CA2235575A1 - Novel human cysteine protease - Google Patents

Abstract

The present invention provides a polynucleotide which identifies and encodes a novel cysteine protease (NCP) expressed in cells of both the adrenal gland and human umbilical vein endothelium. The present invention also provides for antisense molecules designed from the nucleotide sequence or its complement. The invention further provides genetically engineered expression vectors and host cells for the production of purified NCP, antibodies or other molecules specifically binding to NCP and pharmaceutical compositions based on molecules which bind NCP. The invention specifically provides for diagnostic assays based on altered ncp expression and which allow identification of such a condition. These assays utilize probes designed from NCP encoding nucleic acid sequences or antibodies specifically binding NCP.

Description

CA 0223~7~ 1998-04-22 NOVEL HUMAN CYSTEINE PROTEASE

TECHNICAL FIELD
The present invention relates to novel, human cysteine protease and the use of its nucleic acid ~md amino acid sequences in the ~ gn~si~ study, prevention and treatmPnt of autoimmllnP or degen.,~dli~,re ~ ç~ç~

BACKGROUND ART
]l0 Cysteine prote~es are involved in diverse cellular processes ranging from the processing of precursor proteins to intracellular degradation. They may induce vascular perm~bility through activation of the kallikrein/kinin palhw~, complex with various h.-m~gglutinins, activate complement components and destroy serpins. Their endopeptidase activity and "trypsin-like" specificity leads to the speculation that there are many specialized cysteine protease molecules found in various human cells and tissues.
Cysteine proteases are known to be produced by monocytes, macro phages and othercells of the in-mlm~ system. These cells migrate to sites of infl~mm~tion and in their protective role secrete various molecules which clean up damaged tissue. Under other conditions, these same cells may overproduce the same molecules and cause tissuedestruction. This is the case in autoimml~ne ~ e~ce~ such as rheumatoid arthritis, when the secretion of the cysteine protease, cathepsin C, degrades collagen, l~minin, elastin and other structural prol[eins found in the extracellular matrix of bones. Bone weakened by such degradation is more susceptible to tumor invasion and met~t~ic The novel, human cysteine protease of this application was first identified among the sequences of a cDNA library made from human adrenal glands. Human adrenal glands are cap-like strucltures located above each kidney. Each gland consists of the adrenal med~
and the adrenal cortex. The adrenal medulla is made up of chromaffin tissue and mainly secretes norepinephrine (NE) and epinephrine (E). Stim~ tion of the sympathetic nerves to the adrenal med~ releases these two catechol~minPs into the blood. NE constricts blood vessels, stimnl~tes cardiac activity, inhibits the gastrointçstin~l tract, and dilates the pupils of the eyes. E triggers almost the same responses, but it has a stronger effect on cardiac activity CA 0223~7~ 1998-04-22 and a weaker effect on blood vessels. NE and E supplement the effects of the symp~thetic nervous sys'tem but appear to have little effect its function.
The adrenal cortex uses cholesterol to produce a large number of corticosteroidswhich display hormonal activity. The outer layer of the adrenal gland mainly produces the mineralocorticoid, aldosterone. The stim~ tory and inhibitory regulation of aldosterone secretion is governed by potassium level, renin-angiotensin interactions, and secretion of adrenocorticotrophic hormone (ACTH), dopamine, serotonin, and B-endorphin. Aldosterone regulates extracellular fluid volume and sodium/potassium balance by interacting with type-I
mineralocorticoid lec~tol~ in target tissues such as the kidney, salivary gland, and intestin~l lo mucosa.
The inner layers of the adrenal gland are sites of glucocorticoid and androgen, estrogen ancl progesterone biosynthesie The prin~ip~l glucocorticoid is cortisol which functions in the regulation of protein, carbohydrate, lipid, and nucleic acid metabolism, acts as an anti-infl~mm~tory, and plays a biofeedba~ role in ~uppressing endocrine functions.
Androgen, secreted under the regulation of ACTH, is responsible for initiating the development of secondary sexual characteristics (in both sexes), of sex organs in the male, and for m~int~ining lifelong spetm~to~enesis.
Conditions ~liee~eee and disorders of the adrenal gland include chromaffin cell tumors, which are part of the multiple endocrine neoplasia syndromes; Sipple's syndrome, which may be found alone or associated with medullary thyroid carcinoma and parathyroid ~lt?nom~e;
adrenal virilism; Cushing's syndrome; Conn's syndrome; Addison's ~ieeaee~ which is a primary adrenocortical insufficiency; seCon~l~ry adrenocortical insufficiency, and adrenal adenomas, which include benign adrenal cysts, nonfunctional adrenal carcinoma, and tuberculosis of the adrenal gland.
The adrenal gland and its ~li ee~ ees are reviewed, inter alia, in Guyton AC (1991 ) Textbook of Medic~l PhysiolQgy. WB Sa-.n-lers Co, Phil~(lelrlhia PA; Isselbacher, KJ et al (1994) Harrison's Principles of Intern~l Medicin~, McGraw-Hill New York NY; The Merck Manual of Di~n~sis and Th~orapy ( 1992) Merck Research Laboratories, Rahway NJ; and Goodman AG et al. (1993) The Ph~rm~ological Basis of Therapeutics, McGraw-Hill, New York NY.

CA 0223~7~ 1998-04-22 wo 97115592 PCT/US96tl6926 DISCLOSURE OF THE INVENTION
The present invention relates to a novel cysteine protease (NCP) isolated from human - adrenal gland and human umbilical vein endothelial cells (HUVEC) and to the use of this 5 novel protein and its nucleic acid sequences in the diagnosis, study, prevention and tre~tm.?nt of immnn~ rlieP~es~ particularly autoimm--ne and deg~ cldli~e ~i~e~ces.
The subject invention provides a unique nucleotide sequence (SEQ ID NO l) which encodes a novel human cysteine protease. This cysteine protease (ncp) was first identified as a partial nucleotide sequence, Incyte Clone 100877, via computer search for local sequence o ~lig.. ~ among the cDNAs of an adrenal gland library. The pertinent amino acid residues which allow this molecule to be characterized as a cysteine protease are Q48, C52 and Hl50.
Partial nucleotide sequence was also identified in Incyte Clone 66931 (SEQ ID No 4) from a human umbilical vein endothelial cell cDNA library. A modified XL-PCR procedure,specially designt d oligonucleotides and adrenal and HUVEC libraries were used to extend Incyte Clones 100877 and 66931 to obtain the full length sequence. Partial nucleotide sequences (SEQ ID NO: 3 and 5-24) disclosed herein, have been identified in several other libraries which appear to share certain features. These features include cell lines or tissues which are innmortal (ly,lJphol"a and leukemic cell lines), infl~med (adenoid and rheumatoid synovium li braries), or involved in systemic cleanup or defense through either the harboring 20 or production of cells such as monocytes or macro phages (bone marrow, kidney, lung, placenta anal small int~stine libraries). The assembled nucleotide and arnino acid sequences shown in Figure 1 l~lesenl a new human cysteine protease.
An additional c-"n~Lel search for local sequence ~ nment~ of the amino acid sequence of the novel cysteine protease described herein showed that the most closely related 25 molecule, approximately 50% amino acid similarity, is the hemoglobinase from Schistosoma japonicum (GenBank Accession X70967; Merckelbach A et al (1994) Trop Med Parasitol 45:193 198) The cysteine protease of'this application shows 50% amino acid sequence similarity with hemoglobinase from ~. japonicum.
Based on the conserved cysteine protease residues, Q48, C52 and H,50 of the catalytic 30 region; similarity to the closely related molecule, hemoglobinase; and the presence of the CA 0223~7~ 1998-04-22 novel cysteine protease in cells and tissues which are immortal, infl~mPd or involved in systemic cleanup or defense; the novel cysteine protease is involved in proteolysis, in systemic cleanup and defense, and is therefore useful in the diagnosis, study, prevention and treatm~nt c~f autoimmune or degenerative tii~e~ec Further aspects of the present invention include ~nti~en.ce molecules of ncp which are useful in flimini~hing or elimin~ting ~x~iession of the genomic nucleotide sequence.
The present invention also relates, in part, to polynucleotide sequences and expression vectors encoding NCP and methods for the production and recovery of NCP from host cells.
The ncp nucleic acid sequences disclosed herein may be used in diagnostic assays to o detect and quantify levels of ncp mRNA in cells and tissues. For example, a ncp nucleic acid sequence may be used in PCR or hybridization assays of biopsied fluids or tissues to ~ gnose abnormalities in gene expression associated with an immlme disorder. The invention further relates to diagnostic kits for the detection of NCP or nucleic acid sequences encoding NCP
comprising NCP, antibodies specific to NCP or nucleic acid sequences encoding NCP. Such diagnostic kits may be used for the detection of any condition, disorder, or disease state related to aberrant ~.ession of NCP, including but not limited to: ~nPmi~ arteriosclerosis, ~cthm~ bronchitis, cancers, emphysema, gingivitis, infl~mm~tory bowel disease, insulin-dependent diabetes mellitus, lenkemi~ osteoarthritis, osteoporosis, pulmonary fibrosis, rheumatoid arthritis, septic shock syndromes, and systemic lupus eryth~m~tosus. Steps for testing a biological sample with nucleotide probes based on the ncp nucleotide sequence or antibodies produced against the purified NCP protein are provided.
Antibodies may be used for theldl)e--lic as well as diagnostic purposes, eg, in neutralizing the activity of an NCP associated with an immune disorder such as rhe..m~toid arthritis. The present invention also relates in part to proteins, peptides, and organic 25 molecules capable of modl-l~ting activity of NCP which may be used therapeutically in the tre~tm~nt of disease states associated with aberrant expression of an NCP. The present invention also relates to ph~rm~ceutical compositions for the treatment of disease states associated with aberrant expression of ncp comprising NCP, nucleic acid sequences encoding NCP, anti-NCP antibodies, anti NCP or other proteins, peptides or organic molecules capable 30 of modl-l~ting NCP expression.

CA 0223~7~ 1998-04-22 wo 97/1559~2 Pcrluss6/l6926 BRIEF DESCRIPTION OF DRAWINGS
Figure I displays an alignm~nt of cDNA sequences which encompass the coding region of nc:p. ~lignment~ shown in this and the following figures were produced using the multisequence Alignm~nt program of DNASTARTM software (DNASTAR Inc, Madison WI) Figures 2A, 2B, 2C and 2D show the nucleic acid and amino acid ~lignm~ntc of NCP.
Figures 3A and 3B show the amino acid Ali~nm~nt~ bcLw~ell NCP and hemoglobinase from Schi~t~n~ ma japonirllm (Gel RAnk Accession X70967).
Figure 4 displays the DNASl AR analysis of NCP a regions (A), ~' regions (B), turn regions (T), coil regions (C), hydrophilicity plot (H), a ~ .hipAlhic regions (AA), 1~
0 Alllpl~irAI~lic regions (BA), antigenic index (AI) and surface probability plot (S) based on the predicted acid amino sequence and col.l~osi~ion.

