U.S. patent application number 09/967796 was filed with the patent office on 2002-10-24 for novel human cysteine protease.
This patent application is currently assigned to Incyte Pharmaceuticals, Inc.. Invention is credited to Braxton, Scott Michael, Delegeane, Angelo M., Diep, Dinh.
Application Number | 20020155535 09/967796 |
Document ID | / |
Family ID | 24267445 |
Filed Date | 2002-10-24 |
United States Patent
Application |
20020155535 |
Kind Code |
A1 |
Diep, Dinh ; et al. |
October 24, 2002 |
Novel human cysteine protease
Abstract
The present invention provides a polynucleotide (ncp) which
identifies and encodes a novel cysteine protease (NCP) expressed in
cells of both the adrenal gland and human umbilical vein
endothelium. The present invention also provides for antisense
molecules and oligomers designed from the nucleotide sequence or
its antisense. The invention further provides genetically
engineered expression vectors and host cells for the production of
purified NCP peptide, antibodies capable of binding to NCP,
inhibitors which bind to NCP and pharmaceutical compositions based
on NCP specific antibodies or inhibitors. The invention
specifically provides for diagnostic assays based on altered ncp
expression and which allow identification of such a condition.
These assays utilize probes designed from ncp encoding or
controlling nucleic acid sequences or antibodies specific for the
NCP.
Inventors: |
Diep, Dinh; (San Francisco,
CA) ; Braxton, Scott Michael; (San Mateo, CA)
; Delegeane, Angelo M.; (Hayward, CA) |
Correspondence
Address: |
INCYTE GENOMICS, INC.
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Pharmaceuticals,
Inc.
|
Family ID: |
24267445 |
Appl. No.: |
09/967796 |
Filed: |
September 28, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09967796 |
Sep 28, 2001 |
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09449422 |
Nov 23, 1999 |
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09449422 |
Nov 23, 1999 |
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09193524 |
Nov 17, 1998 |
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6007997 |
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09193524 |
Nov 17, 1998 |
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08928613 |
Sep 12, 1997 |
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5840562 |
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08928613 |
Sep 12, 1997 |
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08567506 |
Dec 5, 1995 |
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Current U.S.
Class: |
435/69.1 ;
435/226; 435/252.3; 435/320.1; 435/325; 536/23.2; 800/8 |
Current CPC
Class: |
C12N 9/6472
20130101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 435/252.3; 800/8; 435/226; 536/23.2 |
International
Class: |
C12P 021/02; C12N
001/21; C12N 005/06; A01K 067/00; C07H 021/04; C12N 009/64 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of.
a) a polypeptide comprising an amino acid sequence of SEQ ID NO: 2,
b) a polypeptide comprising a naturally occurring polypeptide
comprising an amino acid sequence at least 90% identical to an
amino acid sequence of SEQ ID NO: 2, c) a biologically active
fragment of a polypeptide having an amino acid sequence of SEQ ID
NO: 2, and d) an immunogenic fragment of a polypeptide having an
amino acid sequence of SEQ ID NO: 2.
2. An isolated polypeptide of claim 1, having a sequence of SEQ ID
NO: 2.
3. An isolated polynucleotide encoding a polypeptide of claim
1.
4. An isolated polynucleotide encoding a polypeptide of claim
2.
5. An isolated polynucleotide of claim 4, having a sequence of SEQ
ID NO: 1.
6. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim
6.
8. A transgenic organism comprising a recombinant polynucleotide of
claim 6.
9. A method for producing a polypeptide of claim 1, the method
comprising: a) culturing a cell under conditions suitable for
expression of the polypeptide, wherein said cell is transformed
with a recombinant polynucleotide, and said recombinant
polynucleotide comprises a promoter sequence operably linked to a
polynucleotide encoding the polypeptide of claim 1, and b)
recovering the polypeptide so expressed.
10. A method of claim 9, wherein the polypeptide has the sequence
of SEQ ID NO: 2.
11. An isolated antibody which specifically binds to a polypeptide
of claim 1.
