleukin-l~t Convertin~ Enzvme The isolelectric point of ICE was estimated using a Bio-Rad Rotofor solution phase isoelectric focusing apparatus. Ten ml of partially purifiled ICE enzyme containing approximately 1,260 units of I;CE ~a unit being defined as that amount of enzyme required to reslIlt in the cleavage of 40 pmoles of P14mer substrate per minute - - ~ in a bu~fer containing 10% sucrose, 0.1 % CHAPS, 20 mM HEPES7 - pH 7.4 200 mM P14mer substrate incubated at 25C for 2 hour~s.) t ! was diluted into 40 ml of a 10% Sucrose, 0.1% CHAPS, 0.2%
Ampholytes (Bio-Lyte, pH 5 - 8 range) buffer and then subjected to ~ ~ 30 isoelectric focusing using a Bio-Rad Rotofor electrofocusing - ~ ~ apparatu~. ~Electrophoresis was performted according to the mamlfacturers instructions. The electrofocusing gradient was eluted ~: ~

~" ~` 2 1 3 6 9 8 1 PCr/US93/~5687 and fractions tested for ICE activity as described above using the Pl4mer pep~ide based assay and authentic pre-IL-l ~ as substrate in the gel-based cleavage assay. ln both assays the peal~ of ICE activity focused to a portion of the gradient with a measured pH of 6.3 unit.s, 5 Figure 5.
EXAMPLE l9 Kinetic Evidence For Oligomeric Structure Of Interleukin-l,B
Convertin~ Enzvme _ ---Original titration studie~s with partially purified ICE
suggested that ICE cleavage activity was not strictly linear with enzyme concentration at low enzyme level.s, a finding consistent with an oligomeric enzyme structure. Similarly, ICE activity is known to 5 decrease following dilution into buf~er in a time- dependant manner and will ultimately achieve a stable steady- sta~e enzyme velocity.
After this steady is achieved, the en~yme can then be reconcentrated to its original volume and, after a 24 hour incubation penod, can be demonstrated to have the identical starting enzyme activity (Figure 20 6). This eurve was obtained by diluting a standard stock solution of partially purified ICE (4 units/ul), lO00-fold into a standard assay mixture (50 uM substrate (l*Km), lO0 mM HEPES, 10% sucrose, 0.1% CHAPS, lO mM DTT, 1 mg/ml BSA). The solid line is theoretical ~or a kinetic model that describes a first-order loss of 25 enzyme activity with a rate constant,kobs = 0.004 min-l, corresponding to a half-life of 2.g hours at 25C. The model also dete~nines values for the initial reaction velocity (vo = 0.006~ uM
i~ AMC/min~ and the velocity obtained at in~mite time (vs = 0.0004 uM AMC/min). These parameters are also represented by bars in 30 the insert of Fig. 6. If the experiment is repeated using saturating levels of substrate (l mM, 20*Km), no time-dependant inactivation is observed, indicating that the enzyme-substrate complex is kinetically WO 94/001~4 2 1 3 6 9 8 1 PCl/US93/0~687 ,~
.. ` . .

- ~6 -stabilized. This dissociation is 100% reversable and complete reassociation of the complex is achieved if the diluted enzyme is reconcentr~ted 1000-fold to the original volume. These data sugge~st that loss of ICE activity is complete'y reversible and the sensitivity 5 of ICE activity to dilution is due to the establishment of a new equilibrium between two dis.sociable species, this association being stabilized by saturating levels of substrate.

Stabilization Or Destabilization of lnterleukin~ onverting Enzvme Oli~omer Following Treatrnent With Mixed Disulfide~s;
In an effort to generate reversible active site modifying reagents to facilitate purification and characterization of the enzyme, 5 a variety of mixed disulfides were tested for their ability to reversibly inllibit ICE activity through generation of a mixed disulfide with the active site thiol. Two that were tested exhibited markedly dissimilar abilities to inhibit ICE in a DTT reversible manner. Oxidized glutathione (GSSG) and cystamine (2-amino-20 ethanedithiol) were tested for their ability to inhibit ICE in acontinuous fluorometric assay. ICE treated with 1 mM GSSG wa~s inhibited with a tl/2 of ~ minutes. Addition of 10 mM DlT after the enzyme reaches its plateau of inactivation inst~tly reactivates the enzyme to 9~% of starting activity, indicating a completely 25 reversible process (Figure 7). The e~fects of GSSG treatment on the observed dissocia~ion of ICE upon dilution was tested by treating concentrated ICE with GSSG, then diluting into substrate-free ICE
assay buffer. The treated enzyme was allowed to equilibrate at room temperature then assayed for remaining ICE activity. Surprisingly, 30 GSSG stabilized ICE activity, increasing the half-tirne of dissociation from 90 minutes to 2800 minutes, an increase of over 30 fold relative to untreated enzyme (Figure ~s). When compared with `'.''? 94/001~4 ` PCI ~US93/056~7 -; 2136981 GSSG, cystamine did not appear to stabilize ICE activity. This was demollstrated in experiments designed to assess the extent of reversibility of cystamine treatment. Following treatment of ICE
with cystamine, the enzyme exhibits a t~me dependant decrea~se in 5 activity with a half-time of 19 minutes at 5 mM cystamine. Upon addition of DlT after achievement of the inactivation plateau only 23% of enzyme activity could be recovered, suggesting the formation of irreversibly inactivated enzyme. That the apparently "irreversibly" inactivated enzyme was in fact dissociated monomer was shown by treating highly concentrated ICE (3 units/ml) with 5 mM cystamine followed by a 3 day equilibration at 4C. Following addition of DTT to the concentrated enzyme, recover,v of activity was monitored by removing aliquots, diluting them into ICE assay buffer and measuring the initial velocity of the reaction. Complete 15 recovery of activity was observed and wa.s achieved with a halflife of 36 minutes (Figure 9). This suggests that cystarnine dissociates ICE
into monomeric components which, if suitably concentrated, can reversibly associate ~ollowing treatment with DTT.

Size Exclusion Chromatography Of Oxidized Glutathione Or Cystamine Modi~ied Interleukin-l,B
Since the data in Example 20 suggests that the 25 association of ICE oligiomers can be influenced by pretreatment with either GSSG or cystamine, experiments were carried out to discern the molecular weight of the stabilized and destabilized enzyme. As previously noted, Example 5, ICE elutes with an apparent Mr of 23,000 on TSK G2000 or 30,000 on TSK G3000 columns in the 30 presence of Dl~. When concentrated ICE is first pretreated with GSSG or cystamine for 24 hrs then passaged over a TSK G2000 size exclusion column, the enzyme activity elutes from the column with wo 94/001~4 2 1 3 6 9 g 1 PCI /us93/0s687 I~

5~
two separable and distinct molecular weights. GSSG treated enzyme elutes from the column with an apparent Mr 39,Q00 while cystamine treated enzyme had an apparent Mr of 22,000 (Figure l0). When SDS-PAGE gels of column fractions are analyzed for protein content S by ~silver staining, it can be observed that the 22 and l0 kD proteins coelute with ICE activity and the elution position of these two proteins change~ with the change in molecular weight observed following GSSG or cystamine treatment. This data indicates that the 22 and l0 kD proteins comprise two subunits of the ICE molecule and that the apparently reversible association of these protein~s can account for the oligomeric beh~vior of ICE in solution.

5 Affinity ChromatographY of lnterleukin-lB Converting Enzvme Preparation of a chromatographic matrix from Compound A
:~ An affinity column for interleukin-l converting enzyme was prepared from the potent peptide aldehyd~ inhibitor Acetyl-Tyr-20 Val-Lys-Asp-CHO (Compound A), coupled via a l2-atom bis-oxirane spacer to SEPHAROSE CL-4B through the Iysine residue.

HJ~H~o J ---`~'? 94/0~1~4 213 6 9 81 PCI`/US93~05687 ', , , Synthesis Of Affinitv Matrix Step A: Epoxy-activated SEPHAROSE CL-4B
Epoxy-activated SEPHAROSE CL-4B was prepared a.~j described in the literature (Sundberg, L., and Porath, J. (1974) J. ::
Chromatogr. 90, ~s7-9~). Specifically, a slu~Ty consisting of 100 gm suction-dried SEPHAROSE CL-4B, 100 ml of 1,4-butanedio~
diglycidyl ether (a nominal 70% solution), and 100 ml 0.6M NaOH
containing 2 mg/ml NaBHa, 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 4C.

15 Step B: Coupling of Peptide Aldehvde Dimethvl Acetal J~Nf ~ ~N f~/ ~OMe CO2t-Bu A' : : !
The blocked aspartyl-t-butyl ester, dimethyl acetal 30 (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 WO 94/001~4 ~ PCr/lJS93/056X7 ~';

to pH 11.00 with HCI, to give a 50% methanol solution containing 2 mM inhibitor and 200 mM carbonate buffer. This solution (10 ml) wa~s mixed with the suction-dried cake (10 gm) of epoxy-activated SEPHAROSE CL-4B, and the slurry was stirred by rotation at 37C
for 3 days. The resulting affinity matrix was washed thoroughly with lM KCl and water, and was stored a,s a slurry at 4C. The incorporation, based on result~s with ~14-C~-lisinopril (Bull, H.G., Thornberry, N.A., and Cordes, E.H. (19~g5) J. Biol. Chem. 260~ -2963-2972), is estimated to be 1 umol/ml packed affinity matrix.