MODES FOR CARRYING OUT THE INVENTION
Definitit)n~
The present invention relates to a novel cysteine protease which is e~pl. ssed in the adrenal glarld, human umbilical vein endothelial cells, lymphoma and leukemic cell lines, adenoid, rh~llmAtoid synoviurn, bone marrow, kidney, lung, placenta and small intestine. As used herein, the abbreviation for the novel cysteine protease in lower case (ncp) refers to a gene, cDNA, RNA or nucleic acid sequence while the upper case version (NCP) refers to a protein, polypeptide, peptide, oligopeptide, or arnino acid sequence.
As used herein, NCP is a terrn which refers to NCP from any species, including bovine, ovin,e, porcine, equine, murine and preferably human. It refers to naturally occurring or variant form and NCP from any source whether natural, semi-synthetic, synthetic or recombinant. A pl~r~..ed NCP is one having at least 80% amino acid sequence similarity, a 25 more ~ef~ ;d variant is one having 90% amino acid sequence similarity, and a most pr~;r~l.ed variant is one having 95% amino acid sequence .~imil~rity to the NCP amino acid sequence ilhlstrated in Figure 1.
An "oligonucleotide" or "oligomer" is a stretch of nucleotide residues which has a sufficient number of bases to be used in a polymerase chain reaction (PCR). These short 30 sequences are based on (or ~leci~n~od from) genomic or cDNA sequences and are used to CA 0223~7~ 1998-04-22 amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 50 nucleotides, preferably about 15 to 30 nucleotides. They are chemically synthPci7P~l and may be used as 5 probes.
"Probes" are nucleic acid sequences of variable length, preferably between at least about 10 and as many as about 6,000 nucleotides. They are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to o hybridize thcm oligonucleotides. They may be single- or double-stranded and are carefully designed to have specificity in PCR, hybridization membrane-based, or ELISA-liketechnologies.
"Reporter" molecules are chemical moieties used for labeling a nucleic or amino acid sequence. They include, but are not limited to, radionuclides, enzymes, fluolesc~
chemiluminescent, or chromogenic agents. Reporter molecules associate with, establish the presence of, and may allow quantification of a particular nucleic or amino acid sequence.
A "portion" or "fragment" of a polynucleotide or nucleic acid compri~es all or any part of the nucleotide sequence having fewer nucleotides than about 6 kb, preferably fewer than about 1 kb which can be used as a probe. Such probes may be labeled with reporter :20 molecules using nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. After pretesting to OptillliZt~ reaction conditions and to elimin~te false positives, nucleic acid probes may be used in Southern, northern or in situ hybridizations to determine whether DNA or RNA encoding the protein is present in a biological sample, cell type, tissue, organ or org~ni~m ,~5 "Recombinant nucleotide variants" are polynucleotides which encode a protein. They may be synthesized by making use of the "recl--n-l~ncy" in the genetic code. Various codon substitutions, such as the silent changes which produce specific restriction sites or codon usage-specific mutations, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic host system, respectively.
~;o "Linkers" are synthPsi7~ palindromic nucleotide sequences which create internal CA 0223~7~ 1998-04-22 restriction endonuclease sites for ease of cloning the genetic material of choice into various vectors. "Polylinkers" are Pnginçered to include multiple restriction enzyme sites and provide for the use of both those enzymes which leave 5' and 3 ' ov~rh~ng.e such as BamHI, EcoRI, PstI, KpnI and Hind III or which provide a blunt end such as EcoRV, SnaBI and StuI.
s "Co:ntrol element.~" or "regulatory sequences" are those nontr~n~l~te~l regions of the gene or DNA such as enh~nrers, promoters, introns and 3' untr~n~1atecl regions which interact with cellular proteins to carry out replication, transcription, and translation. They may occur as boundary sequences or even split 1he gene. They function at the molecular level and along with regulatory genes are very hlll,o~ l in development, growth, di~lenliation and aging 0 processes.
"Chimeric" molecules are polynucleotides or polypeptides which are created by combining one or more of nucleotide sequences of this invention (or their parts) with additional nucleic acid sequence(s). Such combined sequences may be introduced into an a~l~fiate vector and expressed to give rise to a chimeric polypeptide which may be expected to be di~.enl from the native molecule in one or more of the following charact~rictics: cellular location, distribution, ligand-binding affinities, hltel.,hain affinities, degradation/'turnover rate, ~ign~1ine, etc.
"Active" refers to those forms, fr~gment~ or domains of an amino acid sequence which display the biologic and/or immnnngenic activity characteristic of the naturally occurring peptide.
"Nat-urally occurring NCP" refers to a polypeptide produced by cells which have not been genetically en~in~ered or which have been genetically enginPered to produce the same sequence as that naturally produced. Specifically contemplated are various polypeptides which arise i.rom post-translational modifications. Such modifications of the polypeptide include but are not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
"Derivative" refers to those polypeptides which have been chemically modified bysuch techniques as ubiquitination, labeling (see above), pegylation (derivatization with polyethylene glycol), and ch~mic~1 insertion or substitution of amino acids such as ornithine which do nol: norrnally occur in human proteins.

CA 0223~7~ 1998-04-22 "Recombinant polypeptide variant" refers to any polypeptide which differs from naturally occurring NCP by amino acid insertions, deletions and/or substitutions, created using recombinant DNA techniques. Guidance in d~ g which amino acid residuesmay be replaced, added or deleted without abolishing char~ctçri.ctics of interest may be found 5 by col.~p~ g the sequence of NCP with that of related polypeptides and ~ ing the number of amino acid sequence changes made in highly conserved regions.
Amino acid "substitutions" are defined as one for one amino acid repl~cementc.. They are conservative in nature when the substituted amino acid has similar structural and/or chemical pro~ ies. Examples of conservative rep1~rçment~ are substitution of a leucine o with an isoleucine or valine, an aspartate with a gl11t~m~tç, or a threonine with a serine.
Amino acid "insertions" or "deletions" are changes to or within an amino acid sequence. l hey typically fall in the range of about l to 5 amino acids. The variation allowed in a particular amino acid sequence may be t;A~I ;...ent~lly determin~i by producing the peptide synthetically or by systematically making insertions, deletions, or substitutions of nucleotides in the ncp sequence using recombinant DNA techniques.
A "signal or leader sequence" is a short amino acid sequence which or can be used, when desired, to direct the polypeptide through a membrane of a cell. Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous sources by recombinant DNA techniques.
An "oligopeptide" is a short stretch of amino acid residues and may be expressed from an oligonucleotide. It may be functionally equivalent to and either the same length as or considerably shorter than a "fragment ", "portion ", or "segment" of a polypeptide. Such sequences comprise a stretch of amino acid residues of at least about 5 amino acids and often about l 7 or more amino acids, typically at least about 9 to l 3 amino acids, and of sufficient length to display biologic and/or immunogenic activity.
An "inhibitor" is a substance which retards or prevents a chemical or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, antagonists and their derivatives.
A "standard" is a 4u~li~1ive or qualitative measurement use for comparison.
Preferably, il: is based on a statistically applup~;ate number of sarnples and is created to use as CA 0223~7~ 1998-04-22 WO 97/15592 PCTtUS96/16926 a basis of comparison when performing diagnostic assays, running clinical trials, or following patient tre~ment profiles. The samples of a particular standard may be normal or similarly abnormal.
"Animal" as used herein may be defined to include human, domestic (cats, dogs, etc), 5 agricultural (cows, horses, sheep, goats, chicken, fish, etc) or test species (frogs, mice, rats, rabbits, ~imi~nc, etc).
"Conditions" includes cancers, disorders or ~lice~ces in which ncp activity may be implicated. These specifically include, but are not limited to, ~n~nni~ arteriosclerosis, asthma, bronchitis, elllphyse.lla, gingivitis, infl~mm~tory bowel fiice~ce, insulin-dependent o diabetes mellitus le~l~emi~ multiple endocrine neoplasias, osteoarthritis, osteoporosis, pulmonary fibrosis, rh.o~lnn~toid arthritis, septic shock syndromes, and systemic lupus erythPm~tosus.
Since the list oftechnical and scientific terms cannot be all encor.,l.Ac~ g, any nnrlefinPd terms shall be construed to have the sarne m~ning as is commonly understood by one of skill in the art to which this invention belongs. Furthermore, the singular forms "a", "an" and '~le" include plural ~ef~ unless the context clearly dictates otherwise. For example, reference to a "restriction enzyme" or a "high fidelity enzyme" may include mixtures of such enzymes and any other enzymes fitting the stated criteria, or reference to the method includes reference to one or more methods for obtaining cDNA sequences which will 20 be known to those skilled in the art or will become known to them upon reading this specification.
Before the present sequences, variants, formulations and methods for making and using the invention are described, it is to be understood that the invention is not to be limited only to the particular sequences, variants, formulations or methods described. The 25 sequences, variants, formulations and methodologies may vary, and the terminology used herein is for the purpose of describing particular embo-lim~ntc The terminology and definitions are not int~led to be limiting since the scope of protection will ultimately depend upon the claims.

