Note: Descriptions are shown in the official language in which they were submitted.
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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
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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.
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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
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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.
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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
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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
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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.
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"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
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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
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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
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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
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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
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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
14
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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
16
CA 0223~7~ 1998-04-22
WO 97115592 PCT/US96/16926
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
WO 97/15592 PCT/US96/16926
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
19
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
22
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
23
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
24
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
WO 97/15592 PCT/US96/16926
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
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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
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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-
28
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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
29
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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
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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
34
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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.
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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.
(B) STREET: 3174 Porter Drive
(C) CITY: Palo Alto
(D) STATE: CA
(E) COUNTRY: USA
(F) ZIP: 94304
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(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:
(A) TELEPHONE: 415-855-0555
(B) TELEFAX: 415-845-4166
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1855 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: Adrenal
(B) CLONE: 100877
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
36
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AATTCGGCA(' GAGGCCTGCC ACAGGTGTCT GCAATTGAAC TCCAAGGTGC AGAATGGTTT 60
GGAAAGTAGT TGTATTCCTC AGTGTGGCCC TGGGAATTGG TGCCGTTCCT ATAGATGATC 120
CTGAAGATGG AGGCAAGCAC TGGGTGGTGA TCGTGGCAGG TTCAAATGGC TGGTATAATT 180
ATAGGCACCA GGCAGACGCG TGCCATGCCT ACCAGTTCAT TCACCGCAAT GGGATTCCTG 240
CCGAACAGAT CGTTGTGATT ATGTACGATG ACATAGCTTA CTCTGAAGAC AATCCCACTC 300
CAGGAATTGT GATCAACAGG CCCAATGGCA CAGATGTCTA TCAGGGAGTC CCGAAGGACT 360
ACACTGGAGA GGATGTTACC CCACAAAATT TCCTTGTTGT GTTGAGAGGC GATGCAGAAG 420
CAGTGAAGGG TATAGGATCC CGCAAAGTCC TGAAGAGTGG TCCCCAGGAT CACGTGTTCA 480
TTTATTTCA(, TGACCATGGA TCTTCTGGAA TACTGGTTTT CCCCAATGAA GATCTTCATG 540
TAAAGGACCT GATTAAGACC ACCCATTACA TTTTCAAAAA CAAAATGTAC CGAAAGATGG 600
TGTTCTACAT TGAGGCCTGT GAGTCTGGGT CCATGATGAA CCACCTGCCG GATAACATCA 660
ATGTTTATG(' AACTACTGCT GCCAACCCCA GAGAGTCGTC CTACGCCTGT TACTATGATG 720
AGAAGAGGTC CACGTACCTG GGGGACTGGT ACAGCGTCAA CTGGATGGAA GACTCGGACG 780
TGGAAGATCT GACTAAAGAG ACCCTGCACA AGCAGTACCA CCTGGTAAAA TCGCACACCA 840
ACACCAGCCA CGTCATGCAG TATGGAAACA AAACAATCTC CACCATGAAA GTGATGCAGT 900
TTCAGGGTAl' GAAACGCAAA GCCAGTTCTC CCGTCCCCCT ACCTCCAGTC ACACACCTTG 960
ACCTCACCC(' CAGCCCTGAT GTGCCTCTCA CCATCATGAA AAGGAAACTG ATGAACACCA 1020
ATGATCTGGA GGAGTCCAGG CAGCTCACGG AGGAGATCCA GCGGTATCTG GATGCCAGGC 1080
ACCTCATCC(; AGGTGAGGTG GAGCAGCTCC TGTCCGAGAG AGCCCCGCTC ACGGGGCACA 1140
GCTGCTACC(' AGAGGTCCTG TTGTACTTCC GGACCCACTG CTTCAACTGG TACTCCCCCA 1200
CGTACGAGTT ATGTGTTGAG ACATTTTGTA CGTGTTGGTC AACCTTTGTG AGAAGGCGCT 1260
TCCACTTCA(' AGGATATAAT TGTCCATGGC CCACGTGTGC CTTGGTCACT ACTGAAGAGC 1320
TGCCTCCTG(; AAGCTTTTCC CAAGTGTGAG CGCCCCCACC GGCTGTGTTC TTGATCAAGA 1380
GACTGGAGAG GTGGAGTGAG AAGTCTCCGC TGCTCGGGCC CTCCTGGGGG ACCCCCCGCT 1440
CCAGGGCTCG CTCCAGGACC TTCTTCACAA GATGACTTGC TCGCTGTTAC CTGCTTCCCC 1500
AGTCTTTTCT GAAAAACTAC AAATTAGGGT GGGAAAAGCT CTGTATTGAG AAGGGTCATA 1560
TTTGCTTTCT AGGAGGTTTG TTGTTTTGCC TGTTAGTTTT GAGGAGCAGG AAGCTCATGG 1620
GGGCTTCTGT AGCCCCTCTC CAAAGGAGTC TTTATTCTGA GAATTTGAAG CTGAAACCTC 1680
TTTAAATCTT CAGAATGATT TTATTGAAGA GGGCCGCAAG CCCCAAATGG AAAACTGTTT 1740
TTAGAAAATA TGATGATTTT TGATTGCTTT TGTATTTAAT TCTGCAGGTG TTCAAGTCTT 1800
AAAAAATAAA GATTTATAAC AGAACCCCAA AAAAAAAAAA A}~u~AAA AAAAA 1855
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(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: q31 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Val Trp Lys Val Val Val Phe Leu Ser Val Ala Leu Gly Ile Gly