12. An isolated polynucleotide comprising a sequence selected from
the group consisting of: a) a polynucleotide comprising a
polynucleotide sequence of SEQ ID NO: 1, b) a naturally occurring
polynucleotide comprising a polynucleotide sequence at least 90%
identical to a polynucleotide sequence of SEQ ID NO: 1, c) a
polynucleotide having a sequence complementary to a polynucleotide
of a), d) a polynucleotide having a sequence complementary to a
polynucleotide of b) and e) an RNA equivalent of a)-d).
13. An isolated polynucleotide comprising at least 60 contiguous
nucleotides of a polynucleotide of claim 12.
14. A method for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) hybridizing the sample with a
probe comprising at least 20 contiguous nucleotides comprising a
sequence complementary to said target polynucleotide in the sample,
and which probe specifically hybridizes to said target
polynucleotide, under conditions whereby a hybridization complex is
formed between said probe and said target polynucleotide or
fragments thereof, and b) detecting the presence or absence of said
hybridization complex, and, optionally, if present, the amount
thereof.
15. A method of claim 14, wherein the probe comprises at least 60
contiguous nucleotides.
16. A method for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) amplifying said target
polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b) detecting the presence or absence of said
amplified target polynucleotide or fragment thereof, and,
optionally, if present, the amount thereof.
17. A composition comprising a polypeptide of claim 1 and a
pharmaceutically acceptable excipient.
18. A composition of claim 17, wherein the polypeptide has an amino
acid sequence of SEQ ID NO: 2.
19. A method for treating a disease or condition associated with
decreased expression of functional NCP, comprising administering to
a patient in need of such treatment the composition of claim
17.
20. A method for screening a compound for effectiveness as an
agonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting agonist activity in the sample.
21. A composition comprising an agonist compound identified by a
method of claim 20 and a pharmaceutically acceptable excipient.
22. A method for treating a disease or condition associated with
decreased expression of functional NCP, comprising administering to
a patient in need of such treatment a composition of claim 21.
23. A method for screening a compound for effectiveness as an
antagonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting antagonist activity in the sample.
24. A composition comprising an antagonist compound identified by a
method of claim 23 and a pharmaceutically acceptable excipient.
25. A method for treating a disease or condition associated with
overexpression of functional NCP, comprising administering to a
patient in need of such treatment a composition of claim 24.
26. A method of screening for a compound that specifically binds to
the polypeptide of claim 1, the method comprising: a) combining the
polypeptide of claim 1 with at least one test compound under
suitable conditions, and b) detecting binding of the polypeptide of
claim 1 to the test compound, thereby identifying a compound that
specifically binds to the polypeptide of claim 1.
27. A method of screening for a compound that modulates the
activity of the polypeptide of claim 1, said method comprising: a)
combining the polypeptide of claim 1 with at least one test
compound under conditions permissive for the activity of the
polypeptide of claim 1, b) assessing the activity of the
polypeptide of claim 1 in the presence of the test compound, and c)
comparing the activity of the polypeptide of claim 1 in the
presence of the test compound with the activity of the polypeptide
of claim 1 in the absence of the test compound, wherein a change in
the activity of the polypeptide of claim 1 in the presence of the
test compound is indicative of a compound that modulates the
activity of the polypeptide of claim 1.
28. A method for screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a polynucleotide sequence of claim 5, the
method comprising: a) exposing a sample comprising the target
polynucleotide to a compound, under conditions suitable for the
expression of the target polynucleotide, b) detecting altered
expression of the target polynucleotide, and c) comparing the
expression of the target polynucleotide in the presence of varying
amounts of the compound and in the absence of the compound.
29. A method for assessing toxicity of a test compound, the method
comprising: a) treating a biological sample containing nucleic
acids with the test compound, b) hybridizing the nucleic acids of
the treated biological sample with a probe comprising at least 20
contiguous nucleotides of a polynucleotide of claim 12 under
conditions whereby a specific hybridization complex is formed
between said probe and a target polynucleotide in the biological
sample, said target polynucleotide comprising a polynucleotide
sequence of a polynucleotide of claim 12 or fragment thereof, c)
quantifying the amount of hybridization complex, and d) comparing
the amount of hybridization complex in the treated biological
sample with the amount of hybridization complex in an untreated
biological sample, wherein a difference in the amount of
hybridization complex in the treated biological sample is
indicative of toxicity of the test compound.