30 Description of th~ Invention CA 0223~7~ 1998-04-22 W O 97/15592 PCT~US96/16926 The present invention provides for a purified polynucleotide which encodes a novel cysteine protease homolog which is expressed in human cells or tissue. The novel cysteine protease (ncp; Incyte Clone 100877) was first identified among the cDNAs from an adrenal gland cDNA library. The amino acid residues which allow this molecule to be characterized as a cysteine protease are Q48, C52 and H~50. The number of nucleotides sepal~ling the Q and C residues in this novel cysteine protease is fewer than found in other cysteine proteases, such as papain.
In addition, the novel cysteine protease is expressed in human umbilical vein endothelial cells (HUVEC) . The full length ncp sequence was obtained by sequencing o clones from both the adrenal and the HUVEC cDNA libraries. The molecule most closely related to this cysteine protease is hemoglobinase cloned from the blood fluke, Schistosoma japonicum~ GenBank Accession X70967 (Merckelbach A et al (1994) Trop Med Parasitol 45:193 198). The ncp of the present application may well be human hemoglobinase even though it has not been found in either spleen or liver libraries where a hemoglobinase would likely be acl:ive. It was, however, found in numerous tissues in which systemic cleanup or defense had been activated and where hemoglobinase or an NCP might be expected to act proteolytically to clean up the contents of injured or dying red blood cells.
Transcripts which did align with some portion of the ncp molecule were found in other Incyte cDNA libraries. Thirty-two segm~nt~ of cDNAs from dirr~lc,ll Incyte Clones are shown as an overlapping assemblage in Figure 1. Twenty three of these Incyte Clones including cDNAs from U937 cell, THP-1 cell, rheumatoid synovium, bone marrow, kidney, lung, infl~me-l adenoid, placenta, and small intestine libraries are presented in the Sequence T.i~ting In lact, ncp is fairly common in libraries where the normal tissue functions in, and the role of the molecule, proteolytic activity, would appear to be associated with systemic cleanup and defense.
Purified nucleotide sequences, such as ncp, have numerous applications in techniques known to those skilled in the art of molecular biology. These techniques include their use as PCR or hybIidization probes, for chromosome and gene mapping, in the production of sense or ~nti~çn~e nucleic acids, in screening for new th~.d~ulic molecules, etc. These examples are well known and are not intçn(led to be limiting. Furthermore, the nucleotide sequences CA 0223~7~ 1998-04-22 disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on plu~l lies of nucleotide sequences that are elllly kmown, including but not limited to such properties as the triplet genetic code and specific base pair interactions.
s As a result of the degeneracy of the genetic code, a multitude of NCP-encoding nucleotide sequences may be produced and some of these will bear only minim~l homology to the endogenous sequence of any known and naturally occurring cysteine ~urotease sequence. 1 his invention has specifically contemplated each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon o choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring NCP and all such variations are to be considered as being specifically disclosed.
Although the ncp nucleotide sequence and its derivatives or variants are preferably capable of identifying the nucleotide sequence of the naturally occl.rring NCP under optimized conditions, it may be advantageous to produce NCP-encoding nucleotide sequences posse~ing a subst~nti~lly different codon usage. Codons can be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accorda~ce with the frequency with which particular codons are utilized by the host. Other reasons for subst~nti~lly altering the nucleotide sequence encoding the NCP without altering the encoded amino acid sequence include the production of RNA
transcripts having more desirable properties, such as a longer half-life, than transcripts produced from the naturally occurring sequence.
Nucleotide sequences encoding NCP may be joined to a variety of other nucleotidesequences by means of well established recombinant DNA techniques (Sambrook J et al (1989) Molecular Clonir~: A Laborator,v Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor NY, or Ausubel FM et al (1989) Current Protocols in Molecular Biologv~ John Wiley & SOI1S, New York City). Useful sequences for joining to ncp include an assortment of cloning vectors such as plasmids, cosmids, lambda phage derivatives, phagemids, and the like. Vector;s of interest include vectors for replication, expression, probe generation, sequencing, and the like. In general, vectors of interest may contain an origin of replication CA 0223~7~ 1998-04-22 functional in at least one org~ni.cm, convenient restriction endonuclease sensitive sites, and selectable rnarkers for one or more host cell systems.
PCR as described in US Patent Nos. 4,683,195; 4,gO0,195; and 4,965,188 provides additional uses for oligonucleotides based upon the ncp nucleotide sequence. Such oligomers 5 are generally chemically synth~ci7~-1 but they may be of recombinant origin or a mixture of both. Oligomers generally comprise two nucleotide sequences, one with sense orientation (5'->3') and one with antisense (3' to 5') employed under optimized conditions for identification of a specific gene or ~i~gnostic use. The same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less aill;llgt;llL
o conditions for identification and/or 4~ ion of closely related DNA or RNA sequences.
Full length genes may be cloned l-tili~ing partial nucleotide sequence and various methods known in the art. Gobinda et al (1993; PCR Methods Applic 2:318-22) disclose "restriction-site PCR" as a direct method which uses universal primers to retrieve unknown sequence ~dj~cçnt to a known locus. First, genomic DNA is amplified in the presence of 15 primer to linker and a primer specific to the known region. The amplified sequences are subjected to a second round of PCR with the same linker primer and another specific primer intern~l to the first one. Products of each round of PCR are transcribed with an a~lo~l;ate RNA polymerase and sequenced using reverse transcriptase. Gobinda et al present data concçrning l actor IX for which they identified a conserved stretch of 20 nucleotides in the 3' 20 noncoding region of the gene.
Inverse PCR is the first method to report cllccessful acquisition of unknown sequences starting with primers based on a known region (Triglia T et al(1988) Nucleic Acids Res 16:8186). The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and 2s used as a PCR template. Divergent primers are clesi~n~d from the known region. The multiple row1ds of restriction enzyme digestions and ligations that are n~cess~ry prior to PCR
make the procedure slow and expensive (Gobinda et al, supra).
Captllre PCR (Lag~l~Llol.l M et al (1991) PCR Methods Applic 1 ~ 19) is a method for PCR amplification of DNA fr~gment.c adjacent to a known sequence in hwnan and YAC
:30 DNA. As noted by Gobinda et al (supra), capture PCR also requires multiple restriction CA 0223~7~ 1998-04-22 WO 97tlS5'~2 PCT/US96/16926 enzyme digestions and ligations to place an enginPered double-stranded sequence into an unknown portion of the DNA molecule before PCR. Although the restriction and ligation reactions are carlied out ~iml-lt~neously, the requirements for extension, immobilization and two rounds of PCR and purification prior to sequencing render the method cumbersome and time consuming.
Park~er JD et al (1991; Nucleic Acids Res 19:3055-60), teach walking PCR, a method for targeted gene walking which permits retrieval of unknown sequence. In this same vein, PromoterFi.nderTM a new kit available from Clontech (Palo Alto CA) uses PCR and primers derived from p53 to walk in genomic DNA. Nested pl;lll~lS and special PromoterFinder o libraries are used to detect U~SIl-,dlll sequences such as promoters and regulatory elements.
This proces;s avoids the need to screen libraries and is useful in finding intron/exon junctions.
Another new PCR method, "Improved Method for Obt~inin~ Full Ler~th cDNA
Seq~-.on~Pc" by Guegler et al, Patent Application Serial No 08/487,112, filed June 7, 1995 and hereby incorporated by reference, employs XL-PCRTM (Perkin-Elmer, Foster City CA) to amplify and extend partial nucleotide sequence into longer pieces of DNA. This method was developed to allow a single researcher to process multiple genes (up to 20 or more) at one time and to obtain an çxten-lecl (possibly full-length) sequence within 6-10 days. This new method replaces methods which use labeled probes to screen plasmid libraries and allow one lesealchel to process only about 3-5 genes in 14-40 days.
In the first step, which can be performed in about two days, any two of a plurality of primers are decign~d and synthesized based on a known partial sequence. In step 2, which takes about six to eight hours, the sequence is extended by PCR amplification of a selected library. Steps 3 and 4, which take about one day, are purification of the amplified cDNA and its ligation into an a~lo~l;ate vector Step 5, which takes about one day, involves transforming~ and growing up host b~ct~ri~ In step 6, which takes a~ illlately five hours, PCR is used to screen bacterial clones for extended sequence. The final steps, which take about one day, involve the ~ ~dlion and sequencing of selected clones.
If the full length cDNA has not been obtained, the entire procedure is repeated using either the original library or some other preferred library. The l,ref~.led library may be one that has been size-selectecl to include only larger cDNAs or may consist of single or CA 0223~7~ 1998-04-22 WO 97/155g2 PCT/US96/16926 combined collmlel~;ially available libraries, eg. lung, liver, heart and brain from Gibco/BRL
(Gaithersburg MD). The cDNA library may have been pl~al~d with oligo d(T) or random priming. R;andom primed libraries are ~,er~.l. d in that they will contain more sequences which contain 5' ends of genes. A randomly primed library may be particularly useful if an s oligo d(T) library does not yield a complete gene. It must be noted that the larger and more complex the protein, the less likely it is that the complete gene will be found in a single plasmid.
A new method for analyzing either the size or the nucleotide sequence of PCR
products is capillary electrophoresis. Systems for rapid sequencing are available from Perkin lo Elmer (Foster City CA), Bec~m~n Instru~nents (Fullerton CA), and other co~ ies Capillary sequencing employs flowable polymers for electrophoretic separation, four dirr~,ent fluolescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled devise camera. Outputllight intensity is converted to electr c~1 signal using a~plc.l";ate software (eg. Genot,vperTM and Sequence NavigatorTM from Perkin Elmer) and the entire process from loading of samples to conll)u analysis and electronic data display is co,nl)ul.,l controlled. Capillary ele~;l,ophoresis provides greater resolution and is many times faster than standard gel based procedures. It is particularly suited to the sequencing of small pieces of DNA which might be present in limited amo1mts in a particular sample. The reproducible sequencing of up to 350 bp of Ml 3 phage DNA in 30 min has been reported (Ruiz-Martinez MC et al (1993) Anal Chem 65:285 1 -8).
Another aspect of the subject invention is to provide for ncp hybridization probes which are capable of hybridizing with naturally occurring nucleotide sequences encoding NCP. The slringency of the hybridization conditions will determine whether the probe 2s identifies only the native nucleotide sequence of ncp or sequences of other closely related cysteine protease molecules. If degenerate ncp nucleotide sequences of the subject invention are used for lhe detection of related cysteine protease encoding sequences, they should preferably contain at least 50% of the nucleotides of the sequences presented herein.
Hybridization probes ofthe subject invention may be derived from the nucleotide sequences of the SEQ ID NO: 1 and 5-24 or from surrounding genomic sequences comprising WO 97/1~5"2 PCT/US96/16926 untr~n~l~tecl regions such as promoters, enh~ncers and introns. Such hybridization probes may be labeled with al)propl;ate reporter molecules.
Means for producing specific hybridization probes for cysteine proteases includeoligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
Alternatively, the cDNA sequence may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synth~si7~ RNA probes in vitro by addition of an al.~ro~l;ate RNA polymerase such as T7, T3 OI SP6 and labeled nucleotides. A number of companies (such as Ph~rm~ci~
Biotech, Pisca~w~y NJ; Promega, Madison WI; US Bioch~rnic~l Corp, Cleveland, OH; etc.) o supply commercial kits and protocols for these procedures.
It is ,also possible to produce a DNA sequence, or portions thereof, entirely bysynthetic ch,~mi~tTy. Sometimes the source of information for producing this sequence comes from the known homologous sequence from closely related org~ni~m~ After synthesis, the nucleic acid sequence can be used alone or joined with a pre-existing sequence and inserted into one of the many available DNA vectors and their respective host cells using techniques well known in the art. Moreover, synthetic ch~mi~try may be used to introduce specific mutations into the nucleotide sequence. Alternatively, a portion of sequence in which a mutation is desired can be synth~i7t?~i and recombined with a portion of an existing genomic or recombin;mt sequence.
The llCp nucleotide sequences can be used individually in a diagnostic test or assay to detect disorder or disease processes associated wit]h abnormal levels of ncp expression. The nucleotide sequence is added to a sample (fluid, cell or tissue) from a patient under hybridizing conditions. After an incubation period, the sample is washed with a compatible fluid which optionally contains a reporter molecule which will bind the specific nucleotide.
After the cornpatible fluid is rinsed off, the reporter molecule is quantitated and compared with a standard for that fluid, cell or tissue. If ncp ~ ,s~ion is significantly di~l~nl from the standard, the assay indicates the presence of disorder or disease. The form of such qualitative or qua ~ e methods may include northern analysis, dot blot or other membrane-b;lsed technologies, dip stick, pin or chip technologies, PCR, ELISAs or other multiple sample format technologies.