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

~,OH ~NH2 2 5 H o H o ~
~ CO2t-Bu Synthesis of N-(N-Acetyl-tyrosinyl-valinyl-lysinyl)-3-amino-4-30 oxobutanoic acid dimethvl ace~al ~-t-butvl ester.

wo 94/001~4213 6 9 81 Pcr/uss3/os6s7 -6~-Step A: N~allyloxycarbonyl-3-arnino-4-hyroxybutanoic a~id ter~-buty! ester ~O~N OH

CO2t-Bu oTo 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 0C, was added N-methyl morpholine (NMM, 8~5 rnL, 8.05 mmol) followed by isobutyl chloroformate (IBCF, 997 mL, 7.6~ mmol). After lS min, this mixture wa.~ added to a1 su~spension of sodillm borohydride (550 mg, 14.55 mmol) in 50 mL of THF and 12.5 mL of methanol at -45C. After 30 min at -45C, the mixture was warmed to 0C and held at that temperature for 30 min. ~he reaction was quenched with acetic acid, diluted with l:l ethyl acetate:hexane, and wa~shed 3 times with dilute sodium bicarbonate.
20 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: lH NMR (200 MHz, CD30D) ~ 5.9 (m, lH), 5.2~ (br d, lH, J = 17 Hz), 5.lS (br d, lH, J = 9 Hz), 4.52 (br d, 2H, J - 6 Hz), 25 3-9~ (m, lH), 3.4~ (ABX, 2H, J = 5, 6, l l Hz), 2.53 (dd, lH, J = 5, 16 Hz), 2.32 (dd, lH, J = 9, 16 Hz)9 1.43 ~, 9H).

WO 94~001~4 2 13 6 9 ~1 PCr/USg3~05687 ,-~

Step B: N-allyloxycarbonyl-3-amino-4-oxobutanoic acid ~-tert-but~l es_er dimethyl acetal _ ___ _ -H OCH3 `
~ ,0~N OCH3 CO2t-Bu o To a solution of dimethyl sulfoxide (757 mL, 10.67 mmol) in 10 mL of dichloromethnane at 45C was added oxalyl `~
chloride (50~ mL, 5.~2 mmol). After S min, a solution of N-allyloxycarbonyl-3-amino-4-hyroxybutanoic ~cid tert-butyl es~er (1.25 gt 4.~5 mmol) in 10 mL of dichlorornethane was added. After 5 15 min, triethyl arrline (2.03 mL, 14.55 mmol) was added. After 30 min, the mixture was warmed to -23C 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, filtered7 and concentrated.
20 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 S ~imes with dilute 25 sodium bicarbonate. The ether layer was dried over magnesium sulfate, ~lltered, and concentrated to afford the title compound as a colorless oil: lH NMR ~200 MHz, CD30D) ~ 5.9 (m, lH), 5.26 (br d, lH,J= 17Hz),5.14(brd, lH,J= lOHz),4.51 (brd,2H,J=
5.33 Hz), 4.25 (d, lH, J = 4.79 Hz), 4.11 (m, lH), 3.40 (s, 3H), 3.39 30 (s, 3H), 2.52 (dd, lH, J = 4.P~6, 15.27 Hz), 2.30 (dd, lH, J = 9.00, 15.2~ Hz), 1.43 (s, 9H).

-"'O 94/001~4 ~ PCl`/US93/056~7 213 69`81`

Step C: 3-Amino-4-oxobutanoic acid ~-tert-butyl ester di~ethyl acetal H2N J~OCH3 CO2t-Bu To a solution of N-allyloxycarbonyl-3-amino-4-- oxobutanoic acid ~-tert-butyl e~ter dimethyl aGetal (312 mg, 1.03mmol) in 10 mL of THF was added molpholine ( ~97 rnL, 10.3 mmol) and tetrakis triphenylphosphine palladium (100 mg). After 3 - hours, the mixture was diluted with 1:1 ethyl acetate/hexane and washed 5 times wi~ dilute sodium bicarbonate. The orgains were dr,ied over sodium sulfate, filtered, and concentrated. The resulting oil was purified by MPLC on silica-gel (22x300 mm column, liIlear gradient of dichloromethane to 1% ammonia and 10 % methanol in dichloromethane) to afford the title compound as a pale-yellow oil:
lH NMR ~200 MHz, CD30D) ~ 4.15 (d, lH, J = 5.67 Hz), 3.41 (s, 3H), 3'.40 (s, 3H), 3.19 (m~, lH), 2.47 (dd, lH, J = 4.~, 16.06 Hz), ' ' 2.22'(dd, lH, J = 7.86, 16.16 Hz), 1.45 (s, 9H).

:~ 25 WO 94~001~4 PCI /US93/05687 ~
21369~1 `I `

Step D: N-(N-Acetyl-tyro.sinyl-valinyl-(e-CBZ-ly~sinyl))-~-amino-4-oxobutanoic acid ,B-tert-butyl ester dimethyl acetal s ~ ~;

H~NH~ H3 A CO2t-Bu ' To a solution of 3-Amino-4-oxobutanoic acid ~-tert-butyl ester dimethyl acetal (23~ mg, 1.09 mrnol) in 5 mL of DMF at ;
0C was added N-methyl rnorpholine (599 mL, 5.45 mrnol) followed 20 se4uentially by N-Acetyl-tyrosinyl-v~iinyl-e-CBZ-lysine (735 mg, 1.09 mmol), hydroxybenzotriazole (221 mg, 1.64 mmol), and dicyclohexylcarbodiimide (225 mg, 1.09 mmol). After 16 hours at arnbient temperature, the mixture wa~s filtered and purified by Sephadex" LH-20 chromatography (lM x 50 mm column, methanol 2s 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 a~s a colorles~s ~solid: lH
N~R (200 MHz, CD30D) ~ 7.31 (br s, 5H), 7.04 (br d, 2H, J = ~.35 30 Hz), 6.67 (br d, 2H, J = 8.45 Hz), 5.04 (s, 2H), 4.61 (m, 1 H), 4.44-4.25 (m, 3H), 4.17 (d, lH, J = 7.27 Hz), 3.39 (s, 3H), 3.38 (s, 3H), 3.1-2.9 (m, 3H), 2.75 (dd, lH, J = 9.2P~, 14.12 Hz), 2.53 (dd, lH, J =

'~'') 94/001'4 PCr/VS93/0~687 ~- 2136981 ~,~
5.47, 15.5~ Hz), 2.33 (dd, lH, J - 7.96, 15.53 Hz), 2.04 (m, 1~1), (s, 3H~ s-1.2 (m, 6H), 1.41 (s, 9H), 0.94 (d, 6H, J = 6.74 Hz).

S Step E: N-(N-Acetyl-tyrosinyl-valinyl-lysinyl)-3-amino-4-oxobutanoic acid dimethy! acetal ~-t-butyl e~ster J~N~ ~N~ -~OCH3 CO2t-Bu A Isolu$ion of N-(N-Acetyl-tyrosinyl-valiny]-e-CBZ-lysinyl)-3-amino^4-oxobutanoic acid ~-tert-butyl ester dimethyl acetal (15.6 mg) was di.solved in 2 mL of methanol and 10 mg of Pearlman's catalyst (pd(oH)2 on Carbon) was added. After 30 min 20 under hydrogen, the mixture was filtered and concentrated to give the title compound: lH NMR (200 MHz, CD30D) ~ 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.~-4.0 (m, 4H), 3.38 (s, 3H), 3.34 (s, 3H), 3.02 (dd, lH, J - 5.17, 13.81 Hz), 2.75 (dd, lH, J = 9.23, 14.06 Hz), 2.66 (t, 2s 2H, ~ - 7.08 Hz), 2.53 (dd, lH, J = 5.47, 15~5~ Hz), 2.34 (dd, lH, J
- 7.91, 15.57 Hz), 2.03 (m, lH), 1.~ (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).

Af~lnitY Chromatographv Procedure The starting enzyme preparation was purified about 100-fold from THP-l cell Iysate by anion exchange chromatography as described in Examples 2, 3, 5-8 and/or 10.

wo s4/001~4 2 1 3 6!9 8'1 Pcr/ussVoS6s 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 s dimensions were equilibrated with l O column volume~s of the chromatography buffer ( l 00 mM hepe~s, l 0% sucrose, and 0. l % 3-~(3-cholamidopropyl)dimethylammonio]- l -propanesulfonate (CHAPS) at pH 7.50) supplemented with 1 mM dithiothreitol. The enzyme solution (lS ml, lS0,000 unit~s, lS0 mg protein) was applied 0 through the guard column and run onto the ~ffinity column at a flow rate of 0.022 ml/min at 4C, and washed through with an additional 10 ml chromatography buffer at the sarne flow rate. During loading, ~% 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. ~ ml/min at 4C.
No enzymatic activity was detected in the wash fractions.