30. A diagnostic test for a condition or disease associated with
the expression of NCP in a biological sample, the method
comprising: a) combining the biological sample with an antibody of
claim 11, under conditions suitable for the antibody to bind the
polypeptide and form an antibody:polypeptide complex, and b)
detecting the complex, wherein the presence of the complex
correlates with the presence of the polypeptide in the biological
sample.
31. The antibody of claim 11, wherein the antibody is: a) a
chimeric antibody, b) a single chain antibody, c) a Fab fragment,
d) a F(ab').sub.2 fragment, or e) a humanized antibody.
32. A composition comprising an antibody of claim 11 and an
acceptable excipient.
33. A method of diagnosing a condition or disease associated with
the expression of NCP in a subject, comprising administering to
said subject an effective amount of the composition of claim
32.
34. A composition of claim 32, wherein the antibody is labeled.
35. A method of diagnosing a condition or disease associated with
the expression of NCP in a subject, comprising administering to
said subject an effective amount of the composition of claim
34.
36. A method of preparing a polyclonal antibody with the
specificity of the antibody of claim 11, the method comprising: a)
immunizing an animal with a polypeptide having an amino acid
sequence of SEQ ID NO: 2, or an immunogenic fragment thereof, under
conditions to elicit an antibody response, b) isolating antibodies
from said animal, and c) screening the isolated antibodies with the
polypeptide, thereby identifying a polyclonal antibody which binds
specifically to a polypeptide having an amino acid sequence of SEQ
ID NO: 2.
37. An antibody produced by a method of claim 36.
38. A composition comprising the antibody of claim 37 and a
suitable carrier.
39. A method of making a monoclonal antibody with the specificity
of the antibody of claim 11, the method comprising: a) immunizing
an animal with a polypeptide having an amino acid sequence of SEQ
ID NO: 2, or an immunogenic fragment thereof, under conditions to
elicit an antibody response, b) isolating antibody producing cells
from the animal, c) fusing the antibody producing cells with
immortalized cells to form monoclonal antibody-producing hybridoma
cells, d) culturing the hybridoma cells, and e) isolating from the
culture monoclonal antibody which binds specifically to a
polypeptide having an amino acid sequence of SEQ ID NO: 2.
40. A monoclonal antibody produced by a method of claim 39.
41. A composition comprising the monoclonal antibody of claim 40
and a suitable carrier.
42. The antibody of claim 11, wherein the antibody is produced by
screening a Fab expression library.
43. The antibody of claim 11, wherein the antibody is produced by
screening a recombinant immunoglobulin library.
44. A method of detecting a polypeptide having an amino acid
sequence of SEQ ID NO: 2 in a sample, the method comprising: a)
incubating the antibody of claim 11 with a sample under conditions
to allow specific binding of the antibody and the polypeptide, and
b) detecting specific binding, wherein specific binding indicates
the presence of a polypeptide having an amino acid sequence of SEQ
ID NO: 2 in the sample.
45. A method of purifying a polypeptide having an amino acid
sequence of SEQ ID NO: 2 from a sample, the method comprising: a)
incubating the antibody of claim 11 with a sample under conditions
to allow specific binding of the antibody and the polypeptide, and
b) separating the antibody from the sample and obtaining the
purified polypeptide having an amino acid sequence of SEQ ID NO:
2.
46. A microarray wherein at least one element of the microarray is
a polynucleotide of claim 13.
47. A method of generating a transcript image of a sample which
contains polynucleotides, the method comprising: a) labeling the
polynucleotides of the sample, b) contacting the elements of the
microarray of claim 46 with the labeled polynucleotides of the
sample under conditions suitable for the formation of a
hybridization complex, and c) quantifying the expression of the
polynucleotides in the sample.
48. An array comprising different nucleotide molecules affixed in
distinct physical locations on a solid substrate, wherein at least
one of said nucleotide molecules comprises a first oligonicleotide
or polynucleotide sequence specifically hybridizable with at least
30 contiguous nucleotides of a target polynucleotide, and wherein
said target polynucleotide is a polynucleotide of claim 12.
49. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 30
contiguous nucleotides of said target polynucleotide.
50. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 60
contiguous nucleotides of said target polynucleotide.
51. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to said target
plynucleotide.