CA 0223~7~ 1998-04-22 WO 97/lS592 PCT/US96/16926 This same assay, combining a sample with the nucleotide sequence, is applicable in evaluating lhe efficacy of a particular the~ Lic treat~nent regime. It may be used in animal studies, in c:linical trials, or in monitoring the tre~tm~nt of an individual patient. First, standard expression must be established for use as a basis of comparison. Second, samples 5 from the ~nim~l~ or patients affected by a disorder or disease are combined with the nucleotide sequence to evaluate the deviation from the standard or normal profile. Third, an existing the.ayeulic agent is ~flminictered, and a llc;~ profile is generated. The assay is evaluated to determine whether the profile progresses toward or returns to the standard pattern. Successive tre~tm-?nt profiles may be used to show the efficacy of Ll.. ~ lr. ll over a 0 period of several days or several months.
The nucleotide sequence for ncp can also be used to generate probes for mapping the native genornic sequence. The sequence may be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques. These include in situ hybridization to chromosomal spreads (Verma et al (1988) Human Chromosomes: A Manual 5 of Basic T~ niques~ Pergamon Press, New York City), flow-sorted chromosomal yl~ydl~lions~ or artificial chromosome constructions such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P I constructions or single chromosome cDNA libraries.
In _L hybridization of chromosomal ple~,~dlions and physical mapping techniques 20 such as linkage analysis using established chromosomal markers are invaluable in extc n~ing genetic maps. Examples of genetic maps can be found in Science (1994; 265:1981f). Often the placeme nt of a gene on the chromosome of another m~mm~ n species may revealassociated markers even if the number or arm of a particular human chromosome is not known. New sequences can be ~sign~d to chromosomal arms, or parts thereof, by physical 25 mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once a disease or syndrome, such as ataxia telangiectasia (AT), has been crudely localized by genetic linkage to a particular genomic region, for example, AT to 1 lq22-23 (Gatti et al (1988) Nature 336:577-580), any sequences mapping to that area may represent associated or regulatory genes for 30 further investigation. The nucleotide sequence of the subject invention may also be used to CA 0223~7~ 1998-04-22 detect dir~ lces in the chromosomal location due to translocation, inversion, etc. between normal and carrier or affected individuals.
The nucleotide sequence encoding NCP may be used to produce an amino acid sequence using well known methods of recombinant DNA technology. Goeddel (1990, Gene Ex~ression T~hnr)lo~y. Methods ~nfl F.n7ymology~ Vol 185, Acad~rnic Press, San Diego CA) is one among many publications which teach ~p.es~ion of an isolated, purified nucleotide sequence. The amino acid or peptide may be expressed in a variety of host cells, either prokaryotic or eukaryotic. Host cells may be from the same species from which the nucleotide sequence was derived or from a different species. Advantages of producing an amino acid sequence or peptide by recombinant DNA technology include obtaining adequate amounts for purification and the availability of simplified purification procedures.
Cell s transformed with ncp nucleotide sequence may be cultured under conditionssuitable for the ~ cssion and recovery of peptide from cell culture. The peptide produced by a recombinant cell may be secreted or may be col,tah~ed intracellularly depending on the sequence and/or the vector used. In general, it is more convenient to prepare recombinant proteins in secreted form, and this is accomplished by lig~ting ncp to a recombinant nucleotide sequence which directs its movement through a particular prokaryotic or eukaryotic cell membrane. Other recombinant constructions may join ncp to nucleotide sequence encoding a polypeptide domain which will facilitate protein purification (Kroll DJ
et al (1993) :DNA Cell Biol 12:441-53).
Direct peptide synthesis using solid-phase techniques (Stewart et al (1969) Solid-Phase Pel~tide Synth~si~ WH Freeman Co, San Francisco CA; Merrifield J (1963) J
Am Chem Soc 85:2149-2154) is an alternative to recombinant or chimeric peptide production. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer in accordance with the instructions provided by the m~nnf~turer.
Additionally NCP or any part thereof may be mutated during direct synthesis and combined using chemical methods with other cysteine protease sequences or any part thereof.
Although an amino acid sequence or oligopeptide used for antibody induction doesnot require biological activity, it must be imrnunogenic. NCP used to induce specific antibodies may have an amino acid sequence con~i~ting of at least five arnino acids and CA 0223~7~ 1998-04-22 WO 97/15~92 PCT/US96/16926 preferably clt least 10 arnino acids. Short stretches of amino acid sequence may be fused with those of another protein such as keyhole limpet hemocyanin, and the chimeric peptide used for antibody production. Alternatively, the peptide may be of sufficient length to contain an entire ~lom~in s Antibodies specific for NCP may be produced by inoculation of an alJpn)pl;ate animal with an antigenic fragment of the peptide. An antibody is specific for NCP if it is produced against an epitope of the polypeptide and binds to at least part of the natural or recombinant protein. AIltibody production includes not only the stimulation of an i ..., ..l...e response by injection into ~nim~lc, but also analogous processes such as the production of synthetic o antibodies, the screening of recombinant imrnunoglobulin libraries for specific-binding molecules (Orlandi R et al (1989) PNAS 86:3833-3837, or Huse WD et al (1989) Science 256: 1275-1281), or the in vitro stim~ tion of Iymphocyte populations. Current technology (Winter G and Milstein C (1991) Nature 349:293-299) provides for a number of highly specific binding reagents based on the principles of antibody formation. These techniques may be ada~ted to produce molecules which specifically bind NCP. Antibodies or other app.opl;ate molecules generated against a specific immlmogenic peptide fragment or oligopeptide can be used in Western analysis, enzyme-linked imrnunosorbent assays (ELISA) or similar tests to establish the presence of or to quc~l~iLdLe amounts of NCP active in normal, rli~e~e~ or the,dl~eu~ically treated cells or tissues.
The exarnples below are provided to illustrate the subject invention. These examples are provided by way of illustration and are not included for the purpose of limiting the inventlon.

INDUSTRIAL APPLICABILITY
:~s I Adrenal Gland cDNA Library Construction Although both the adrenal gland and human umbilical vein endothelial cell cDNA
libraries were employed to clone the full length gene; for purposes of exarnple, the adrenal gland cDNA library construction will be described.
The adrenal gland cDNA library was constructed from a pooled sample of five, whole, 30 normal adrenal glands from C~ r<~ n males and females who r mged in age from 10 to 46 CA 0223~7~ 1998-04-22 years. The poly A+ RNA was obtained from Clontech Laboratories Inc (Catalogue #6571-2;
Palo Alto CA) Stratagene (La Jolla CA) made the cDNA library using this poly A+ RNA. The cDNA
synthesis was primed using both oligo d(T) and random hex~mers, and the two cDNAs libraries were treated separately. Synthetic adapter oligonucleotides were ligated onto the ends of the cDNAs enabling their insertion into the Uni-ZAPTM vector system (Stratagene).
The pBluescriptTM phagemid (Stratagene) cDNA clones were obtained by the in vivoexcision process, and phagemids from the two cDNA libraries were combined into a single library by mlixing equal numbers of bacteriophage. The latter were used to transform E. coli 0 host strain XLl-BlueTM (Stratagene). Enzymes from both pBluescript and a cotransformed fl helper phage nicked the DNA, initiated new DNA synthesis, and created the smaller, single-stranded circular phagemid DNA molecules which contained the cDNA insert. The phagemid DNA was released, purified, and used to reillfe~;l fresh host cells (SOLRTM, Stratagene). Presence of the ~3-la-;L~ ase gene on the phagemid allowed transformed bacteria to grow on rnedium CO~ g ampicillin.

II If D!-tion of cDNA Clones Phagemid DNAs cont~ining the cDNA insert may be purified using the QIAWELL-8TM Plasmid purification system from QIAGEN (Chatsworth CA). This high-throughput 20 method isolaLtes highly purified phagemid DNA from Iysed bacterial cells using QIAGEN
anion-ex~ e resin particles and EMPORETM membrane technology from 3M (Minneapolis MN) in a multiwell format. The DNA was eluted and prepared for DNA sequencing and other analytical manipulations.

2s III Sequencing of cDNA Clones The cDNA inserts from random isolates of the adrenal gland library were sequenced in part. Methods for DNA seqllen~ing are well known in the art and employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE~ (US Biochemical Corp) or Taqpolymerase. Methods to extend the DNA from an oligonucleotide primer annealed to the 30 DNA template of interest have been developed for both single- and double-stranded templates. Chain terrnin~tion reaction products were separated using electrophoresis and CA 0223~7~ 1998-04-22 WO 97/155~2 PCT/US96/16926 detected via their incorporated, labeled precursors. Recent improvements in mech~ni7.?d reaction l.lel.~dtion, sequencing and analysis have permittecl expansion in the number of sequences that can be determinP~I per day. Preferably, the process is ~ntom~ted with m~r.hinPs such as the Applied Biosystems Catalyst 800 and 373 DNA seq--en~ers The quality of any particular cDNA library may be ~l~t~ .. ine~1 by performing a pilot scale analysis of the cDNAs and çhP~ ~ing for p~ ges of clones co~ g vector, lambda or E . ~Qli DNA, mitochondrial or lel,~lilive DNA, and clones with exact or homologous m~tçhPs to public ~~t~b~es The number of unique sequences, those having no known match in any available rl~t~hace~ are then recorded.