Step B: E!ution of Bound ICE
To elute the enzyme, the column was then flooded with 1 column volume of buffer containing 200 mM Acetyl-Tyr-Val-Ly.s(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 ~hen reGovered 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 be~n adequate.

`''~) 94/001~4 PCr/US93/05687 Step C: Reactivation of ICE
The eluted ICE wa~ reactavated using two synergistic chemical approaches: conversion of the inhibitor to its oxime, and oxidation of the active site thiol to it.s mixed disulfide with 5 glutathione by thiol-disulfide interchange.
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 25C. After allowing 10 halflives for the exchange, the inhibitor oxime and excess reagen~s were removed by exhaustive desalting in an AMICON CENTRICON-10 ultrafiltration cell using 15 the chromatography buffer at 4C. When desired, the enzyme-glutathione conjugate was reduced with 10 mM dithiothreitol (halflife < 1 min) to give active enzyme. The purified enzyme i.s stable indefinitely at -~0C. The recovery of enzymatic activity by ;~ affinity chromatography was >90%, and the final pu~fication ~ achieved was about 75,000-fold, as measured by SDS-polyacrylamide gel electrophoresis. The results are summarized on Table 4.

;

WO 94/001~4 2 1 3 6 9 8 1 ~ ~ PCr/US9~/05687 ~, - 6~ -AFFINITY PURIFICATION OF ICE

vol. units/ units/
(ml) lmits ml mg mg/ml mg DEAE sample 15 150,000 10 150 10 10 Affinity Eluate 0.2 140,000 700 0.03* 0.1~S 4.7 x * estimated from silver staining intensity on SDS-PAGE
5 Recovery of ICE was 93% with a purification of 4700-fold.

Synthesis of N-(N-Acetyl-tyrosinyl-Yalinyl-e-CBZ-lysinyl)-3-arnino-4-oxobutanoic acid.
20 Step A:

O

J~N~N'~I

~ 4/001~4 PCI`/US93t~5687 N -(N-Acetyl-tyrosinyl-valinyl-(e-CBZ-lysinyl))-3-arnino-4-oxobutanoic acid ~-tert-butyl ester dimeth~d acetal. To a solution of 3-Amino-4-oxobutanoic acid ~B-tert-butyl ester dimethyl acetal (23~ mg, 1.09 mmol) in 5 mL of DMF at 0C was added N-methyl molpholine (599 rnL, 5.45 mmol) followed sequentially by N-Acetyl-tyrosinyl-valinyl-e-CBZ-lysine (735 mg, 1.09 mmol~, hydroxybenzotriazole (221 mg1 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 ~lM x 50 mm column, methanol eluent).
The resulting product was further purified by MPLC on silica-~el (22 x 300 mm column, eluting with a linear gradient of dichloromethane to 1% ammoinia ~nd 10% methanol in dichloromethane) to give the ~itle compolmd as a colorless solid: lH :;
NMR (200 MHz, CD30D) ~ 7.31 (br s, SH), 7.04 (br d, 2H, J = ~.35 Hz), 6.67 (br d, 2H, J - 8.45 Hz), 5.04 (s, 2H), 4.61 (m, lH), 4.44-4.25 (m, 3H), 4.17 (d, lH, J = 7.27 Hz), 3.39 ~, 3H), 3.38 (s, 3H), 3.1-2.9 (m, 3H), 2.75 (dd, lH, J = 9.2~, 14.12 Hz), 2.53 (dd, lH, J -5.47, 15.58 Hz), 2.33 (dd, lH, J = 7.96, 15.53 Hz), 2.04 (m, lH), 8 (s, 3H), 1.8-1.2 (m, 6H~, 1.41 (s, 9H), 0.94 (d, 6H, J = 6.74 Hz).

i WO 94/001~4 . PCI/US93/05687 i'~
2136981 i~`

Step B:

J~ N~U N~

5 N-(N-Acetyl-tyrosinyl-valinyl-e-(: BZ-lysinyl)-3 amino-4-xobutanoic acid. _ _ A solution of N-(N-Acetyl-tyrosinyl-valinyl-e-CBZ-ly~sinyl)-3-amino-4-oxobutanoic acid ,~-tert-butyl ester dimethyl acetal (14.9 mg) was treated with 1 mL of tr~fluoroacetic acid, a~ed 20 for 15 minutes, ~nd concentrated in vacuo. The residue was dis.solved in 1.0 mL of methanol and 1.0 mL of water containing 20 uL of thionyl chlor~de was added. After 1 hour, the pH of the solution was adjusted to around 5 with sodium acetate to afford a solution of the title compound: lH NMR (200 MHz, CD30D) ~ 7.33 25 (br s, 5H), 7.05 (br d, 2H, J = g.35 Hz), 6.74 (br d, 2H, J = 8.35 Hz), 4.6-3.9 (m, SH), 3.1-2.3 (m, 6H), l.9g (m, lH), 1.92 (s, 3H), 1.g-1.2 (m, 6H), 0.89 (d, 6H, J = 6.60 Hz).

`~'~ 94tO01~4 ~ 1 3 6 9 8 1 P~/US93/05687 Generation of ICE~pecific DNA products by PCR
Degenerate oligonucleotides were designed based on the 5 amino acid sequence from the amino terminu.s and internal region~
of both the 20 kDa and 10 kDa proteins (Figure 17 and 18). For the 20 kDa protein, the amino terminal prirner, GAYCCNGCNATGCCNAC (SEQ.ID.NO.:3), was 12~ fold degenerate while the internal primer, 3'ATRGGNTAl:)TACCTRTT5' (SEQ.ID.NO.:4), was 48 fold degenerate. For the 10 kDa protein, the amino terminal primer, GCNATHAARAARGCNCA (SEQ.ID.NO.:5)9 was 192 fold degenerate while the internal primer, GTYTACGGNTGNTGNCT
(SEQ.ID.NO.:6), was 128 fold degenerate.
Single-stranded THP.l cDNA was synthesized from THP. 1 cellular poly A+ mRNA and used as a PCR template. PCR
- was conducted essentially as described by a modification of the -MOPAC procedure (Lee, et ah, Science 239~ 1288 (1988)). For each PCR, 10 pmol of each primer was added to that quantity of 20 cDNA synthesized for 0.4mg of poly A+ mRNA in a reaction buffer consisting of 50 mM K~l, 10 mM TRlS-HCl (pH 8.3). 1.5 mM
MgCI29 0.01% w/v gelatin, and 200 uM of each dNTP in a final volume of 10 ~11. The PCR program consisted of one cycle of denaturation for 100C for 10 minutes, the addition of 2 units of ~y 25 polymerase, followed by 30 cycles of the following step~s: 95C for 30 seconds, 4RC for 30 seconds, and 70C for 1 minutes. For the 20 kDa protein, a PCR product of 116 bp was synthesized and its - ! ide~tity wag verified by hybridization with an internal inosine-substituted oligonucleotide [ATIGGRTAIATYTCIGCR]
30 (SEQ.ID.NO.:7), while for the 10 kDa protein, a PCR product of 221 bp was synthesized and verified with a similar type of probe ~ATIGARAARGAYTTYATIGC](SEQ.ID.NO.:8). Both PCR

WO 94/001~4 PCI/US93/05687 products were subcloned into Bluescript vectors (Stratagene), ~and ~sequenced by the chain termination method (Sanger, et al., PNAS 74, 5463 ( 1 977)).
The deduced arnino acid sequence for the 20 kDa and 5 the 10 kDa subunit are shown in Table 7, panel A, and panel B, respectively.

o A .
Asp Pro Ala Met Pro Thr Ser Ser Gly Ser Glu Gly Asn Val Lys Leu Cys Ser Leu Glu Glu Ala Gln Arg Ile Trp Lys Gln Lys Ser Ala Glu Ile Tyr Pro Ile Met Asp Lys (SEQ.ID.NO.

B
Ala Ile Lys Lys Ala His Ile Glu Lys Asp Phe Ile Ala Phe Cys Ser Ser Thr Pro Asp Asn Val Ser Trp Arg His Pro Thr Met Gly Ser Val Phe Ile Gly Arg Leu Ile Glu His Met Gln Glu Tyr Ala Cys Ser Cys Asp Val Glu Glu Ile Phe Arg Lys Val Arg Ph~ Ser Phe Glu Gln Pro Asp Gly Arg Ala Gln Met Pro Thr Thr Glu (SEQ.ID.NO.:12) 2s EXAMPLE 24 lsolation of ICE cDNA clone~s from lambda cDNA }ibrarie~s l'he PCR derived products for the lOkda and 20kda ICE
subunits were used as hybridization probes for screening a larnbda 30 gtlO cDNA library from THP.l cells (Clontech~. Plating and plaque lifts of the library were performed by standard methods (T.
Maniatis, E.F. Fritsch, J. Sambrook~ Molecular Cloning: A

`~0 94/001~4 PCl/US93/0~687 2136g81 Laboratory Manual ~Cold Spring Harbor Laboratory, Cold Sp~ring Harbor, New York, 19~2~. The probes were random-primed Iabelled with 32P-dCTP to high specific activity and a separate screening of the library (600,000 plaques per .screen) was conducted with each probe. The probes were added to hybridization buffer (50% fo~namide, SX Denhardts, 6X SSC ~lX SSC = 0.15 M), 0.5%
SDS, 100 ,ug/ml salmon spe~n DNA) at 1 x 106 cpm/ml. Eleven positively hyb~dizing phage were detected using the 10 kDa ~specific probe while seven positively hybridi~ing phage were observed using o the 20 kDa probe.