52. An array of claim 48, which is a microarray.
53. An array of claim 48, further comprising said target
polynucleotide hybridized to a nucleotide molecule comprising said
first oligonucleotide or polynucleotide sequence.
54. An array of claim 48, wherein a linker joins at least one of
said nucleotide molecules to said solid substrate.
55. An array of claim 48, wherein each distinct physical location
on the substrate contains multiple nucleotide molecules, and the
multiple nucleotide molecules at any single distinct physical
location have the same sequence, and each distinct physical
location on the substrate contains nucleotide molecules having a
sequence which differs from the sequence of nucleotide molecules at
another distinct physical location on the substrate.
56. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO: 2.
57. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO: 1.
Description
[0001] This application is a continuation application of U.S. Ser.
No. 09/449,422, filed Nov. 23, 1999, which is a divisional
application of U.S. Ser. No. 09/193,524, filed Nov. 17, 1998, now
U.S. Pat. No. 6,007,997, which is a divisional application of U.S.
Ser. No. 08/928,613, filed Sep. 12, 1997, now U.S. Pat. No.
5,840,562, which is a continuation application of U.S. Ser. No.
08/567,506, filed Dec. 5, 1995, now abandoned, the contents each of
which is hereby incorporated by reference, and each application
entitled "A Novel Human Cysteine Protease".
FIELD OF THE INVENTION
[0002] The present invention relates to novel, human cysteine
protease and the use of its nucleic acid and amino acid sequences
in the diagnosis, study, prevention and treatment of autoimmune or
degenerative diseases.
BACKGROUND OF THE INVENTION
[0003] Cysteine proteases are involved in diverse cellular
processes ranging from the processing of precursor proteins to
intracellular degradation. They may induce vascular permeability
through activation of the kallikrein/kinin pathway, complex with
various hemagglutinins, 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.
[0004] Cysteine proteases are known to be produced by monocytes,
macro phages and other cells of the immune system. These cells
migrate to sites of inflammation 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 tissue destruction. This is the case in
autoimmune diseases such as rheumatoid arthritis, when the
secretion of the cysteine protease, cathepsin C, degrades collagen,
laminin, elastin and other structural proteins found in the
extracellular matrix of bones. Bone weakened by such degradation is
more susceptible to tumor invasion and metastasis.
[0005] The novel, human cysteine protease of this application was
first identified among the sequences of a cDNA library made from
human adrenal glands.
[0006] Human adrenal glands are cap-like structures located above
each kidney. Each gland consists of the adrenal medulla and the
adrenal cortex. The adrenal medulla is made up of chromaffin tissue
and mainly secretes norepinephrine (NE) and epinephrine (E).
Stimulation of the sympathetic nerves to the adrenal medulla
releases these two catecholamines into the blood. NE, constricts
blood vessels, stimulates cardiac activity, inhibits the
gastrointestinal tract, and dilates the pupils of the eyes.
Epinephrine triggers almost the same responses, but it has a
stronger effect on cardiac activity and a weaker effect on blood
vessels. NE and E supplement the effects of the sympathetic nervous
system but appear to have little effect on its function.
[0007] The adrenal cortex uses cholesterol to produce a large
number of corticosteroids which display hormonal activity. The
outer layer of the adrenal gland mainly produces the
mineralocorticoid, aldosterone. The stimulatory and inhibitory
regulation of aldosterone secretion is governed by potassium level,
renin-angiotensin interactions, and secretion of
adrenocortlicotrophic hormone (ACTH), dopamine, serotonin, and
.beta.-endorphin. Aldosterone regulates extracellular fluid volume
and sodium/potassium balance by interacting with type-I
mineralocorticoid receptors in target tissues such as the kidney,
salivary gland, and intestinal mucosa.
[0008] The inner layers of the adrenal gland are sites of
glucocorticoid and androgen, estrogen and progesterone
biosynthesis. The principal glucocorticoid is cortisol which
functions in the regulation of protein, carbohydrate, lipid, and
nucleic acid metabolism, acts as an anti-inflammatory, and plays a
biofeedback role in suppressing 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 maintaining
lifelong spermatogenesis.