IV Homology Searching of cDNA Clones and Their Deduced Pr~t~n~
Each sequence so obtained was compa~d to sequences in GlonR~nk using a search algorithm developed by Applied Biosystems and incorporated into the INHERIT~ 670Sequence Analysis System. In this algorithm, Pattern Specification Language (TRW Inc, Los s Angeles CA) was used to detçrrnine regions of homology. The three parameters that determine how the sequence co~llp~isons run were window size, window offset, and error tolerance. IJsing a combination of these three parameters, the DNA ~t~h~e w~ searched for sequences cont~ining regions of homology to the query sequence, and the a~ ;ate sequences were scored with an initial value. Subsequently, these homologous regions were 20 ex~min~d using dot matrix homology plots to distinguish regions of homology from chance matches. Smith-w~t~rm~n a1ignm~nt~ were used to display the results of the homology search.
Peptide and protein sequence homologies were ascertained using the INHERIT~ 670 Sequence Analysis System in a way similar to that used in DNA sequence homologies.
2~ Pattern Specification Language and parameter windows were used to search protein datab~es for sequences co~ ;..il.g regions of homology which were scored with an initial value. Dot-matrix homology plots were ex~min~l to distinguish regions of significant homology from chance m~tçhPs Altermatively, BLAST, which stands for Basic Local Alignment Search Tool, is used to search for local sequence ~1ignment~ (Altschul SF (1993) J Mol Evol 36:290-300;

CA 0223~7~ 1998-04-22 Wo97/155!~2 PCTAUS96/l6g26 Altschul, Sl~ et al (1990) J Mol Biol 215:403-10). BLAST produces ~ nment.c of both nucleotide and amino acid sequences to rlet~rminP sequence similarity. Because of the local nature of thl lignmPtlt~, BLAST is especially useful in determining exact matches or in identifying ]homologs. While it is useful for m~tçhes which do not contain gaps, it is s hla~plop~ial:e for performing motif-style searching. The filn-l~ment~l unit of BLAST
algorithm output is the High-scoring Segment Pair (HSP).
An HSP consists of two sequence fr~ment~ of ~billdl y but equal lengths whose ~lignmPnt is locally maximal and for which the ~lignmPnt score meets or çxcee~l~ a threshold or cutoff score set by the user. The BLAST approach is to look for HSPs between a query 0 sequence and a ~l~t~h~ce sequence, to evaluate the statistical significance of any m~tchP,s found, and to report only those m~t~l~Ps which satisfy the user-selected threshold of significance. The parameter E establishes the statistically significant threshold for reporting tl~t~h~e sequence m~t~.hPs E is interpreted as the upper bound ofthe expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire ~1~t~b~e search. Any 11~t~b~e sequence whose match satisfies E is reported in the program output.
All the partial ncp molecules presented and claimed in this application-- Incyte Clone 1098 (SEQ lD NO 3) from the U937 (a histiocytic Iymphoma cell line) library, Incyte Clone 75848 (SEQ ID NO 5) from the THP-l (a leukemic monocyte cell line) library, Incyte Clones 77015 (SEQ ID NO 6), 77424 (SEQ ID NO 7), 77645 (SEQ ID NO 8), 77651 (SEQ ID NO
9), and 78547 (SEQ ID NO 10) from the rheumatoid synovium library, Incyte Clone 104286 (SEQ ID NO 11) from the bone marrow library, Incyte Clone 115565 (SEQ ID NO 12) from the kidney library, Incyte Clones 125569 (SEQ ID NO 13) and 125830 (SEQ ID NO 14) from the lung libr~ry, Incyte Clones 158868 (SEQ ID NO 15) and 162199 (SEQ ID NO 16) from the inflamed adenoid library, Incyte Clones 172449 (SEQ ID NO 17) and 174690 (SEQ ID
2s NO 18) from the bone marrow library, Incyte Clones 180594 (SEQ ID NO 19) and 180935 (SEQ ID NO 20) from the placenta library, Incyte Clone 190299 (SEQ ID NO 21) from the rheumatoid synovium library, Incyte Clones 195541 (SEQ ID NO 22) and 197617 (SEQ ID
NO 23) from the kidney library, and Incyte Clone 238970 (SEQ ID NO 24) from the small intestine libn~ry--were identified using the criteria above. The full length nucleic and arnino acid sequencles for this novel human cysteine protease are shown in Fig 2. Fig 3 shows the CA 0223~7~ l998-04-22 WO 97tlS5g2 PCT/US96/16926 Ali~nmPnt between the tr~n~l~ted amino acid sequence for ncp and the closest related cysteine protease, hemoglobinase from Schict~som~ japonicum (GenBank Accession X70967;
Merckelbach A et al (1994) Trop Med Parasitol 45:193 198). As previously described, Fig 4 shows various parameters (hydrophilicity, etc) of the enzyme.

V Extension of cDNAs to Full Length The Incyte clones plescnled here can be and were used to design oligonucleotide primers for e xtension of the cDNAs to full length. Primers are desi~nPfl based on known sequence; one primer is syntheci7P~ to initiate extension in the ~ntiCçnce direction (XLR) and o the other to e xtend sequence in the sense direction (XLF). The primers allow the sequence to be extended "outward" generating amplicons cont~ining new, unknown nucleotide sequence for the gene of interest. The primers may be designPcl using Oligo 4.0(National Biosciences Inc, Plymouth MN), or another applol~l;ate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at tem~ Lures about 15 68 ~-72 ~ C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimeri_ations was avoided.
The adrenal cDNA library was used with XLR = GGT GAA TGA ACT GGT AGG
CAT GG and XLF = AAT CCC ACT CCA GGA ATT GTG ATC primers to extend and amplify Incyte Clone 100877. Using a second set of primers, XLR = ACC CAG ACT CAC
20 AGG CTT CAA TG and XLF = GGG GAC TGG TAC AGC GTC AAC TG and the HUVEC
cDNA library, Incyte Clone 66931 was extended to obtain the rem~ining portion of the cysteine protease sequence.
By following the instructions for the XL-PCR kit and thoroughly mixing the enzyme and reaction mix, high fidelity amplification is obtained. Beginning with 40 pmol of each 25 primer and the recommended concentrations of all other components of the kit, PCR is performed using the Peltier Thermal Cycler (PTC200; MJ Research, Watertown MA) and the following parameters:
Step 1 94~ C for 1 min (initial denaturation) Step 2 65~ C for 1 min :30 Step 3 6~~ C for 6 min Step 4 94~ C for 15 sec Step 5 65~ C for 1 min CA 0223~7~ 1998-04-22 WO 97/1~59:2 PCT/US96/16926 Step 6 68 ~ C for 7 min Step 7 Repeat step 4-6 for 15 additional cycles Step 8 94~ C for 15 sec Step 9 65 ~ C for 1 min Step 10 68~ C for 7:15 min Step 11 Repeat step 8-10 for 1 2 cycles Step 12 72~ C for 8 min Step 13 4~ C (and holding) o A 5- l O ~l aliquot of the reaction mixture is analyzed by electrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gel to ~et~nninP which reactions were sllccç~sful in exten-linf~ the sequence. Although all extensions potentially contain a full length gene, some of the largest products or bands are selected and cut out of the gel. Further purification iinvolves using a CO~ cial gel extraction method such as QIAQuickTM(QIAGEN Inc). After recovery of the DNA, Klenow enzyme is used to trim single-stranded, nucleotide overhangs creating blunt ends which facilitate religation and cloning.
After ethanol ~lecip;l~lion, the products are redissolved in 13 ,~1 of ligation buffer.
Then, l,ul T4-DNA ligase (15 units) and 1~1 T4 polynucleotide kinase are added, and the mixture is incubated at room telllp~ldLul~e for 2-3 hours or overnight at 16~ C. Competent E.
coli cells (in 40 ,ul of appl~p~;ate media) are transformed with 3 ,~1 of ligation mixture and cultured in 80 ,ul of SOC mediurn (Sambrook J et al, supra). After incubation for one hour at 37~ C, the whole transformation mixture is plated on Luria Bertani (LB)-agar (Sambrook J et al, supra) co~ g 2x Carb. The following day, 12 colonies are randomly picked from each plate and cultured in 150 ,ul of liquid LB/2x Carb medium placed in an individual well of an :z5 ~plopl;ate, commercially-available, sterile 96-well microtiter plate. The following day, 5 ,ul of each ovemight culture is transferred into a non-sterile 96-well plate and after dilution 1:10 with water, 5 ,ul of each sample is transferred into a PCR array.
For PCR amplification, 18 ,ul of concentrated PCR reaction mix (3.3x) cont~ining 4 units of rTth DNA polymerase, a vector primer and one or both of the gene specific primers :30 used for the extension reaction are added to each well. Amplification is performed using the following conditions:
Step 1 94~ C for 60 sec Step 2 94 ~ C for 20 sec Step 3 55~ C for 30 sec CA 0223F,.77., 1998 - 04 - 22 WO 9711555~2 PCT/US96/16926 Step 4 72~ C for 90 sec Step 5 Repeat steps 2-4 for an additional 29 cycles Step 6 72 ~ C for 180 sec Step 7 4~ C (and holding) Aliquots of the PCR reactions are run on agarose gels together with molecular weight markers. The sizes of the PCR products are con~ed to the original partial cDNAs, and a~lol~liate clones are selected, ligated into plasmid and sequenced.

VI Diagnostic Assay Using NCP Specific Oligomers o In those cases where a specific condition (see definitions supra) is suspected to involve altered quantities of ncp, oligomers may be design~oA to esf~ h the presence and/or quantity of mRNA expressed in a biological sample. There are several methods ~ cnlly being used to quantitate the expression of a particular molecule. Most of these methods use radiolabeled (Melby PC et al 1993 J Immunol Methods 159:23544) or biotinylated (Duplaa C et al 1993 Anal Biochem 229-36) nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimPnt~l results are interpolated. Qu~~ ion may be spee~le~ up by running the assay in an ELISA format where the oligomer-of-interest is presented in various dilutions and a colorimetric response gives rapid quantitation. For example, NC'P deficiency may result in an abundance ofthe proinfl~mm~tory interleukin molecules, rmuch swelling and discomfort. In like manner, o~lc~ es~ion may causeapoptosis and major tissue damage. In either case, a quick diagnosis may allow health professionals to treat the condition and prevent worsening of the condition. This same assay can be used lo monitor progress of the patient as his/her physiological situation moves toward the normal rcmge during therapy.
VII Sense or Antisense Molecules Knowledge of the correct cDNA sequence of this novel cysteine protease or its regulatory elements enable its use as a tool in sense (Youssoufian H and HF Lodish 1993) Mol Cell Biol 13:98-104) or ~nti.~en~e (Eguchi et al (1991) Annu Rev Biochem 60:631-652) :30 technologies for the investigation or alteration of gene e~les~ion. To inhibit in vivo or in vitro ncp expression, an oligonucleotide based on the coding sequence of a fragment of an CA 0223~7~ 1998-04-22 WO 97/155~2 PCT/US96116926 ncp cle~ign~(l using Oligo 4.0 (National Biosciences Inc) may be used. Alternatively, a fragment of an ncp produced by digesting ncp coding sequence with restriction enzymes selected to digest the ncp at specific restriction sites using Inherit Analysis software (Applied Biosystems ) may be used to inhibit ncp e~ ssion. Furthermore, ~nti~n~e molecules can be s ~lesign~cl to inhibit promoter binding in the u~sl~ nontr~n~l~tecl leader or at various sites along the ncp coding region. Alternatively, ~nti~n~e molecules may be design~ to inhibit translation of an mRNA into polypeptide by pl~d~hlg an oligomer or fragment which will bind in the region sp~nning a~l.roxil,lately -10 to +10 nucleotides at the 5' end of the coding sequence. These technologies are now well known in the art.
o In addition to using fr~gm~ntc constructed to interrupt transcription of the open reading frame, modifications of gene ~ression can be obtained by clecigning ~ntic~n~e sequences to enh~nrers, introns, or ever~ to trans-acting regulatory genes. Similarly, inhibition can be achieved using Hogeboom base-pairing methodology, also known as "triple helix" base pairing. Triple helix pairing co~plu~.ises the ability of the double helix to open sufficiently for the binding of polymerases, lldils~ Jtion factors, or regulatory molecules.
Any of these types of ~nticen~e molecules may be placed in e~ession vectors and used to transform pl~lled cells or tissues. This may include introduction of the ~lession vector into a synovial cavity for transient or short term therapy. Expression of the ~nti~t?n~e sequence wc,uld continue to flood the cell with inhibitory molecules until all copies of the vector were disabled by endogenous nucleases. Such transient e~lession may last for a month or more with a non replicating vector and three months or more if apprûpliate replication elements are used in the transformation or expression system.
Stable transformation of a~,opllate dividing cells with a vector co.~ g the ~nti~n~e molecule can produce a ~ sgellic cell line, tissue or organism (see, for example, Trends in Biotechnol 1 1:155-215 (1993) and US Patent No. 4,736,866, 12 April 1988).
Those cells ~;vhich ~c~imil~te or replicate enough copies of the vector to allow stable integration will also produce enough ~nti~n.~e molecules to co,..plo",ise or entirely elimin~te normal activity of the ncp. Frequently, the function of an ncp can be ascertained by observing behaviors such as lethality, loss of a physiological pathway, changes in 30 morphûlogy, etc. at the cellular, tissue or orp;~ni~m~l level.