1 5 10 15
~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
100 105 110
~he Leu Val Val Leu Arg Gly Asp Ala Glu Ala Val Lys Gly Ile Gly
115 120 125
Ser Arg Lys Val Leu Lys Ser Gly Pro Gln Asp His Val Phe Ile Tyr
130 135 140
Phe Thr Asp His Gly Ser Ser Gly Ile Leu Val Phe Pro Asn Glu Asp
145 150 155 160
~eu His Val Lys Asp Leu Ile Lys Thr Thr His Tyr Ile Phe Lys Asn
165 170 175
~ys Met Ty:r Arg Lys Met Val Phe Tyr Ile Glu Ala Cys Glu Ser Gly
180 185 190
~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
210 215 220
Arg Ser Th:r Tyr Leu Gly Asp Trp Tyr Ser Val Asn Trp Met Glu Asp
225 230 235 240
~er Asp Va:L Glu Asp Leu Thr Lys Glu Thr Leu His Lys Gln Tyr His
245 250 255
~eu Val Lys Ser His Thr Asn Thr Ser His Val Met Gln Tyr Gly Asn
38
CA 0223~7~ l998-04-22
WO 97/lSSg2 PCT/US96/16926
260 265 270
Lys Thr Ile Ser Thr Met Lys Val Met Gln Phe Gln Gly Met Lys Arg
275 280 285
Lys Ala Ser Ser Pro Val Pro Leu Pro Pro Val Thr His Leu Asp Leu
290 295 300
Thr Pro Ser Pro Asp Val Pro Leu Thr Ile Met Lys Arg Lys Leu Met
305 310 315 320
~sn Thr Asn Asp Leu Glu Glu Ser Arg Gln Leu Thr Glu Glu Ile Gln
325 330 335
~rg Tyr Leu Asp Ala Arg His Leu Ile Arg Gly Glu Val Glu Gln Leu
340 345 350
Leu Ser Glu Arg Ala Pro Leu Thr Gly His Ser Cys Tyr Pro Glu Val
355 360 365
Leu Leu Tyr Phe Arg Thr His Cys Phe Asn Trp Tyr Ser Pro Thr Tyr
370 375 380
Glu Leu Cys Val Glu Thr Phe Cys Thr Cys Trp Ser Thr Phe Val Arg
385 390 395 400
~rg Arg Phe His Phe Thr Gly Tyr Asn Cys Pro Trp Pro Thr Cys Ala
405 410 415
~eu Val Thr Thr Glu Glu Leu Pro Pro Gly Ser Phe Ser Gln Val
420 425 430
~2) INFORMATION FOR SEQ ID NO:3:
~i) S:EQUENCE CHARACTERISTICS:
(A) LENGTH: 267 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(E) HAPLOTYPE: U937
(vii) I~MEDIATE SOURCE:
(A) LIBRARY: 001098
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
ATTTGCTTTC TAGGAGGTTT GTTGTTTTGC CTGTTAGTTT TGAGGAGCAG GAAGCTCATG 60
GGGGCTTCTG TAGCCCCTCT CAAAAGGAGT CTTTATTCTG AGAATTTGAA GCTGAAACCT 120
CTTTAATCTT CAGAATGATT TTATTGAAGA GGGCCGCAAG CCCCAAATGG AAAACTGTTT 180
TTAGAAAATA TGATGATTTT TGATTGCTTT TGTATTTAAT TCTGCAGGTG TTCAAGTCTT 240
AAAAAATAAA GATTTATAAC AGAACCC 267
CA 0223~7~ l998-04-22
W O 97/l~g2 PCTAUS96/16926
(2) INFOR~TION FOR SEQ ID NO:4:
(i) ',EQUENCE CHARACTERISTICS:
(A) LENGTH: 195 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vii) ]:MMEDIATE SOURCE:
(A) LIBRARY: Huvec
(B) CLONE: 066931
(xi) 'iEQUENCE DESCRIPTION: SEQ ID NO:9:
ACGGAGGAGA TCAGCGGCAT CTGGATGCAG GCACCTCATT GAGAAGTCAG TGCGTAAGAT 60
CGCTCATTCT GGCAGCGTCC GAGGCTGAGG TGGAGCAGCT CCTGTCCGAG AGAGCCCCGC 120
TCACGGGGAC' AGCTCTACCC AGAGGCCCTG CTGCACTTCG GACCCACTCT TAACTGCACT 180
CCCCCACGTA CGAGT 195
(2) INFORMATION FOR SEQ ID NO:5:
(i) ';EQUENCE CHARACTERISTICS:
(A) LENGTH: 155 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vii) ]:MMEDIATE SOURCE:
(A) LIBRARY: THP-1
(B) CLONE: 075848