[0009] Conditions, diseases 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 adenomas; adrenal virilism; Cushing's syndrome; Conn's
syndrome; Addison's disease, which is a primary adrenocortical
insufficiency; secondary adrenocortical insufficiency, and adrenal
adenomas, which include benign adrenal cysts, nonfunctional adrenal
carcinoma, and tuberculosis of the adrenal gland.
[0010] The adrenal gland and its diseases are reviewed, inter alia,
in Guyton A C (1991) Textbook of Medical Physiology, W B Saunders
Co, Philadelphia Pa.; Isselbacher, K J et al (1994) Harrison's
Principles of Internal Medicine, McGraw-Hill New York N.Y.; The
Merck Manual of Diagnosis and Therapy (1992) Merck Research
Laboratories, Rahway N.J.; and Goodman A G et al. (1993) The
Pharmacological Basis of Therapeutics, McGraw-Hill, New York
N.Y.
SUMMARY OF THE INVENTION
[0011] 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 novel protein and
its nucleic acid sequences in the diagnosis, study, prevention and
treatment of immune diseases, particularly autoimmune and
degenerative diseases.
[0012] The subject invention provides a unique nucleotide sequence
(SEQ ID NO 1) 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 alignments 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 Q.sub.48,
C.sub.52 and H.sub.150. 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 designed 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 immortal
(lymphoma and leukemic cell lines), inflamed (adenoid and
rheumatoid synovium libraries), or involved in systemic cleanup or
defense through either the harboring or production of cells such as
monocytes or macro phages (bone marrow, kidney, lung, placenta and
small intestine libraries). The assembled nucleotide and amino acid
sequences shown in FIG. 1 represent a new human cysteine
protease.
[0013] An additional computer search for local sequence alignments
of the amino acid sequence of the novel cysteine protease described
herein showed that the most closely related 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 S. japonicum.
[0014] Based on the conserved cysteine protease residues, Q.sub.48,
C.sub.52 and H.sub.150 of the catalytic region; similarity to the
closely related molecule, hemoglobinase; and the presence of the
novel cysteine protease in cells and tissues which are immortal,
inflamed 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 treatment of autoimmune or degenerative
diseases.
[0015] Further aspects of the present invention include antisense
molecules of ncp which are useful in diminishing or eliminating
expression of the genomic nucleotide sequence.
[0016] 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.
[0017] The ncp nucleic acid sequences disclosed herein may be used
in diagnostic assays to 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 diagnose abnormalities in gene expression associated with an
immune disorder. The invention farther 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 expression of NCP, including but not limited to: anemia,
arteriosclerosis, asthma, bronchitis, cancers, emphysema,
gingivitis, inflammatory bowel disease, insulin-dependent diabetes
mellitus, leukemia, osteoarthritis, osteoporosis, pulmonary
fibrosis, rheumatoid arthritis, septic shock syndromes, and
systemic lupus erythematosus. 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.
[0018] Antibodies may be used for therapeutic as well as diagnostic
purposes, eg, in neutralizing the activity of an NCP associated
with an immune disorder such as rheumatoid arthritis. The present
invention also relates in part to proteins, peptides, and organic
molecules capable of modulating activity of NCP which may be used
therapeutically in the treatment of disease states associated with
aberrant expression of an NCP. The present invention also relates
to pharmaceutical compositions for the treatment of disease states
associated with aberrant expression of ncp comprising NCP, nucleic
acid sequences encoding NCP, anti NCP or other proteins, peptides
or organic molecules capable of modulating NCP expression.
DESCRIPTION OF THE FIGURES
[0019] FIG. 1 displays an alignment of cDNA sequences which
encompass the coding region of ncp. Alignments shown in this and
the following figures were produced using the multisequence
alignment program of DNASTAR.TM. software (DNASTAR Inc, Madison
Wis.)
[0020] FIGS. 2A, 2B, 2C and 2D show the nucleic acid and amino acid
alignments of NCP.
[0021] FIGS. 3A and 3B show the amino acid alignments between NCP
and hemoglobinase from Schistosoma japonicum (GenBank Accession
X70967).
[0022] FIG. 4 displays the DNASTAR analysis of NCP .alpha. regions
(A), .beta. regions (B), turn regions (T), coil regions (C),
hydrophilicity plot (H), .alpha. amphipathic regions (AA), .beta.
amphipathic regions (BA), antigenic index (AI) and surface
probability plot (S) based on the predicted acid amino sequence and
composition.