CA 0223~7~ 1998-04-22 VIII Expression of NCP
Expression of the NCP may be accomplished by subcloning the cDNAs into a~lo~.;ate vectors and transfecting the vectors into host cells In this case, the cloning vector previously used for the generation of the tissue librar,v also provides for direct e~l,res~ion of the ncp sequence in E. ~Qli U~ n of the cloning site, this vector contains a promoter for 13-galactosidase, followed by sequence cont~ining the amino-terminal Met and the subsequent 7 residues of J~-galactosidase. Immediately following these eight residues is a bacteriophage promoter useful for transcription and a linker contAining a number of unique o restriction sites.
Induction of an isolated, transfected bacterial strain with IPTG using standard methods will produce a fusion protein corresponding to the first seven residues of ~-galactosidase, about 5 to 15 residues which correspond to linker, and the peptide encoded within the ncp cDNA. Since cDNA clone inserts are generated by an escçnti~lly random s process, there is one chance in three that the included cDNA will lie in the correct frarne for proper translation. If the cDNA is not in the proper reading frame, it can be obtained by deletion or insertion of the applopl;ate nurnber of bases by well known methods including in vitro mutagenesis, digestion with exonuclease III or mung bean nuclease, or oligonucleotide linker inclusion.
The cDNA can be shuttled into other vectors known to be useful for ~ e;,~ion of protein in specific hosts. Oligonucleotide linkers cont~ining cloning sites as well as a stretch of DNA sufficient to hybridize to the end of the target cDNA (25 bases) can be synthP~i7ed chemically by standard methods. These primers can then used to arnplify the desired gene frAgment~ by PCR. The resulting fragments can be digested with al,plol),;ate restriction enzymes under standard conditions and isolated by gel electrophoresis. ~ltern~tively, similar gene fr~gment~ can be produced by digestion of the cDNA with a~plopl;ate restriction enz,vmes and filling in the mi~sing gene sequence with chemically synthesized oligonucleotides. Partial nucleotide sequence from more than one cysteine protease homolog can be ligated together and cloned into applopl;ate vectors to optimize expression.
Suitable expression hosts for such chimeric molecules include but are not limited to CA 0223~7~ 1998-04-22 WO 97/1559:2 PCT/US96/16926 m~mm~ n cells such as Chinese Hamster Ovary (CHO) and human 293 cells, insect cells such as Sfg cells, yeast cells such as Sacch~romyces cerevisiae, and bacteria such as E. coli.
For each of these cell systems, a useful expression vector may also include an origin of replication to allow propagation in bacteria and a selectable marker such as the J3-lactamase antibiotic reCict~nre gene to allow selection in bacteria. In addition, the vectors may include a second selectable marker such as the neomycin phosphotransferase gene to allow selection in transfected eukaryotic host cells. Vectors for use in eukaryotic tA~,~,ssion hosts may require RNA proces.cing elements such as 3' polyadenylation sequences if such are not part of the cDNA of interest.
0 If native promoters are not part of the cDNA, other host specific promoters may be specifically c ombined witn the coding region of ncp. They include MMTV, SV40, and metallothionine promoters for CHO cells; trp, lac, tac and T7 promoters for bacterial hosts;
and alpha factor, alcohol oxidase and PGH promoters for yeast. In addition, transcription enh~nrers, such as the rous sarcoma virus (RSV) enhancer, may be used in m~mm~ n host cells. Once homogeneous cultures of recombinant cells are obtained through standard culture methods, large quantities of recombinantly produced peptide can be recovered from the conditioned medium and analyzed using methods known in the art.

IX Isolation of Recombinant NCP
~o NCP may be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification. Such purification facilitating domains include, but are not limited to, metal chel~ting peptides such as histidine- tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS,~5 extension/affinity purification system (Immunex Corp, Seattle WA). The inclusion of a cleavable linker sequence such as Factor XA or enterokinase (Invitrogen) between the purification domain and the ncp sequence may be useful to facilitate ex~leSSiOn of NCP.

X NCP Activib 3,0 The activity of purified or expressed NCP may be tested by mixing a known quantity CA 0223~7~ 1998-04-22 of the enzyme with a proteinaceous matrix material (such as collagen) in a biologically acceptable medium and allowing NCP to carry out digestion for an al)plol"iate period of time. A zymogram, which consists of a non~le~ g polyacrylamide gel soaked in theploteinaceous material onto which various concentrations, preferably between 10 and 100 ng/~l, of NCP are spotted, may be used to demonstrate NCP activity. After 30-60 min, the gel is stained with Coomassie blue. An active enzyme will create spots in which the concentration of protein has been reduced (lighter stain) or completely cleared (Paech et al (1993) Anal Biochem 208:249-54).

o XI Identification of or Prodr-~t;on of NCP Specific Antibodies Purified NCP is used to screen a pre-existing antibody library or to raise antibodies using either polyclonal or monoclonal methodology. In a polyclonal approach, denatured protein from the reverse phase HPLC separation is obtained in quantities up to 75 mg. This denatured protein can be used to i~ lunize mice or rabbits using standard protocols; about 100 micrograms are adequate for immunization of a mouse, while up to 1 mg might be used to i,.""~ , a rabbit. For identifying mouse hybridomas, the denatured protein can be radioiodinaled and used to screen potential murine B-cell hybridomas for those which produce antibody. This procedure requires only small quantities of protein, such that 20 mg would be sufficient for labeling and screening of several thousand clones.
In a monoclonal approach, the amino acid sequence of NCP, as ~leduced from translation of the cDNA, is analyzed to detçnnine regions of high immunogenicity.
Oligopeptides comprising a~l,ropl;ate hydrophilic regions, as shown in Fig. 3, are synthesi7~d and used in suitable immunization protocols to raise antibodies. Analysis to select al~prop1;ate epitopes is described by Ausubel FM et al (supra). The optimal amino acid sequences for immunization are usually at the C-tçnnin-l~, the N-tçrrninns and those intervening, hydrophilic regions of the polypeptide which are likely to be exposed to the ext~rn~l environrnent when the protein is in its natural conformation.
Typically, selected peptides, about 15 residues in length, are synthPci7Pd using an Applied Biosystems Peptide Synth~si~r Model 431A using fmoc-ch~ try and coupled to keyhole limpet hemocyanin (KLH, Sigma) by reaction with M-maleimidobenzoyl- N-WO 97/15592 PCTtUS96/16926 hydroxysuccinimide ester (MBS; Ausubel FM et al, supra). If nt ces.e~ry, a cysteine may be introduced at the N-terrninl-c of the peptide to permit coupling to KLH. Rabbits are .-i7Pd with the peptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for anlip~lide activity by binding the peptide to plastic, blocking with 1%
BSA, reacting with antisera, washing and reacting with labeled (radioactive or fluorescent), affinity purified, specific goat anti-rabbit IgG.
Hyb]idomas may also be l,repdled and screened using standard techniques.
Hybridomas of interest are detected by screening with labeled NCP to identify those fusions producing the monoclonal antibody with the desired specificity. In a typical protocol, wells o of plates (FAST; Becton-Dickinson, Palo Alto, CA) are coated with affinity purified, specific rabbit-anti-mouse antibodies (or suitable anti-species Ig) at 10 mg/ml. The coated wells are blocked with 1% BSA, washed and exposed to supernatants from hybridomas. After inrnb~tion the wells are exposed to labeled NCP, 1 mg/ml. Clones producing antibodies will bind a quantity of labeled NCP which is ~letect~ble above background. Such clones are exl ~n~l~d and subjected to 2 cycles of cloning at limi~ing dilution (1 cell/3 wells). Cloned hybridomas are injected into pristine mice to produce ascites, and monoclonal antibody is purified from mouse ascitic fluid by affinity chromatography on Protein A. Monoclonal antibodies with affinities of at leact 108 /M, preferably 109 to 10~~ or stronger, will typically be made by standard procedures as described in Harlow and Lane (1988) Antibodies: A
Laboratory ~nll~l, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, and in Goding (1986) Monoclonal ~ntibodies: Principles and Practice. ~-~ad~n ic Press, New York City, both incorporated herein by reference.