Subclonin~ and se4uencin~ of ICE cDNA clone~s Several cDNA clones ranging in size from 1.0 kb to 1.6 lcb in length and containing a single open reading frame of 404 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 ICE i~ shown in ` Table S (A), with the 24 kDa (B), 20 kDa (C), and 10 kDa (D) ; - ~ 20 coding regions shown as well. The full length clone was designated clone OCP9. The deduced arnino acid sequence of full length ICE
from the cloned cDNA is shown in Table 6 (A), with the deduced amino acid sequence for the, 24 kDa (B), 20 kDa (C), and 10 kDa (D) subunits sho~,vn as well.
2s lnspection of the deduced amino acid sequence and comparison with the amino acid sequences derived from purified native ICE 20 kDa and 10 kDa subunits (Figure 19 and 20) reveals ; amino acid sequence identity except for one amino acid found at position 120 of the full length nascent ICE protein. According to the cDNA sequence, Asn is encoded at position 120 of the full-length protein. This amino acid position corresponds to the NH2-terminus ~ of the 20 kDa subunit. The amino acid sequence derived from the ,~, , wos4/oolC4 213`~9 ~1 : PCr/Us93/~5687 purified nati~/e 20 kDa subunit detennined the NH2-terminal amino acid to be Asp. This difference between the deduced amino acid se4uence and the known amino acid sequence, the only difference found, may be attributable to deamidation of the NH2-terminal Asn 5 of the 20 kDa subunit tc) fo~n Asp. This type of dearnidation is known to occur and is cornmon when Asn is found at the NH2- ;
terminus. Whether Asn or Asp is found at the NH2-terminus of the 20 kDa subunit, or whether Asn or Asp is found within the ICE
polypeptide at position 120 of the full length protein, or in a polypeptide fragment, is not expected to affect ICE activity significantly, if at all.

TABLE S

A
GCCATGGC CGACAAGGTC CTG.~GGAGA 30 . AAGACAGTTA CCTGGCAGGG ACGCTGGGAC 270 ~O 94/001~4 21~ 6 9 81 - ~ PCr/US93/05687 GCGTAGATGT GA~AAAPAAT CTCACTGCTT 63C

lS CTACAGAAGA GTTTGAGGAT GATGCTATTA 960 : TTATTGGAAG ACTCATTGAA CATATGCA~G lOi30 AATATGCCTG TTCCTGTGAT GTGGAGGAAA lllO

i ACTCTAGGTT TACAGGCTGA GAATCCTTAA 1350 TCCAAA~AAT TCGAATTTTG AAATGCTCTA 1380 TATTCAAAGG AAATGTTTAT TGAAACATTT 1~40 ~AAATTATAG GTTTTTGGAT TAGGGATGCT 1470 WO 94~001~4 2 1 3 6 9 8 1 ` PCl/US93/05687 ~ .

.. ~,. . .

(SEQ.ID.NO.:9) B
TCTCAA GGAGTACTTT
CTTCCTTTCC AGCTCCTCAG GCAGTGCAGG
ACAACCCAGC TATGCCCACA TCCTCAGGCT
CAGAAGGGAA TGTCAAGCTT TGCTCCCTAG
AAGAAGCTCA AAGGATATGG AAACAAAAGT
CGGCAGAGAT TTATCCAATA ATGGACAAGT
CAAGCCGCAC ACGTCTTGCT CTCATTATCT
GCAATGAAGA ATTTGACAGT ATTCCTAGAA
GAACTGGAGC TGAGGTTGAC ATCACAGGCA
TGACAATGCT GCTACAAAAT CTGGGGTACA
GCGTAGATGT GA~AAAAAAT CTCACTGCTT
CGGACATGAC TACAGAGCTG GAGGCATTTG
CACACCGCCC AGAGCACAAG ACCTCTGACA
GCACGTTCCT GGTGTTCATG TCTCATGGTA
TTCGGGAAGG CATTTGTGGG AAGAAACACT
CTGAGCAAGT CCCAGATATA CTACAACTCA
ATGCAATCTT TAACATGTTG AATACCAAGA
ACTGCCCAAG TTTGAAGGAC AAACCGAAGG
TGATCATCAT CCAGGCCTGC CGTGGTGACA
GCCCTGGTGT GGTGTGGTTT AAAGAT
~SEQ.ID.NO.. l~) C

AACCCAGC TATGCCCACA TCCTCAGGCT
CAGAAGGGAA TGTCAAGCTT TGCTCCCTAG

CGGCAGAGAT TTATCCAATA ATGGACAAGT
CAAGCCGCAC ACGTCTTGCT CTCATTATCT

WCI g4/001~4 P~/l)S93/05687 GCAATGAAGA ATTTGACAGT ATTCCTAGAA
GAACTGGAGC TGAGGTTGAC ATCACAGGCA
TGACAATGCT GCTACAAAAT CTGGGGTACA
GCGTAGATGT GAAAAAAAAT CTCACTGCTT
CGGACATGAC TACAGAGCTG GAGGCATTTG
CACACCGCCC AGAGCACAAG ACCTCTGACA
GCACGTTCCT GGTGTTCATG TCTCATGGTA
TTCGGGAAGG CATTTGTGGG AAGAAACACT
CTGAGCAAGT CCCAGATATA CTACAACTCA

ACTGCCCAAG TTTGAAGGAC AAACCGAAGG
TGATCATCAT CCAGGCCTGC CGTGGTGACA
GCCCTGGTGT GGTGTGGTTT AAAGAT
(SEQ.ID.NO.:14) D
GCTATTA
AGAAAGCCCA CATAGAGAAG GATTTTATCG
CTTTCTGCTC TTCCACACCA GATAATGTTT
CTTGGAGACA TCCCACAATG GGCTCTGTTT
TTATTGGAAG ACTCATTGAA CATATGCAAG
AATATGCCTG TTCCTGTGAT GTGGAGGAAA
TTTTCCGCAA GGTTCGATTT TCATTTGAGC
AGCCAGATGG TAGAGCGCAG ATGCCCACCA
CTGAAAGAGT GACTTTGACA AGATGTTTCT
ACCTCTTCCC AGGACAT
tSEQ.ID~NO.:15) WO 94/001~4 PCl`/US93/05687 ~.`

- 7~ -Met Ala Asp Lys Val Leu Lys Glu Lys Arg Lys Leu Phe Ile Arg Ser Met Gly Glu Gly Thr Ile Asn Gly Leu Leu Asp Glu Leu Leu Gln Thr Arg Val Leu Asn Lys Glu Glu Met Glu Lys Val Lys Arg Glu Asn Ala Thr Val Met Asp Lys Thr Arg Ala Leu Ile Asp Ser Val Ile Pro Lvs Gly Ala Gln Ala Cvs Gln Ile Cys Ile Thr Tyr Ile Cys Glu Glu Asp Ser Tyr Leu Ala Gly Thr Leu Gly Leu Ser Ala Asp Gln Thr Ser Gly Asn l~rr Leu Asn Met Gln Asp Ser Gln Gly Val Leu Ser Ser Phe Pro Ala Pro Gln Ala Val Gln Asp Asn Pro Ala Met Pro Thr Ser Ser Gly Ser Glu Gly Asn Val Lys Leu Cys Ser Leu Glu Glu Ala Gln Arg Ile Trp Lys Gln Lys Ser Ala Glu Ile Tyr Pro Ile Met Asp Lys Ser Ser Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Glu Glu Phe Asp Ser Ile Pro Arg Arg Thr C-ly Ala Glu Val Asp Ile Thr Gly Met Thr Met Leu Leu Gln Asn Leu GIy Tyr Ser Val Asp Val Lys Lys Asn Leu Thr Ala Ser Asp Met Thr Thr Glu Leu Glu Ala Phe Ala His Arg Pro Glu His Lys Thr Ser Asp Ser Thr Phe Leu Val Phe Met Ser Elis Gly Ile Arg Glu Gly Ile Cys Gly Lys Lys His Ser Glu Gln Val Pro Asp Ile Leu Gln Leu Asn Ala Ile Phe Asn Met Leu Asn Thr Lys Asn Cys Pro Ser Leu Lys Asp Lys Pro Lys Val Ile Ile Ile Gln Ala Cys Arg Gly Asp Ser Pro Gly Val Val Trp Phe Lys Asp Ser Val Gl~ Val Ser Gly Asn Leu Ser Leu Pro Thr Thr Glu Glu Phe Glu Asp Asp Ala Ile Lys Lys Ala His Ile Glu Lys Asp Phe Ile Ala Phe Cys Ser Ser Thr Pro Asp Asn Val Ser Trp Arg His Pro Thr Met Gly Ser Val Phe Ile Gly Arg Leu Ile Glu His Met Gln Glu Tyr Ala Cys Ser Cys WO 94/001~4 - PC~/U~i93/05687 Asp Val Glu Glu Ile Phe Arg Lys Val Arg Phe Ser Phe Glu Gln Pro Asp Gly Arg Ala Gln Met Pro Thr Thr Glu Arg Val Thr Leu Thr Arg Cys Phe Tyr Leu Phe Pro Gly His (SEQ.ID.NO. :10) S