(xi) ';EQUENCE DESCRIPTION: SEQ ID NO:5:
GAGAAGAGGT CCACGTACCT GGGGGACTGG TACAGCNTCA ACTGGATGGA AGACTCGGAC 60
GTGGAAGATC' TGACTAAAGA GACCCTGCAC AAGCAGTACC ACCTGGTAAA ATCGCACACC 120
AACACCAGCC' ACGTCATGCA GTATGGAAAC AAAAC 155
(2) INFORMATION FOR SEQ ID NO:6:
(i) ';EQUENCE CHARACTERISTICS:
(A) LENGTH: 295 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vii) ]:MMEDIATE SOURCE:
(A) LIBRARY: Rheumatoid Synovium
(B) CLONE: 077015
CA 0223~7~ l998-04-22
WO 97/15592 PCTtUS96tl6926
(xi) ';EQUENCE DESCRIPTION: SEQ ID NO:6:
GAAACGCAAA GCCAGTTCTC CCGTCCCCCT ACCTCCAGTC ACACACCTTG ACCTCACCCC 60
CAGCCCTGAI' GTGCCTCTCA CCATCATGAA AAGGAAACTG ATGAACACCA ATGATCTGGA 120
GGAGTCCAGG CAGCTCACGG NGGAGATCCA GCGGCATCTG GATGNCAGGC ACCTCATTGA 180
GAAGTCAGTG CGTAAGATCG TCTCCTTGCT GGNAGCGTCC GAGGCTGAGG TGGAGCAGCT 240
CCTTA 245
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 185 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: Rheumatoid Synovium
(B) CLONE: 077424
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GATGTGCTCT ACCATCATGA AAAGGAAACT ATGAACACCA ATATCTGGAG GAGTCCAGGC 60
AGCTCACGGA. GGAGATCCAG CGGCATCTGG ATGCCAGGCA CCTCATTGAG AAGTCAGTGC 120
GTAAATCGTT CCTTGCTGGC AGCGTCCGAG GCTGAGGTGG AGCAGCTCCT TCCGAGAGAG 180
CCCCG 185
(2) INFORMATION FOR SEQ ID NO:8
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 232 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: Rheumatoid Synovium
(B) CLONE: 077645
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
AGAGACTGGA GAGGTGGAGT GAGAAGTCTC CGCTGCTCGG GCCTCCTGGG GAGCCCCCGC 60
TCCAGGGCTC GCTCCAGGAC CTTCTTCACA AGATGACTTN NTCGCTGTTA CCTGCTTCCC 120
CAGTCTTTTC TGNAAAACTA CAAATTAGGG TGGGAAAAGC TCTGTATTGA GAAGGGTCAT 180
ATTTGCTTTC TAGGAGGTTT GTTGTTTTGC CTGTAAGTTT TGAGGAGCAG GA 232
CA 0223~7~ 1998-04-22
W O 97/155$2 PCTAUS96/16926
(2) INFORMATION FOR SEQ ID NO:9:
(i) ',EQUENCE CHARACTERISTICS:
(A) LENGTH: 253 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vii) :MMEDIATE SOURCE:
(A) LIBRARY: Rheumatoid Synovium
(B) CLONE: 077651
(xi) ';EQUENCE DESCRIPTION: SEQ ID NO:9:
TGCACAAGCA GTACCACCTG GTAAAATCGC ACACCAACAC CAGCCACGTC ATGCAGTATG 60
GAAACAAAAC' AATCTCCACC ATGAAAGTNA TGCAGTTTCA GGGTATGAAA CGCAAAGCCA 120
GTTCTCCCGT CCCCCTACCT TCAGTCACAC ACCTTGACCT CACCCCCAGC CCTGATGTGC l80
CTCTNACCAT CATGAAAAGG GTAACTGNTG AACACCAATN ATCTTGAGGA GTCCAGGNAG 240
CTTTACGGTC; GTT 253
(2) INFOR~IATION FOR SEQ ID NO:l0:
(i) ';EQUENCE CHARACTERISTICS:
(A) LENGTH: 331 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: Rheumatoid Synovium
(B) CLONE: 078597
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l0:
ACTGGATGGP. AGACTCGGAC GTGGAAGATC TGACTAAAGA GACCCTGCAC AAGCAGTACC 60
ACCTGGTAAP. ATCGCACACC AACACCAGCC ACGTCATGCA GTATGGAAAC AAAACANTCT 120
CCACCATGAA AGTNATGCAG TTTCAGGGTA TGAAACGCAA AGCCAGTTCT CCCGTCCCCC 180
TACCTCCAGI CACACACCTT TGACCCTCAC CCCCAGNCCT GATGTGCCTC TAACCATCAT 240