XII Diagnostic Test Using NCP Specific Antibodies .2s Particular NCP antibodies are useful for the diagnosis of prepathologic conditions, and chronic or acute ~li.ce~cec which are characterized by differences in the arnount or distribution of NCP. To date, NCP has been found in many libraries where it is predomin~ntly associated with organ function, infl~mm~tion or defense.
Diagnostic tests for NCP include methods utili7.ing the antibody and a label to detect ~;o NCP in human body fluids, tissues or extracts of such tissues. The polypeptides and CA 0223~7~ 1998-04-22 WO 97/15592 PCTtUS96/16926 antibodies of the present invention may be used with or without modification. Frequently, the polypeptides and antibodies will be labeled by joining them, either covalently or noncovalently, with a reporter molecule. A wide variety of labels and conjugation techniques are known and have been reported extensively in both the scientific and patent literature.
s Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemil-lminf~scent, or chromogenic agents previously mentioned as well as substrates, cofactors, inhibitors, m~gn.otic particles and the like. Patents tea~l-ing the use of such labels include US PatentNos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149, and 4,366,241. Also, recombinant h~ oglobulins may be produced as shown in US Patent o No. 4,816,567, incorporated herein by rcfe~,ce.
A variety of protocols for measuring soluble or membrane-bound NCP, using eitherpolyclonal or monoclonal antibodies specific for the respective protein are known in the art.
Examples include enzyme-linked immllnQsorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site, monoclonal-based immlmo~ee~y lltili7ing monoclonal antibodies reactive to two non-interfering epitopes on NCP is prefc.l~d, but a co~ ilive binding assay may be employed. These assays are described, among other places, in Maddox, DE et al (1983, J Exp Med 158:1211).

XIII Purification of Native NCP Using Specific Antibodies :~o Native or recombinant NCP can be purified by immunoaffinity chromatography using antibodies specific for that particular NCP. In general, an immunoaffinity column is constructed by covalently coupling the anti-NCP antibody to an activated chromatographic resin.
Polyclonal immunoglobulins are p.epaled from immune sera either by precipitation,!5 with ammonium sulfate or by purification on immobili~d Protein A (Pharmacia Biotech).
Like~vise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate pl~ci~ lion or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently at~ h.od to a chromatographic resin such as CnBr-activated Sepharose (Pharmacia Biotech). The antibody is coupled to the resin, the resin is blocked, ~;o and the derivative resin is washed according to the m~nllf~ctllrer's instructions.

CA 0223~7~ 1998-04-22 Such immnno~ffinity columns may be utilized in the purification of NCP by ~ p~illg a fraction from cells cont~ining NCP in a soluble form. This plelJdldlion may be derived by solubilization of whole cells or of a subcellular fraction obtained via differential centrifugation (with or without addition of detergent) or by other methods well known in the 5 art. ~ltem~tively, soluble NCP co~ a signal sequence may be secreted in useful quantity into the medium in which the cells are grown.
A soluble NCP-co..~ -g ~l~a,dLion is passed over the irnm-lno~ffinity column, and the column is washed under conditions that allow the preferential absorbance of NCP (eg, high ionic strength buffers in the presence of detergent). Then, the column is eluted under 0 conditions that disrupt antibodylNCP binding (eg, a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion), and NCP is collected.

XIV Drug Screening This invention is particularly useful for screening thel~eulic compounds by using 5 binding fr~gn-~ntc of NCP in any of a variety of drug scr~ g techniques. The peptide fragment employed in such a test may either be free in solution, affixed to a solid support, borne on a ce.ll surface or located intracellularly. One may measure, for example, the formation of complexes between NCP and the agent being tested. Alternatively, one can ex~min~ the ~l;".;~ ;on in complex formation between NCP and a receptor caused by the 20 agent being tested.
Methods of s- leenil1g for drugs or any other agents which can affect macrophageactivation comprise cont~ting such an agent with NCP fragment and assaying for the presence of a complex between the agent and the NCP fr~gm~nt In such assays, the NCP
fragment is typically labeled. After suitable incllb~tion, free NCP fragment is sep~dl~d from 25 that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to NCP.
Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the NCP polypeptides and is described in detail in Eulol~eall Patent Application 84/035~4, published on September 13, 1984, 30 incorporated herein by reference. Briefly stated, large numbers of dirr."el~ small peptide test CA 0223~7~ 1998-04-22 WO 97/155~2 PCT/US96/16926 compounds are synth.o~i7~d on a solid substrate, such as plastic pins or some other surface.
The peptide test compounds are reacted with NCP fragment and washed. Bound NCP
fragment is then ~letectçrl by methods well known in the art. Purified NCP can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support.
This invention also contemplates the use of competitive drug screening assays inwhich neutralizing antibodies capable of binding NCP specifically compete with a test compound for binding to NCP fr~mPntc In this manner, the antibodies can be used to detect o the presence of any peptide which shares one or more antigenic determin~nt~ with NCP.

XV Identification of Molecules Which Interact with NCP
The inventive purified NCP is a research tool for identification, characterization and purification of interacting molecules. Appropriate labels are incorporated into NCP by various methods known in the art and NCP is used to capture soluble or interact with membrane-bound molecules. A ~,cLll~d method involves labeling the primary amino groups in NCP with '25I Bolton-Hunter reagent (Bolton, AE and Hunter, WM (1973) Biochem J 133: 529). This reagent has been used to label various molecules without concomitant loss of biological activity (Hebert CA et al (1991 ) J Biol Chem 266: 18989-94;
:20 McColl S et al (1993) J Immunol 150:4550-4555). Membrane-bound molecules areincubated with the labeled NCP molecules, washed to removed unbound molecules, and the NCP complex is quantified. Data obtained using dir~lel~ concentrations of NCP are used to calculate values for the number, affinity, and association of NCP.
Labeled NCP fr~gml-.nt~ are also useful as a reagent for the purification of molecules .!5 with which NCP interacts, specifically including inhibitors. In one embodiment of affinity purification, NCP is covalently coupled to a chromatography column. Cells and their membranes are extracted, NCP is removed and various NCP-free subcomponents are passed over the colurnn. Molecules bind to the column by virtue of their NCP affinity. The NCP-complex is recovered from the column, dissociated and the recovered molecule is subjected to 3,0 N-t-qnnin~l protein sequencing or other identification procedure. If the captured molecule has CA 0223~7~ 1998-04-22 WO 97/1~592 PCT/US96/16926 an amino acid sequence, it can be used to design degenerate oligomers for use in cloning the gene from an appropl;ate cDNA library.
In an alternate method, monoclonal antibodies raised against NCP fr~gmPntc are screened to identify those which inhibit the binding of labeled NCP. These monoclonal 5 antibodies are then used in affinity purification or expression cloning of associated moiecules.
Other soluble binding molecules are identified in a similar manner. Labeled NCP is inc~bat~d ~ith extracts or other a~ropliate m~t-ori~lc derived from lung, kidney or other tissues with activated monocytes or macro phages. After incubation, NCP complexes (which are larger than the lone NCP fragment) are identified by a sizing technique such as size o exclusion chromatography or density gradient centrifugation and are purified by methods known in the art. The soluble binding protein(s) are subjected to N-termin~l sequencing to obtain inforrnation sufficient for ~l~t~b~e identification, if the soluble protein is known, or for cloning, if the soluble protein is unknown.

s XVI Use and Administration of Anti~ or IDhibitors to NCP
The antibodies and inhibitors can provide different effects when ~lmini~tered therapeutically. The antibodies and inhibitors are used to lessen or elimin~te undue damage caused by disorders or ~ e~eC associated with upregulated NCP e~es~ion. Each of these molecules or treatments (TSTs) will be formulated in a nontoxic, inert, ph~rm~eutically 20 acceptable aqueous carrier medium preferably at a pH of about 5 to 8, more preferably 6 to 8, although the pH may vary according to the different characteristics of the peptide, antibody or inhibitor beimg fonn~ t~l and the condition to be treated. Characteristics of TSTs include solubility of the molecule, half-life, antigenicity/irnmunogenicity and the ability of the inhibitor to reach its target(s). These and other characteristics may aid in dçfining an 25 effective carrier. Native human proteins are preferred as TSTs, but recombinant peptides as well as organic or synthetic molecules resulting from drug screens may be equally effective in particular situations.
TSTs may be delivered by known routes of ~mini~tration including but not limited to topical creams and gels; transmucosal spray and aerosol; tr~n~derm~l patch and bandage;
30 injectable, intravenous and lavage formulations; and orally atlrnini~tPred liquids and pills CA 0223~7~ 1998-04-22 WO 971155~2 PCT/US96/16926 particularly form~ tecl to resist stomach acid and enzymes. The particular formulation, exact dosage, and route of ~lmini.~tration will be determined by the ~ttçnrling physician and will vary according to each specific situation.
Such detçrmin~tions are made by considering multiple variables such as the condition 5 to be treated, the TST to be ~lmini~tered, and the pharmacokinetic profile of the particular TST. Additional factors which may be taken into account include disease state (eg. severity) of the patient, age, weight, gender, diet, time and frequency of ~imini~tration~ drug combination, reaction sensitivities, and tolerance/response to therapy. Long acting TST
forrnulations might be ~mini~tçred every 3 to 4 days, every week, or once every two weeks l o depending OII half-life and clearance rate of the particular TST.
Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of ~rlmini~tration. Guidance as to particular dosages and methods of delivery is provided in the literature. See US Patent No. 4,657,760;
5,206,344; or 5,225,212. Those skilled in the art will employ different formulations for 5 different TS1 s. A~lminictration to lung cells may necessitate delivery in a manner di~~ t from that to kidney or other cells.
It is contemplated that conditions associated with altered NCP expression are treatable with TSTs. l hese conditions, which specifically include, but are not limited to, ~nemi~
arteriosclerosis, ~cthm~ bronchitis, emphysema, gingivitis, infl~mm~tory bowel disease, 20 insulin-dependent diabetes mellitus, lellkemi~ multiple endocrine neoplasias, osteoarthritis, osteoporosis, pulmonary fibrosis, rheumatoid arthritis, septic shock syndromes, and systemic lupus erythPm~tosus may be specifically diagnosed by the tests discussed above. In addition, such tests may be used to monitor tre~,tm~nt All publications and patents mentioned in the above specification are herein 25 incorporated by reference. Various modifications and variations of the described method and system of the invention will be ap~ent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of 30 the above-deseribed modes for carrying out the invention which are obvious to those skilled WO 97/15~;92 PCT/US96116926 in the field of molecular biology or related fields are int~nflecl to be within the scope of the following cklims.