B

Asp Ser Gln Gly Val Leu Ser Ser Phe Pro Ala Pro Gln Ala Val Gln Asp Asn Pro Ala Met Pro Thr Ser Ser Gly l O Ser Glu Gly Asn Val Lys Leu Cys Ser Leu Glu Glu Ala Gln Arg Ile Trp Lys Gln Lys Ser Ala Glu Ile Tyr Pro Ile Met Asp Lys Ser Ser Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Glu Glu Phe Asp Ser Ile Pro Arg Arg Thr Gly Ala Glu Val Asp Ile Thr Gly Met Thr Met Leu Leu 15 Gln Asn Leu Gly Tyr Ser Val Asp Val Lys Lys Asn Leu Thr Ala Ser Asp Met Thr Thr Glu Leu Glu Ala Phe Ala His Arg Pro Glu ~lis Lys Thr Ser Asp Ser Thr Phe Leu Val Phe Met Ser His Gly Ile Arg Glu Gly Ile Cys Gly Lys Lys His Ser Glu Gln Val Pro Asp Ile Leu Gln Leu 20 Asn Ala Ile Phe Asn Met Leu Asn Thr Lys Asn Cys Pro Ser Leu Lys Asp Lys Pro Lys Val Ile Ile Ile Gln Ala Cys Arg Gly Asp Ser Pro Gly Val Val Trp Phe Lys Asp (SEQ.ID.NO. :16) Asn Pro Ala Met Pro Thr Ser Ser Gly Ser Glu Gly Asn Val Lys Leu Cys Ser Leu Glu Glu Ala Gln Arg Ile Trp Lys Gln Lys Ser Ala Glu Ile Tyr Pro Ile Met Asp Lys Ser Ser Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Glu 30 Glu Phe Asp Ser Ile Pro Arg Arg Thr Gly Ala Glu Val Asp Ile Thr Gly Met Thr Met Leu Leu Gln Asn Leu Gly Tyr Ser Val Asp Val Lys Lys Asn Leu Thr Ala Ser Asp WO94/00154 2~369~ PCr/US93/05687 ~-- ~0-Met Thr Thr Glu Leu Glu A.l~ Phe Ala His Arg Pro GluHis Lys Thr Ser Asp Ser Thr Phe Leu Val Phe Met Ser His Gly Ile Arg Glu Gly Ile Cys Gly Lys Lvs His Ser Glu Gln Val Pro Asp Ile Leu G~.n Leu A~n Ala Ile Phe 5 Asn Met Leu Asn Thr Lys Asn Cys Pro Ser Leu Lys Asp Lys Pro Lys Val Ile Ile Ile Gln Ala Cys Arg Gly Asp Ser Pro Gly Val Val Trp Phe Lys Asp (SEQ.ID.NO.:17) D

Ala Ile Lys Lys Ala His Ile Glu Lys Asp Phe Ile Ala Phe Cys Ser Ser Thr Pro Asp Asn Val Ser Trp Arg His Pro Thr Met Gly Ser Val Phe Ile Gly Arg Leu Ile Glu His Met Gln Glu Tyr Ala Cys Ser Cys Asp Val Glu Glu 15 Ile Phe Arg Lys Val Arg Phe Ser Phe Glu Gln Pro Asp Gly Arg Ala ~,ln Met Pro Thr Thr Glu Arg Val Thr Leu hr Arg Cys Phe Tyr Leu Phe Pro Gly His (SEQ.ID.NO.:18) , - - :

,~ ~
Clonin of the ICE cDNA into E. coli Expres~sion Vectors Recombinant ICE is produced in E. coli following the transfer of the ICE expression cassette into E. coli expression 25 vectors, including but not limited to, the pET series (Novagen). The pET vectors place ICE expression under control of the tightly regulated bacteriophage T7 promoter. Following transfer of this ' . ', f ' i construct into an E. coli host which contains a chromo~somal copy o f the T7 RNA polymerase gene driven by the inducible lac promoter, 30 expression of ICE is induced when an approriate lac substrate (IPTG) is added to the culture. The levels of expressed ICE are dete~nined by the assays described above.

`~'0 ~4/001C;4 2 1 ~ 6 9 8 1 Pcr/usg3/0s6g7 The cDNA encoding ~he entire open reading frarne for p45 was inserted into the Ndel site of pET 1 1 a. Constructs in the positive orientation were identified by sequence analysis and used to transfo~n the expression host strain BL21. Transformants were 5 then used to inoculate cultures for the production of ICE protein.
Cultures may be grown in M9 or ZB media, whose forrnulation is known to those skilled in the art. After growth to an OD600= 1.5, expression of ICE~ was induced with 1 mM IPTG for 3 hours at 37C. Authentic ICE enzymatic activity was found in the insoluble inclusion body fraction from these cells. Soluble ICE was extracted from the inclusion body fraction with 5 M guanidine-HCI in a buffer containing 50 mM Tris-HCI (pH ~) and 100 mM dithiothreitol.
Active ICE was generated from this extract following dialysis against 100 volumes of 25 mM HEPES (pH 7.5), 5 mM dithiothreitol, 10%
5 sucrose.

EXAMPLE ~7 In Vitro Translation of ICE mRNA by Xenopus Oocyte 20 Microiniection Vector and Expres~sion in Mammalian Cells ICE cDNA constructs are ligated into in vitro transcription vectors (the pGEM series, Promega) for the production of synthetic m~As.
Synthetic mRNA is produced in sufficient quantity in 2s vitro by cloning double stranded DNA encoding ICE mRNA into a plasmid vector containing a bacteriophage promoter, linearizing the plasmid vector containing ~e cloned ICE-encoding DNA, ~nd transcribing ~e cloned DNA in vitro using a DNA-dependent ~NA
polymerase from a bacteriophage that specifilcally recognizes the 30 bacteriophage promoter on the plasmid vector.
Various plasmid vectors are available containing a bacteriophage promoter recognized by a bacteriophage DNA-W094/00~4 ~13~!~811; PCI'/US93/05687,,~

dependent RNA polymerase, including but not limited to plas~nids pSP64, pSP65, pSP70, pSP71, pSP72, pSP73, pGEM-3Z, pGEM-4Z, pGEM-3Zft pGEM-SZf, pGEM-7Zf, pGEM-9Zf, and pGEM-llZf, the entire series of plasmid~s 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 ~CE-encoding DNA in the proper orientation is identified and isolated, i~ 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 eo T3, T7, and 2s SP6 RNA polymerase. The synthetic ICE m~NA is then i~solated and purified.
It may be advantageous to synthesize mRNA containing a 5' terminal cap structure and a 3' poly A tail tn improve mRNA
stability. A cap structure, or 7-methylguanosine, may be incorporated at the ~'terrninus 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 ~'0 94~001 ~4 PCI /US93/0~i687 2 1 3 6 9 8 1 i - ~3 at the ~' te~ninus a~s it synthesizes the mRNA. The poly A tai~i~s found narurally occuning in many cDNA's but can be added to the 3' terminu~s of the mRNA by simply inserting ~ 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 reticulocy$e Iysate and wheat gelm extracts (both commercially available from Promega and New England Nuclear) or in a cell based system, including but not limited to microinjection into Xenopus oocyte~s, with microinjection into Xenopus oocytes being preferred.
Xenopus oocytes are microinjected with a ~suff1cient arnount of synthetic ICE mRNA to produce ICE protein. The microinjected oocytes are incubated to allow translation of the ICE
mRNA, fo~ning 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: 370-3~6, (19~3)].
Oocytes will be harvested and analyzed for ICE expression as described below.

EXAMPLE 2~

Clonin~ of ICE cPNA into a Mammalian Expression Vector ~, . .
ICE cDNA expr~ssion cassettes are ligated at appropriate restriction endonuclease sites to the following vectors containing strong, universal mammalian promoters: pBC12BI
[Cullen, B.~. Methods in Enzymol. 152: 684-704 19~8], and pEE12 (CellTech EP O 338,841) and its derivatives pSZ9016-1 and p9019.
p9019 represents the construction of a mam~nalian expression vector containing the hCMVIE prm, polylinker and SV40 polyA element with a selectable marker/amplification system comprised of a mutant W~ 94/001~4 , ~ PCI`/US93/05687 ~`
2 1 3 ?