DETAILED DESCRIPTION OF THE INVENTION
[0023] Definitions
[0024] The present invention relates to a novel cysteine protease
which is expressed in the adrenal gland, human umbilical vein
endothelial cells, lymphoma and leukemic cell lines, adenoid,
rheumatoid synovium, 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 amino acid
sequence.
[0025] As used herein, NCP is a term which refers to NCP from any
species, including, bovine, ovine, 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 preferred NCP variant is one having at least 80%
amino acid sequence similarity, a more preferred variant is one
having 90% amino acid sequence similarity, and a most preferred
variant is one having 95% amino acid sequence similarity to the NCP
amino acid sequence illustrated in FIG. 1.
[0026] 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 sequences
are based on (or designed from) genomic or cDNA sequences and are
used to 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 synthesized and may be used as 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 hybridize than oligonucleotides. They may be single- or
double-stranded and are carefully designed to have specificity in
PCR, hybridization membrane-based, or ELISA-like technologies.
[0027] "Reporter" molecules are chemical moieties used for labeling
a nucleic or amino acid sequence. They include, but are not limited
to, radionuclides, enzymes, fluorescent, chemiluminescent, or
chromogenic agents. Reporter molecules associate with, establish
the presence of, and may allow quantification of a particular
nucleic or amino acid sequence.
[0028] A "portion" or "fragment" of a polynucleotide or nucleic
acid comprises 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 molecules using nick translation, Klenow fill-in reaction,
PCR or other methods well known in the art. After pretesting to
optimize reaction conditions and to eliminate 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
organism.
[0029] "Recombinant nucleotide variants" are polynucleotides which
encode a protein. They may be synthesized by making use of the
"redundancy" 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.
[0030] "Linkers" are synthesized palindromic nucleotide sequences
which create internal restriction endonuclease sites for ease of
cloning the genetic material of choice into various vectors.
"Polylinkers" are engineered to include multiple restriction enzyme
sites and provide for the use of both those enzymes which leave 5'
and 3' overhangs such as BamHI, EcoRI, PstI, KpnI and Hind III or
which provide a blunt end such as EcoRV, SnaBI and StuI.
[0031] "Control elements" or "regulatory sequences" are those
nontranslated regions of the gene or DNA such as enhancers,
promoters, introns and 3' untranslated regions which interact with
cellular proteins to carry out replication, transcription, and
translation. They may occur as boundary sequences or even split the
gene. They function at the molecular level and along with
regulatory genes are very important in development, growth,
differentiation and aging processes.
[0032] "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
appropriate vector and expressed to give rise to a chimeric
polypeptide which may be expected to be different from the native
molecule in one or more of the following characteristics: cellular
location, distribution, ligand-binding affinities, interchain
affinities, degradation/turnover rate, signaling, etc.
[0033] "Active" refers to those forms, fragments, or domains of an
amino acid sequence which display the biologic and/or immunogenic
activity characteristic of the naturally occurring peptide.
[0034] "Naturally occurring NCP" refers to a polypeptide produced
by cells which have not been genetically engineered or which have
been genetically engineered to produce the same sequence as that
naturally produced. Specifically contemplated are various
polypeptides which arise from post-translational modifications.
Such modifications of the polypeptide include but are not limited
to acetylation, carboxylation, glycosylation, phosphorylation,
lipidation and acylation.
[0035] "Derivative" refers to those polypeptides which have been
chemically modified by such techniques as ubiquitination, labeling
(see above), pegylation (derivatization with polyethylene glycol),
and chemical insertion or substitution of amino acids such as
ornithine which do not normally occur in human proteins.
[0036] "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 determining which amino
acid residues may be replaced, added or deleted without abolishing
characteristics of interest may be found by comparing the sequence
of NCP with that of related polypeptides and minimizing the number
of amino acid sequence changes made in highly conserved
regions.
[0037] Amino acid "substitutions" are defined as one for one amino
acid replacements. They are conservative in nature when the
substituted amino acid has similar structural and/or chemical
properties. Examples of conservative replacements are substitution
of a leucine with an isoleucine or valine, an aspartate with a
glutamate, or a threonine with a serine.