GNAAAGGA~A. CTGGATGGAC ACCAATGATC TGGGAGGAGT CCAGGGAAGG NTCACGGAGG 300
GNGATCCCAG CGGGNATCTG GGATTCCCAN N 331
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 219 base pairs
42
CA 0223~7~ l998-04-22
W O 97/15592 PCTAUS96/16926
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vii) ]:MMEDIATE SOURCE:
(A) LIBRARY: Bone Marrow
(B) CLONE: 104286
(xi) ';EQUENCE DESCRIPTION: SEQ ID NO:11:
GTTTATGCAA CTNCTGCTGC CAACCCCAGA GAGTCGTCCT ACGCCTGTNA CTATGATGAG 60
AAGAGGTCCP. CGTACCTGGG GGACTGGTAC AGCGTCAACT GGATGGAAGA CTCGGACGTG 120
GAAGATCTGP. CTAAAGAGAC CCTGCACAAG CAGTACCACC TGGTAAAATC GCACACCAAC 180
ACCAGCCACG TCATGCAGTA TGGAAACAAA ACANTCTCC 219
(2) INFORMATION FOR SEQ ID NO:12:
(i) 'EQUENCE CHARACTERISTICS:
(A) LENGTH: 271 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: Kidney
(B) CLONE: 115565
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
TCACTACTGA AGAGCTGCCT CCTGGAAGCT TTTCCAAGTN TGAGCGCCCC ACCGACTGTT 60
TGCTGATCAN AGACTGGAGA GGTGGAGTGA GAAGTCTCCG CTGCTCGGGC CCTCCTGGGG 120
AGCCCCCGCT CCAGGGCTCG CTCCAGGACC TTNTTCACAA GATGACTTGC TCGCTGTTAC 180
CNGCTTCCCC AGTCTTTTNT GAAAAACTAC AAATTAGGGT GGGAAAAGCT CTGTATTGAG 240
AAGGGTCATA TTTNCTTTCT AGGAGGTTTT T 271
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 229 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: Lung
CA 0223~7~ l998-04-22
W O 97115592 PCTAUS96116926
(B) CLONE: 125569
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GTTCTACATT GANGCCTGTG AGTCTGGGTC CATGATGAAC CACCTNCCGG ATAACATCAA 60
TGTTTATGCA ACTACTGCTG CCAACCCCAG AGAGTCGTCC TACGCCTGTT ACTATGATGA 120
GAAGAGGNCC ACGTACCTGG GGGACTNGTA CAAAGTNAAA NTNGATGGAA GAATTCAGAC 180
GAGGAAGATC TNNCTAAAAN AGAACCTTAA CAAANCANTA ACNCCTAAG 229
(2) INFOR~TION FOR SEQ ID NO:l9:
~i) SEQUENCE CHARACTERIST:ECS:
(A) LENGTH: 207 base pairs
(B) TYPE: nuclelc acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: cDNA
~vii) II~MEDIATE SOURCE:
~A) LIBRARY: Lung
~B) CLONE: 125830
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
CCAGAGNCCT GCNGCACTTC CGGACCCA('T GCTTCAACTG GCACTCCCCC ACGTACGAGT 60
ATGCNTTGAG ACATTTGTAC GTGCTGGTCA ACCTTTGTNA GAAGCCGTAT CCACTTCACA 120
GGATAAAATT GTCCATGGAC CACGTGTGCC TTGGTCACTA CTGANGAGCT GCCTCCTGGA 180
AGCTTTTCCA AGTNTGAGCG CCCCACC 207
~2) INFOR~TION FOR SEQ ID NO:15:
~i) SE.QUENCE CHARACTERISTICS:
'A) LENGTH: 220 base pairs
'B) TYPE: nucleic acid
'C) STRANDEDNESS: slngle
D) TOPOLOGY: linear
~ii) MOLECULE TYPE: cDNA
~vii) I~EDIATE SOURCE:
'A) LIBRARY: Kidney
B) CLONE: 158868
~xi) SE'QUENCE DESCRIPTION: SEQ ID NO:15:
ATGAACCACC TGCCGGATAA CATCAATGTT TATGCAACTA CTGCTGCCAA CCCCAGAGAG 60
TCGTCCTACG CCTGTAACTA TGATGAGAAG AGGTCCACGT ACCTGGGGGA CTGGTACAGC 120
GTCAACTGGA TGGAAGACTC GGACGTGGAA GATCTGACTA AAGAGACCCT GCACAAGCAG 180
CA 0223~7~ l998-04-22
W O 97/15592 PCTAUS96/16926
TACCACCTGG TAAAATCGCA CACCAACACC AGCCACGTTG 220
(2) INFORMATION FOR SEQ ID NO:16:
(i) SF.QUENCE CHARACTERISTICS:
(A) LENGTH: 254 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: Adenoid
(B) CLONE: 162199
(xi) Si.QUENCE DESCRIPTION: SEQ ID NO:16:
GTACCACCTG GTAAAATCGC ACACCAACAC CAGCCACGTC ATGCAGTATG GAAACAAAAC 60
AATCTCCACC ATGAAAGTGA TGCAGTTTCA GGGTATGAAA CGCAAAGCCA GTTCTCCCGT 120
CCCCCTACCT CCAGTCACAC ACCTTGACCT CACCCCCAGC CCTGATGTGC CTCTCACCAT 180
CATGAAAAGG AAACTGATGA ACACCAATGA TCTGGAGGAG TCCAGGCAGC TCACGGGAGG 240
AGATCCAGCG GCAT 254
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 218 base pairs
~B) TYPE: nucleic acid
'C) STRANDEDNESS: single
'D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vii) IM~iEDIATE SOURCE:
~A) LIBRARY: Bone Marrow
l:B) CLONE: 172499
(xi) SE.Q~ENCE DESCRIPTION: SEQ ID NO:17:
AAATTGTCCA TGGACCACGT GTGCCTTGGT CACTACTGAA GAGCTGCCTC CTGGAAGCTT 60
TTCCAAGTGT GAGCGCCCCA CCGACTGTNT GCTGATCAGA GACTGGAGAG GTGGAGTGAG 120
AAGTCTCCGC TGCTCGGGCC CTCCTGGGGA GCCCCCGCTC CAGGNCTCGC TCCAGGACCT 180
TCTTCACAAG ATGACTTGCT CGCTGTTACC TGCTTCCG 218
(2) INFORM~TION FOR SEQ ID NO:18:
(i) SE:QUENCE CHARACTERISTICS:
I:A) LENGTH: 199 base pairs
l:B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 0223~7~ l998-04-22
W O 97/15592 PCT~US96/16926
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vii) :[MMEDIATE SOURCE:
(A) LIBRARY: Bone Marrow
(B) CLONE: 174690
(xi) ',EQUENCE DESCRIPTION: SEQ ID NO:18:
GNGACTGGTA CAGCNTCAAC TGGNTGGAAG NCTNGGACGT GGAAGATCTG ACTAANGAGA 60
CCCTGCACAA GCAGTACCAC CTGGTAAAAT CGNACANCAA NACCAGCCAC GTCATGCAGT 120
ATGGGACAAN NCAATCTCCA CCATGAAAGT GATGCAGTTT CAGGGTATGA AACGCAGAGC 180
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:
GGCCCGGGCA GCGGAGACTT CTCACTCCAC CTCTCCAGTC TCTGATCAGC AC 52
(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:
GGCCGGGCAG CGGAGACTTC TCACTCCACC TCTCCAGTCT CTGATCAGCA C 51
(2) INFOR~ATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 234 base pairs
46
CA 0223~7~ l998-04-22
W O 97115592 PCTAUS96/16926
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
TGCACTTCCG GACCCACTGC TTCAACTGGC ACTCCCCCAC GTACGAGTAT GCNTTGAGAC 120
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:
CTGGTTTTNC CCAATGAAGA TCTTCATGTA AAGGACCTGA NTGAGACCAT CCATTACATG 60
TACAAACACA AAATGTACCG AAAGATGGTG TTCTACATTN AGGCCTGTNA GTCTGGGTCC 120
ATGTTGANCC ACCTGCCGGN NANCATCAAN GTTNNTGCAA CTACTGNTNC CAACCCCTGA 180
GAGTNGTCCG ANGNCTGTNA CTATGT 206
(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
47
CA 0223~7~ l998-04-22
W O 97/15592 PCTAUS96/16926
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CTAAAGAGAC CCTGCACAAG CAGTACCACC TGGTAAAATC GCACACCAAC ACCAGCCACG 60
TCATGCAGTA TGGAAACAAA ACAATCTNCA CCATGAAAGT NATGCAGTTT CAGGGTATGA 120
AACGCAAAGC CAGTTCTCCC GTCCCCCTAC CTCCAGTCAC ACACCTTGAC CTCACCCCCN 180
T 181
(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
CCCCCGCTCC AGGGCTCGCT CCAGGACCTT NTTCACAAGA TGACTTGCTC GCTGTTACCT 180
GCTTCCCCAC, TCTTTTCTGA AAAACTA('AA AA 212
48