- ~4 -gene for dehydrofolate reducta~se (mDHFR) (Simonsen, C.C. ~d Levinson, A. D. Proc. Natl. Acad. Sci USA ~0: 2495-2499 [19~3]) 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 Shalp? Nucl.
Acid Res. 13: 841-857 [1985~) as template. The resulting 0.25 Kb PCR product was digested with ClaI and SpeI and ligated into the 6.7 Kb fragment of pEE12 which had been likewise digested. The resultant plasmid was designated p901~. p901~ was digested with o BglII and SfiI to liberate the 3' portion of the SV40 early promoter and the GScDNA from the vector. A 0.73 Kb Sfil-XhoII fragment isolated from plasmid pFR400 (Simonsen, C.C. and Levinson, A. D.
Proc. Natl. Acad. Sci USA 80: 2495-2499 [19~3]) 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. pSZ9016-1 is identical to p9019 except for the substitution of the HIV LTR for the huCMVIE promoter. This vector was constructed by digesting p9019 with XbaI and Mlul to remove the huMVIE promoter. The HIV LTR promoter, from residue - 117 to +gO (as found in the vector pCD23 containing the portion of the HIV-l LTR (Cullen, Cell 46:973 [1986]) was PCR arnplified 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 III and Xba I sites were appended on the 3' side.
2s Following the digestion of the resulting 0.2 kb PCR product with the enzymes MluI and Xba I the fragment was agarose gel purified and ligated int~ the 4.3 Kb promoter~ess DNA fragment to generate the ~ I vector pSZ90;16~
- Cassettes containing the ICE cDNA in the positive 30 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 .uo94/001~4 ~1 36981 PCl/US93/05687 ~5 introduced into various fiboblastic host cells: [COS-7 (ATCC#
CRL1651), CV-l tat [Sackevitz et al., Science 23~: 1575 (19~7)], -293, L (ATCC# CRL6362)] by standard methods including but not limited to electroporation,or chemical procedures (cationic 5 liposomes, DEAE dextran, calcium phosphate). Transfected cells and cell culture supernatants can be harvested and analyzed for ICE
expression as described below.
All of the vectors used for mammalian transient expression can be used to establish stable cell lines expressing ICE.
Unaltered ICE cDNA constructs cloned into expression vectors will be expected to program host cells to make intracellular ICE protein.
In addition, ICE is expressed extracellularly as a secreted protein by ligahng ICE cDNA constructs to DNA encoding the signal sequence of a secreted protein such as the human growth horrnone or human 15 Iysozyme. The transfection host cells include, but are not limited to, CV-l-P [Sackevitz et al., Science 23~s: 1575 (1987)~, tk-L [Wigler, et al. Cell 1 1: 223 (1977)], NS/0, and dHFr- CHO [Kaufman and Sharp, J. Mol. Biol. 159: 601, (19P~2)].
Co-transfection of any vector containing ICE cDNA
20 with a drug selection plasmid (included, but not limited to G41~, aminoglycoside phosphotransferase, pLNCX [Miller, A.D. and Rosman G. J. Biotech News 7: 9~0-990 (19P~9)]; hygromycin, hygromycin-B phospholransferase, pLG90 ~Gritz. L. and Davies, J., GENE 25: 179 (1983)]; APRT, xar.thine-guanine pho,sphoribosyl-2s transferase, p~AM (Clontech~ [Murray, et ah, Gene 31: 233 (19~S4)]will allow for the selection of stably transfected clones. Levels of ICE are quantitated by the assays described above.
' ! ' i f ICE cDNA constructs are ligated into vectors containing amplifiable drug-resistance markers for the production of 3 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 agen$, and WO 94/001~4 . ,.; ! ., PCI/US93/05687 ~ . `.

, - ~6 -isolation of an over-expressing clone with a high copy numbe~ of the plasmid is accomplished by selection in increasing doses of the agènt.
The following systems are utilized: the 9016 or the 9019 plasmid containing ~he mutant DHFR gene [Simonson, C. and Levinson, A., Proc. Natl. Acad. Sci. USA 80: 2495 ~19~3)], transfected into dHFR-CHO cells and selected in methotrexate; the pEE12 pla~smid containing the glutamLrle' synthetase gene, tran~sfected into NSio cells and selected in methionine sulfoximine (CellTech lnternational 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 ' amplffied~with~ HAT (100 uM;hypoxanthine, 0.4 uM ~aminopterin, 16 5 ~uM~ thymldlne3~
The~ expression of recombinant IM~ was achleved by transfection of full~-}ength ICE cDNA, including the comple~e ORF
of the~ 45 ~kDa-~lCE preprotein (Fig. 23), into a mammalian host cell.
-Th~ .6 kbEçQRI fragment containing the~full length ICE cDNA
2~0 ~ was cloned into vector pSZ-90~16. 6 ,ug of this DNA along with 0.6 ;ug'of pX8TAT,~a~mammalian expression vector which places the trans-a~lvating~protein~TAT) of HIV under the control of the SV40 early promoter,~ ~was~ trans~cted into COS-7 cells by the cationic-1 iposome~;method~ Cells ~were~h~arvested 4~ hours later and Iysed in " ~ ` 2s ~ detergent ~ r~mM`HEPES pH 7, 1% Triton-X-100, lmM
p ~ E~I~,~rn~M D~I, 10~ug/ml'aprotinin, 10 ug/ml leupeptin, 10 ug/ml pèpstatin, and 2 mM PMSF). Cell Iysates were incubated with i ràdiolabeled IL~ precursor to measure ICE activity. Cleavage i `
products of IL-l,B were analyzed by immunoprecipitation with IL-I,B
30 ~ antibody and fractionation on SDS polyacrylamide gels. The L- l precursor;~was cleaved to the mature, 17 kDa form by cells t ransfected with'lCE cDNA,~ but not by cells transfected with the ",~

i,, ~, ,,~ ~, .

wo 94/001~4 ~ . i PCI /US93/05687 - ~7 -expression plasmid alone. The cleavage product comigrated withmature IL-l~ produced by incubation of substrate with affinity purified ICE and was completely inhibited by the specific ICE
inhibitor (L-709,04g). The substrate specificity of the expressed 5 ICE activity was verified by incubating lysate~s with an IL-1 ,B
precursor cleavage site mutant (Alal 16) which cannot be cleaved by ICE. As with native ICE, the activity from the transfectants cleaves the rnutant protein to a 2~s kDa product but not to the 17 kDa form (Fig. 25).
Recombinant ICE activity was not observed upon transient transfection of individual constructs expressing the p20 or plO subunits, both singly or in combination. These re.sults suggest that p20 and plO when exp~ressed in this manner may not be folded properly and thus ~e active conformation of ICE may only be lS generated upon proteolysis of the p45 proenzyme. ~n addition, mutation of the catalytic Cys (residue 285) to Ala abolishes ICE
activity while deletion of the predomain (pl 4) demonstrate~s that it is not required for ICE activity.

.
Cloning of ICE cDNA into a Baculovirus Expression Vector for Expression in Insect (~ll~s ~ ~ _ - ~ Baculovirus vector~s, which are derived from the genome of the AcNPV virus, are designed to provide high level expression of cDNA in ~e Sf9 line of insect cells (ATCC CRL#
171 1). Recombinant baculoviruses expressing ICE cDNA is 'prcduced~by ~e following standard methods (InVitrogen Maxbac Manual): ~e ICE cDNA constructs are ligated into the polyhedrin gene iI1 a variety of baculovirus transfer vectors, including the pAC360 and the BlueBac vector (InVitrogen). Recombinant baculoviruses are generated by homologous recombination following : : .

WO 94/00154 2 1 3 6;~ 8 1`; PCr/US93/0~687 ,~

co-transfection of the baculovirus tran~sfer vector and linearized AcNPV genomic DNA [Kitts, P.A., Nuc. Acid. Res. 18: 5667 (1990)] into Sf9 cells. Recombinant pAC360 viruses are identified by the absence of inclusion bodies in infected cells and recornbinant 5 pBlueBac viruses 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 0 p45 was inserted irlto the BarnHI site of pBlueBacII. Constructs in the positive orientation were identified by sequence analysis and used to transfect Sf9 cells in the presence of linear AcNPV mild type DNA.
Authentic,;enzymatically-active ICE was found in the 5~ cytoplasm~of;i~lfècted eells. ~Active~lCE was extracted from infected cells under~native conditions by hypotonic or detergent lysis.

.,, ~
,, 20 Cloning of ~lCE~o a-yeas~ression vector ; Recomb:i.qant ICE is produced in the yeast S cerevisiae fol1O~g~ in~ertion~ of the optimal ICE cDNA cistron into expression~vectors~`~designéd to direct the intracellular or extracéll~ ex~ssion~heterologous proteins. In ~e case of 2s~ intrac~ar~expression, ;vectors- such as Err~Lyex4 or the like are lig~ted to:the~lCE~cistron [Rinas, U. et al, Biotechnology 8: 543-545 (1990); Horowitz B~ et aL, J~ Biol. Chem. 265: 41894192 (1989)~
r ~ For extrace~llularlexpression, the ICE cistron is ligated into yeast -~ ~ - expression vectors w~hich fuse a secretion signal (a yeast or 30 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)].
',_,,. :. ' ' ~ :
'-" ,~" ~ ',' ' ' ' ' "'','~: ' ~ -~.~0 94/001~4 213 6 9 ~1 PCI/US93/0~687 ~9 These vectors include, but are not limited to pAV~El>6, which fuses the human serum albumin signal to the expressed cDNA[Steep O. Biotechnology ~: 42-46 (1990)], and the vector pL~PL which fuses the human lysozyme signal to the expressed cDNA [Yarnamoto, Y., Biochem. 2~: 272~-2732)]. In addition, ICE
is expressed in yeast as a fusion protein conjugated to ubiquitin utilizing the vector pVEP ~Ecker7 D. J., J. Biol. Chem. 264: 7715-7719 (1989), Sabin, E. A., Biotechnology 7: 705-709 (19~9), McDonnell D. P., Mol. Cell Biol. 9: 5517-5523 (19~9)]. The level~s of expressed ICE are determined by the assays described above.