CA 0223~7~ l998-04-22 W O 97/15592 PCTrUS96/16926 SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: INCYTE PHARMACEUTICALS, INC.
(ii) TITLE OF INVENTION: NOVEL HUMAN CYSTEINE PROTEASE
(iii) NUMBER OF SEQUENCES: 24 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: INCYTE PHARMACEUTICALS, INC.
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(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) PCT APPLICATION NUMBER: To Be Assigned (B) FILING DATE: Herewith (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/005,799 (B) FILING DATE: 23-OCT-1995 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Billings, Lucy J.
(B) REGISTRATION NUMBER: 36,749 (C) REFERENCE/DOCKET NUMBER: PF-0048 PCT
(ix) TELECOMMUNICATION INFORMATION:
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CA 0223~7~ 1998-04-22 AATTCGGCA(' GAGGCCTGCC ACAGGTGTCT GCAATTGAAC TCCAAGGTGC AGAATGGTTT 60 TTTATTTCA(, TGACCATGGA TCTTCTGGAA TACTGGTTTT CCCCAATGAA GATCTTCATG 540 ATGTTTATG(' AACTACTGCT GCCAACCCCA GAGAGTCGTC CTACGCCTGT TACTATGATG 720 TTCAGGGTAl' GAAACGCAAA GCCAGTTCTC CCGTCCCCCT ACCTCCAGTC ACACACCTTG 960 ACCTCACCC(' CAGCCCTGAT GTGCCTCTCA CCATCATGAA AAGGAAACTG ATGAACACCA 1020 ACCTCATCC(; AGGTGAGGTG GAGCAGCTCC TGTCCGAGAG AGCCCCGCTC ACGGGGCACA 1140 GCTGCTACC(' AGAGGTCCTG TTGTACTTCC GGACCCACTG CTTCAACTGG TACTCCCCCA 1200 TCCACTTCA(' AGGATATAAT TGTCCATGGC CCACGTGTGC CTTGGTCACT ACTGAAGAGC 1320 TGCCTCCTG(; AAGCTTTTCC CAAGTGTGAG CGCCCCCACC GGCTGTGTTC TTGATCAAGA 1380 AAAAAATAAA GATTTATAAC AGAACCCCAA AAAAAAAAAA A}~u~AAA AAAAA 1855 CA 0223~7~ l998-04-22 (2) INFORMATION FOR SEQ ID NO:2:
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Met Val Trp Lys Val Val Val Phe Leu Ser Val Ala Leu Gly Ile Gly ~la Val Pro Ile Asp Asp Pro Glu Asp Gly Gly Lys His Trp Val Val Ile Val Ala Gly Ser Asn Gly Trp Tyr Asn Tyr Arg His Gln Ala Asp Ala Cys His Ala Tyr Gln Phe Ile His Arg Asn Gly Ile Pro Ala Glu Gln Ile Val Val Ile Met Tyr Asp Asp Ile Ala Tyr Ser Glu Asp Asn ~ro Thr Pr~ Gly Ile Val Ile Asn Arg Pro Asn Gly Thr Asp Val Tyr ~ln Gly Val Pro Lys Asp Tyr Thr Gly Glu Asp Val Thr Pro Gln Asn ~he Leu Val Val Leu Arg Gly Asp Ala Glu Ala Val Lys Gly Ile Gly Ser Arg Lys Val Leu Lys Ser Gly Pro Gln Asp His Val Phe Ile Tyr Phe Thr Asp His Gly Ser Ser Gly Ile Leu Val Phe Pro Asn Glu Asp ~eu His Val Lys Asp Leu Ile Lys Thr Thr His Tyr Ile Phe Lys Asn ~ys Met Ty:r Arg Lys Met Val Phe Tyr Ile Glu Ala Cys Glu Ser Gly ~er Met Met Asn His Leu Pro Asp Asn Ile Asn Val Tyr Ala Thr Thr 19.~ 200 205 Ala Ala Asn Pro Arg Glu Ser Ser Tyr Ala Cys Tyr Tyr Asp Glu Lys Arg Ser Th:r Tyr Leu Gly Asp Trp Tyr Ser Val Asn Trp Met Glu Asp ~er Asp Va:L Glu Asp Leu Thr Lys Glu Thr Leu His Lys Gln Tyr His ~eu Val Lys Ser His Thr Asn Thr Ser His Val Met Gln Tyr Gly Asn CA 0223~7~ l998-04-22 WO 97/lSSg2 PCT/US96/16926 Lys Thr Ile Ser Thr Met Lys Val Met Gln Phe Gln Gly Met Lys Arg Lys Ala Ser Ser Pro Val Pro Leu Pro Pro Val Thr His Leu Asp Leu Thr Pro Ser Pro Asp Val Pro Leu Thr Ile Met Lys Arg Lys Leu Met ~sn Thr Asn Asp Leu Glu Glu Ser Arg Gln Leu Thr Glu Glu Ile Gln ~rg Tyr Leu Asp Ala Arg His Leu Ile Arg Gly Glu Val Glu Gln Leu Leu Ser Glu Arg Ala Pro Leu Thr Gly His Ser Cys Tyr Pro Glu Val Leu Leu Tyr Phe Arg Thr His Cys Phe Asn Trp Tyr Ser Pro Thr Tyr Glu Leu Cys Val Glu Thr Phe Cys Thr Cys Trp Ser Thr Phe Val Arg ~rg Arg Phe His Phe Thr Gly Tyr Asn Cys Pro Trp Pro Thr Cys Ala ~eu Val Thr Thr Glu Glu Leu Pro Pro Gly Ser Phe Ser Gln Val ~2) INFORMATION FOR SEQ ID NO:3:
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GAAACAAAAC' AATCTCCACC ATGAAAGTNA TGCAGTTTCA GGGTATGAAA CGCAAAGCCA 120 GTTCTCCCGT CCCCCTACCT TCAGTCACAC ACCTTGACCT CACCCCCAGC CCTGATGTGC l80 CTTTACGGTC; GTT 253 (2) INFOR~IATION FOR SEQ ID NO:l0:
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AAGAGGTCCP. CGTACCTGGG GGACTGGTAC AGCGTCAACT GGATGGAAGA CTCGGACGTG 120 GAAGATCTGP. CTAAAGAGAC CCTGCACAAG CAGTACCACC TGGTAAAATC GCACACCAAC 180 (2) INFORMATION FOR SEQ ID NO:12:
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CATNTTCTCC' CGTTCNACT 199 (2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 52 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) ~IOLECULE TYPE: cDNA
(vii) I:MMEDIATE SOURCE:
(A) LIBRARY: Placenta (B) CLONE: 180599 (xi) ';EQUENCE DESCRIPTION: SEQ ID NO:19:

(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: Placenta (B) CLONE: 180935 (xi) CEQUENCE DESCRIPTION: SEQ ID NO:20:

(2) INFOR~ATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 234 base pairs CA 0223~7~ l998-04-22 tB) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: Rheumatoid Synovium (B) CLONE: 190299 (xi) ';EQUENCE DESCRIPTION: SEQ ID NO:21:
AGCAGCTCCI' GTCCGANAGA GCCCCGCTCA CGGGGCACAG CTGCTACCCA GAGGCCCTGC 60 ATTTGTACGI' GCTGGTCAAC CTTTGTNAGA AGCCGTATCC GCTTCANAGG ATAAAATTGT 180 CCATGGACCP, CGTGTGCCTT GGTCACTACT GAAGAGCTGC CTCCTGGAAG CTTT 239 (2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 206 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) M:OLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: Kidney (B) CLONE: 195541 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 181 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:
(A) LIBRARY: Kidney (B) CLONE: 197617 CA 0223~7~ l998-04-22 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

(2) INFORI~ATION FOR SEQ ID NO:24:
(i) 'iEQVENCE CHARACTERISTICS:
(A) LENGTH: 212 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: cDNA
(vii) :.MMEDIATE SOURCE:
(A) LIBRARY: Small Intestine (B) CLONE: 238970 (xi) ';EQUENCE DESCRIPTION: SEQ ID NO:24:
ACTACTGAA(; AGCTGCCTCC TGGAAGCTTT TCCAAGTGTG AGCGCCCCAC CGACTGTTTG 60 CTGATCAGAC, ACTGGAGAGG TNGAGTNAGA AGTCTCCGCT GCTCGNGCCC TCCTGGGGAG 120 GCTTCCCCAC, TCTTTTCTGA AAAACTA('AA AA 212

Claims (20)

1. A substantially purified novel cysteine protease (NCP) comprising the amino acid sequence of SEQ ID NO:2 or fragments thereof.

2. An isolated and purified polynucleotide sequence encoding the novel cysteine protease of claim 1.

3. An isolated and purified polynucleotide sequence comprising SEQ ID NO:1 or fragments thereof.

4. A polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of claim 3.

5. An oligonucleotide of the polynucleotide of claim 3.

6. A diagnostic test for a condition associated with altered ncp expression comprising the steps of:
a) providing a biological sample;
b) combining the biological sample and the oligonucleotide of claim 5 in a solution;
c) amplifying the ncp sequence;
d) measuring ncp expression; and e) comparing measured expression with standard expression, thereby establishing the presence of the condition.

7. The diagnostic test of claim 6 wherein the condition is selected from the group consisting anemia, arteriosclerosis, asthma, bronchitis, emphysema, gingivitis, inflammatory bowel disease, insulin-dependent diabetes mellitus, leukemia, multiple endocrine neoplasias, osteoarthritis, osteoporosis, pulmonary fibrosis, rheumatoid arthritis, septic shock syndromes, and systemic lupus erythematosus.

8. An expression vector comprising the polynucleotide of claim 3.

9. A host cell transformed with the expression vector of claim 8.

10. A method for producing and purifying a biologically active or immunogenic polypeptide, said method comprising the steps of:
a) culturing the host cell of claim 9 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.

11. A pharmaceutical composition comprising the peptide of claim 1 and a pharmaceutically acceptable excipient.

12. A method for treating diseases associated with altered NCP expression by appropriate administration of the pharmaceutical composition of claim 11.

13. An antibody specific for the purified polypeptide of claim 1.

14. A diagnostic test for a condition associated with altered NCP expression comprising the steps:
a) providing a biological sample;
b) combining the biological sample and the antibody of claim 13;
c) measuring complex formation between the antibody and NCP; and d) comparing expression with standard expression, thereby establishing the presence of the condition.

15. The diagnostic test of claim 14 wherein the condition is selected from the group consisting of anemia, arteriosclerosis, asthma, bronchitis, emphysema, gingivitis, inflammatory bowel disease, insulin-dependent diabetes mellitus, leukemia, multiple endocrine neoplasias, osteoarthritis, osteoporosis, pulmonary fibrosis, rheumatoid arthritis, septic shock syndromes, and systemic lupus erythematosus.

16. A pharmaceutical composition comprising the antibody of claim 13 and a pharmaceutically acceptable excipient.

17. A method of treating a condition associated with altered NCP expression comprising administering the pharmaceutical composition of claim 16.

18. A method of screening a plurality of compounds for specific binding affinity with the polypeptide of claim 1 comprising the steps of:
a) providing a plurality of compounds;
b) combining the compounds with the polypeptide in a manner and for a time sufficient to bind NCP; and c) detecting and recovering bound NCP; thereby identifying the compound.

19. A pharmaceutical composition comprising the compound of claim 18 and a pharmaceutically acceptable excipient.

20. A method of treating a condition associated with altered NCP expression comprising administering the pharmaceutical composition of claim 17 to a subject in need of such treatment.