~ .

Purification of Recombinant ICE
Recombinantly produced ICE may be purified by any one or combination of purification procedures of Examples 1-3, and 5-7. In addition~ recombinantly produced 20 kDa subunit, lOkDa subunit and nascent full-length ICE polypeptides may be individually purified using the appropriate monospecific antibodies.

W O 94/001i4 PCT/US93/05687 ,.~.
~ ~ 3 6 9 8 1 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Howard, Andrew D
Molineaux, Susan M
Tocci, Michael J
Calaycay, Jimmy Miller, Douglas K.
(ii) TITLE OF INVENTION: COMPLIMENTARY DNA ENCODING PRECURSOR
INTERLEUKIN lB CONVERTING ENZYME
(iii) NUMBER OF SEQUENCES: 19 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Merck & Co., Inc.
(B) STREET: P.O. Box 2000, 126 E. Lincoln Avenue (C) CITY: Rahway ~D) STATE: New Jersey (E) COUNTRY: USA
(F) ZIP: 07065 (v) COMPUTER READABLE FORM:
(A) NEDIUM TYPE: Fioppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
~ii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 746,g54 (B) FILING DATE: 16 Aug 1991 (~iii) ATTORNEY/AGENT INFORMATION:
(A) NANE: Wallen, John W. III
(Bj REGISTRATION NUMBER: P-35403 (C) REFERENCE/DOCKET NUMBER: 18498IC
(ix) TELECOMMnNICATION INFORNATION:
(A) IELEPHONE: (908) 59~-3905 (C) TELEX: (908) 594-4720 (2) INFOR~ATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
~:~ (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~) 94/001~4 PCI/US93/05687 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Asp Pro Ala Met Pro Tyr Ser Ser Gly Ser Glu Gly Asn Val (2) INFORMATION FOR SEQ ID NO:2:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Ala Ile Lys Lys Ala His Ile Glu Lys Asp Phe Ile Ala Phe Cys Ser Ser (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) ~OLECULE TYPE: c~NA

(xi) 5EQUENCE DESCRIPTION: SEQ ID NO:3:

(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs ' (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: cDNA

(xi) SE~UENCE DESCRIPTION: SEQ ID NO:4:
ATRGG~TADT ACCTRTT 17 (2) INFORMATION FOR SEQ ID NO:5:

W O 94/001~4 2 I 3 6 9 8I PCT/US93/05687 ~ ;

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GCNATHAARA ARGCNCA l7 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQU~NCE DESCRIPTION: SEQ ID NO:6:
,:
GTYTACGGNT GNTGNCT l7 ~`
: ~2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
ATNGGRTANA TYTCNGCR l8 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 20 base pairs (L) TYPE: nucleic acid tC) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

ATNGARAARG AY m ATNGC 20 `~1O 94/001~4 PCT/Us93/05687 21369~1 (2) INFORMATION FOR SEO ID NO:9:
(i) SEQUENCE CHA~ACTERISTICS:
(A) LENGTH: 1490 base pairs (B) '~YPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE T}'PE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CGGCCATGGC CGACAAGGTC CTGAAGGAGA AGAGAA~GCT G'MTATCCGT TCCATGGGTG 60 AAGACAGTTA CCTGGCAGGG ACGC'~GGGAC TCTCAGCAGA TCAAACATCT GGAAATTACC 300 TTAATATGCA AGACTCTCAA GGAGTACTTT CTTCCT'rTCC AGCTCCTCAG GCAGTGCAGG 360 TTCGGGAAGG CA m GTGGG AAGAAACACT CTGAGCAAGT CCCAGATATA CTACAACTCA 780 WO 94~001 ~4 PCl /US93/Q5687 ;~. .
2 ~ !

GTATGGTCGG GAGTGTGGGA AGGTTGAGGA AAGGGTACTG AAAGTCCATT TGAGTCAAG~ 1~20 CAAATTATAG GTTTTTGGAT TAGGGATGCT AA~CCAGTAA GTATACAGCT 1490 (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 404 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) S~QUENCE DESCRIPTION: SEQ ID NO:10:
Met Ala Asp Lys ~al Leu Lys Glu Lys Arg Lys Leu Phe Ile Arg Ser ;~ Met Gly Glu Gly Thr Ile Asn Gly Leu Leu Asp Glu Leu Leu Gln Thr Arg Val Leu Asn Lys Glu Glu Met Glu Lys Val Lys Arg Glu Asn Ala Thr Val Met Asp Lys Thr Arg Ala Leu Ile Asp Ser Val Ile Pro Lys Gly Ala Gln Ala Cys Gln Ile Cys Ile Thr Tyr Ile Cys Glu Glu Asp Ser Tyr Leu Ala Gly Thr Leu Gly Leu Ser Ala Asp Gln Thr Ser Gly g5 Asn Tyr Leu Asn Met Gln Asp Ser Gln Gly Val Leu Ser Ser Phe Pro Ala Pro Gln Ala Val Gln Asp Asn Pro Ala Met Pro Thr Ser Ser Gly 9er Glu Gly Asn Val Lys Leu Cys Ser Leu Glu Glu Ala Gln Arg Ile 130 135 lg0 Trp Lys Gln Lys Ser Ala Glu Ile Tyr Pro Ile Met Asp Lys Ser Ser lg5 150 155 160 Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Glu Glu Phe Asp Ser Ile Pro Arg Arg Thr Gly Ala Glu Val Asp Ile Thr Gly Met Thr Met Leu yVO 94/001~4 PCT~US93/05687 ; ~136981 9s - Leu Gln Asn Leu Gly Tyr Ser Val Asp Val Lys Lys Asn Leu Thr Ala 195 200 20~ ~
Ser Asp Met Thr Thr Glu Leu Glu Ala Phe Ala His Arg Pro Glu His Lys Thr Ser Asp Ser Thr Phe Leu Val Phe Met Ser His Gly Ile Arg Glu Gly Ile Cys Gly Lys Lys His Ser Glu Gln Val Pro Asp Ile Leu Gln Leu Asn Ala Ile Phe Asn Met Leu Asn Thr Lys Asn Cys Pro Ser Leu Lys Asp Lys Pro Lys Val Ile Ile Ile Gln Ala Cys Arg Gly Asp Ser Pro Gly Val Val Trp Phe Lys Asp Ser Val Gly Val Ser Gly Asn Leu Ser Leu Pro Thr Thr Glu Glu Phe Glu Asp Asp Ala Ile Lys Lys Ala His Ile Glu Lys Asp Phe Ile Ala Phe Cys Ser Ser Thr Pro Asp Asn Val Ser Trp Arg His Pro Thr Met Gly Ser Val Phe Ile Gly Arg : Leu Ile Glu His Met Gln Glu Tyr Ala Cys Ser Cys Asp Val Glu Glu Ile Phe Arg Lys Val Arg Phe Ser Phe Glu Gln Pro Asp Gly Arg Ala 370 . 375 380 Gln Met Pro Thr Thr Glu Arg Val Thr Leu Thr Arg Cys Phe Tyr Leu 385 390 395 . 400 Phe Pro Gly His ~2) INFORMATION FOR SEQ ID NO~
(i) SEQUENCE CHARACTERISTICS:
' 1 i (A) LENGTH: 39 amino acids (B) TYPE: amino acid tC) STRANDEDNESS: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
Asp Pro Ala Met Pro Thr Ser Ser Gly Ser Glu Gly Asn Val Lys Leu WO 94~001~i4 ~ PCT'/USg3/05687 `-~136981 !`.~

Cys Ser Leu Glu Glu Ala Gln Arg Ile Trp Lys Gln Lys Ser Ala Glu Ile Tyr Pro Ile Met Asp Lys (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 74 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Ala Ile Lys Lys Ala His Ile Glu Lys Asp Phe Ile Ala Phe Cys Ser Ser Thr Pro Asp Asn Val Ser Trp Arg His Pro Thr Met Gly Ser Val 20 25 30 ~.
Phe Ile Gly Arg Leu Ile Glu His Met Gln Glu Tyr Ala Cys Ser Cys Asp Val Glu Glu Ile Phe Arg Lys Val Arg Phe Ser Phe Glu Gln Pro Asp Gly Arg Ala Gln Met Pro Thr Thr Glu (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 582 base p~irs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

CTqGCTCTCA TTATCTGCAA TGAAGAATTT GACAGTATTC CTAGAAGAAC TGGAGCTGAG 240 ~4/001~4 2~ 1 3 6 9 8 1 ; PCT/US~3/05687 (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 53g base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

~(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
AACCCAGCTA TGCCCACATC CTCAGGCTCA GAAG~GAATG TCAAGCTTTG CTCCCTAGAA 60 AGC~GCACAC GTCTTGCTCT CATTATCTGC AATGAAGAAT TTGACAGTAT TCCTAGAAGA 180 ACTGGAGCTG AGGTTGACAT CACAGGCATG ACAA~GCTGC TACAAAATCT GGGGTACAGC 240 GCAATCTTTA ACAT¢TTGAA TACCAAGAAC TGCCCAAGTT TGAAGGACAA ACCGAAGGTG 480 (2) INFORMATION FOR SEQ ID NO:15:
ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26g base pairs tB) TYPE: nucleic acid (C) STRANDEDNESS: single tD) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

WO 94/001~4 2 1 3 6 9 8 1 ` ` PCr/US93/05687 GCTATTAAGA AAGCCCACAT AGAGAAGGAT rrTATCGCTT TCTGCTCTTC CACACCAGAT 6 0 AATGrrrCTT GGAGACATCC CACAATGGGC TCTGT rrTTA TTGGAAGACT CATTGA~CAT 12 0 ATGCAAGAAT ATGCCIY~TTC CTGTGATGTG GAGGAAATTT TCCGCAAGGT TCGATI~I`TCA 18 0 TTTGAGCAGC CAGATGGTAG AGCGCAGATG CCCAC:CACTG AAAGAGIGAC TTMACAAGA - 2~0 (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 195 amino acids (B) TYPE: amino acid ( C ) STRANDEDNESS: s ing 1 e ( D ) TOPOLOGY: l inear (ii) MOLECULE ~YPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Asp Ser Gln Gly Val Leu Ser Ser Phe Pro Ala Pro Gln Ala Val Gln Asp Asn Pro Ala Met Pro Thr Ser Ser Gly Ser Glu Gly Asn Val Lys Leu Cys Ser Leu Glu Glu Ala Gln Arg Ile Trp Lys Gln Lys Ser Ala Glu Ile Tyr Pro Ile Met Asp Lys Ser Ser Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Glu Glu Phe Asp Ser Ile Pro Arg Arg Thr Gly Ala Glu Val Asp Ile Thr Gly Met Thr Met Leu Leu Gln Asn Leu Gly Tyr Ser Val Asp Val Lys Lys Asn Leu Thr Ala Ser Asp Met Thr Thr Glu Leu Glu Ala Phe Ala His Arg Pro Glu His Lys Thr Ser Asp Ser Thr Phe Leu Val Phe Met Ser His Gly Ile Arg Glu Gly Ile Cys Gly Lys Lys His Ser Glu Gln Val Pro Asp Ile Leu Gln Leu Asn Ala Ile Phe 145 150 15`5 160 Asn Met Leu Asn Thr Lys Asn Cys Pro Ser Leu Lys Asp Lys Pro Lys ~ 94/001~4 2 1~ 6 9 8 1 P~T/US93/05687 -_ 99 _ Val Ile Ile Ile Gln Ala Cys Arg Gly Asp Ser Pro Gly Val Val Trp Phe Lys Asp (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 178 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Asp Pro Ala Met Pro Thr Ser Ser Gly Ser Glu Gly Asn Val Lys Leu Cys Ser Leu Glu Glu Ala Gln Arg Ile Trp Lys Gln Lys Ser Ala Glu Ile Tyr Pro Ile Met Asp Lys Ser Ser Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Glu Glu Phe Asp Ser Ile Pro Arg Arg Thr Gly Ala Glu Val Asp Ile Thr Gly Met Thr Met Leu Leu Gln Asn Leu Gly Tyr Ser Val Asp Val Lys Lys Asn Leu Thr Ala Ser Asp Met Thr Thr Glu Leu ~; 85 90 95 Glu Ala Phe Ala His Arg Pro Glu His Lys Thr Ser Asp Ser Thr Phe Leu Val Phe Met Ser His Gly Ile Arg Glu Gly Ile Cys Gly Lys Lys - ~ 115 120 125 His Ser Glu Gln Val Pro Asp Ile Leu Gln Leu Asn Ala Ile Phe Asn ! ~130 ~ 135 ~ 140 Met Leu Asn Thr Lys Asn Cys Pro Ser Leu Lys Asp Lys Pro Lys Val Ile Ile Ile Gln Ala Cys Arg Gly Asp Ser Pro Gly Val Val Trp Phe Lys Asp (2) IN~ORMATION FOR SEQ ID NO:18:

W O 94/001~4 ~j. ' P ~ /US93~05687 `~`

~oo (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 88 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO-18:
Ala Ile Lys Lys Ala His Ile Glu Lys A~p Phe Ile Ala Phe Cys Ser Ser Thr Pro Asp Asn Val Ser Trp Arg His Pro Thr Met Gly Ser Val Phe Ile Gly Arg Leu Ile Glu His Met Gln Glu Tyr Ala Cys Ser Cys A~p Val Glu Gl~ Ile Phe Ars Lys Val Arg Phe Ser Rhe Glu Gln Pro Asp Gly Arg Ala Gln Met Pro Thr Thr Glu Arg Val Thr Leu Thr Arg ~5 70 75 80 Cys Phe Tyr Leu Phe Pro Gly His (2) INFORMATION FOR SEQ ID NO:l9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:
Ser Gln Gly Val Leu Ser Xaa Phe Pro Ala Pro Gln Ala Gln Asp Asn Pro Ala Met Pro Thr

Claims (20)

WHAT IS CLAIMED IS:

1. A DNA molecule which encodes the complete unmodified form of precursor interleukin 1 beta converting enzyme, having the nucleotide sequence:
.
(SEQ.ID.NO.:9)

2. A DNA molecule which encodes the 22 kDa protein of precursor interleukin 1 beta converting enzyme, having the nucleotide sequence:

.
(SEQ.ID.NO.:13)

3. A DNA molecule which encodes the 20 kDa subunit of precursor interleukin 1 beta converting enzyme, having the nucleotide sequence:

.
(SEQ.ID.NO.:14)

4. A DNA molecule which encodes the 10 kDa subunit of precursor interleukin 1 beta converting, having the nucleotide sequence:
.
(SEQ.ID.NO.:15)

5. 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:

(SEQ.ID.NO.:9) , or , or (SEQ.ID.NO.:13) , or (SEQ.ID.NO.:14) (SEQ.ID.NO.:15)

6. 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:
(SEQ.ID.NO.:9), or , or (SEQ.ID.NO.:13) , or (SEQ.ID.NO.:14) .

(SEQ.ID.NO.:15)

7. A recombinant host cell expressing recombinant interleukin 1 beta and containillg one or more recombinant genes selected from the group consisting of:
GCCATGGC CGACAAGGTC CTGAAGGAGA

(SEQ.ID.NO.:9) , or , or (SEQ.ID.NO.:13) , or (SEQ.ID.NO.:14) .

(SEQ.ID.NO.:15)

8. A protein, in substanitally pure form which can specifically cleave precursor IL-1 beta to form mature IL-1 beta, having an amino acid sequence:

.
(SEQ.ID.NO.:10)

9. A protein in substantially pure form which corresponds to the 22 kDa subunit of precursor interleukin 1 beta converting enzyme, having the amino acid sequence:
, (SEQ.ID.NO.:16)

10. A protein, in substantially pure form which corresponds to the 20 kDa subunit of presursor interleukin 1 beta converting enzyme, having an amino acid sequence:
, (SEQ.ID.NO.:17) wherein R is Asp or Asn.

11. A protein, in substantially pure form which corresponds to the 10 kDa subunit of precursor interleukin 1 beta converting enzyme, having an amino acid sequence:

.
(SEQ.ID.NO.:18)

12. A composite protein molecule, in substantially pure form which can specifically cleave precursor interleukin 1 to form mature interlukin 1 beta, comprising:

(a) a polypeptide having the amino acid sequence:
, (SEQ.ID.NO.:17) wherein R is Asp or Asn, and (b) a polypeptide having the amino acid sequence:

.
(SEQ.ID.NO. :18)

13. A composite protein molecule, in substantially pure form which can specifically cleave precursor interleukin 1 beta to form mature interleukin 1 beta, comprising:
(a) a polypeptide having the amino acid sequence:
(SEQ.ID.NO.:16) and (b) a polypeptide having the amino acid sequence:

.
(SEQ.ID.NO.:18)

14. A monospecific antibody immunologically reactive with monomeric precursor interleukin 1 beta converting enzyme.

15. The antibody of Claim 11, wherein the antibody blocks the activity of precursor interleukin 1 beta converting enzyme, and the antibody is a monoclonal antibody.

16. A monospecific antibody immunologically reactive with the 20 kDa subunit of precursor interleukin 1 beta converting enzyme.

17. The antibody of Claim 13, wherein the antibody blocks the activity of precursor interleukin 1 beta converting enzyme, and the antibody is a monoclonal antibody.

18. A monospecific antibody immunologically reactive with the 10 kDa subunit of precursor interleukin 1 beta converting enzyme.

19. The antibody of Claim 15, wherein the antibody blocks the activity of precursor interleukin 1 beta converting enzyme, and the antibody is a monoclonal antibody.

20. A recombinant microorganism having the designation ATCC 68655.