[0038] Amino acid "insertions" or "deletions" are changes to or
within an amino acid sequence. They typically fall in the range of
about 1 to 5 amino acids. The variation allowed in a particular
amino acid sequence may be experimentally determined by producing
the peptide synthetically or by systematically making insertions,
deletions, or substitutions of nucleotides in the ncp sequence
using recombinant DNA techniques.
[0039] A "signal or leader sequence" is a short amino acid sequence
which 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.
[0040] 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 17 or more
amino acids, typically at least about 9 to 13 amino acids, and of
sufficient length to display biologic and/or immunogenic
activity.
[0041] 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.
[0042] A "standard" is a quantitative or qualitative measurement
used for comparison. Preferably, it is based on a statistically
appropriate number of samples and is created to use as a basis of
comparison when performing diagnostic assays, running clinical
trials, or following patient treatment profiles. The samples of a
particular standard may be normal or similarly abnormal.
[0043] "Animal" as used herein may be defined to include human,
domestic (cats, dogs, etc.), agricultural (cows, horses, sheep,
goats, chicken, fish, etc.) or test species (frogs, mice, rats,
rabbits, simians, etc.).
[0044] "Conditions" includes cancers, disorders or diseases in
which ncp activity may be implicated. These specifically include,
but are not limited to, anemia, arteriosclerosis, asthma,
bronchitis, emphysema, gingivitis, inflammatory bowel disease,
insulin-dependent diabetes mellitus leukemia, multiple endocrine
neoplasias, osteoarthritis, osteoporosis, pulmonary fibrosis,
rheumatoid arthritis, septic shock syndromes, and systemic lupus
erythematosus.
[0045] Since the list of technical and scientific terms cannot be
all encompassing, any undefined terms shall be construed to have
the same meaning as is commonly understood by one of skill in the
art to which this invention belongs. Furthermore, the singular
forms "a", "an" and "the" include plural referents 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 be known to
those skilled in the art or will become known to them upon reading
this specification.
[0046] 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 sequences, variants, formulations and methodologies may vary,
and the terminology used herein is for the purpose of describing
particular embodiments. The terminology and definitions are not
intended to be limiting since the scope of protection will
ultimately depend upon the claims.
[0047] Description of the Invention
[0048] 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 Q.sub.48,
C.sub.52 and H.sub.150. The number of nucleotides separating the Q
and C residues in this novel cysteine protease is fewer than found
in other cysteine proteases, such as papain.
[0049] In addition, the novel cysteine protease is expressed in
human umbilical vein endothelial cells (HUVEC). The full length ncp
sequence was obtained by sequencing 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 active. 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.
[0050] Transcripts which did align with some portion of the ncp
molecule were found in other Incyte cDNA libraries. Thirty-two
segments of cDNAs from different Incyte Clones are shown as an
overlapping assemblage in FIG. 1. Twenty-three of these Incyte
Clones including cDNAs from U937 cell, THP-1 cell, rheumatoid
synovium, bone marrow, kidney, lung, inflamed adenoid, placenta,
and small intestine libraries are presented in the Sequence
Listing. In fact, 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.
[0051] 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
hybridization probes, for chromosome and gene mapping, in the
production of sense or antisense nucleic acids, in screening for
new therapeutic molecules, etc. These examples are well known and
are not intended to be limiting. Furthermore, the nucleotide
sequences disclosed herein may be used in molecular biology
techniques that have not yet been developed, provided the new
techniques rely on properties of nucleotide sequences that are
currently known, including but not limited to such properties as
the triplet genetic code and specific base pair interactions.
[0052] 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 minimal homology to the endogenous
sequence of any known and naturally occurring cysteine protease
sequence. This invention has specifically contemplated each and
every possible variation of nucleotide sequence that could be made
by selecting combinations based on possible codon 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.
[0053] Although the ncp nucleotide sequence and its derivatives or
variants are preferably capable of identifying the nucleotide
sequence of the naturally occurring NCP under optimized conditions,
it may be advantageous to produce NCP-encoding nucleotide sequences
possessing a substantially 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
accordance with the frequency with which particular codons are
utilized by the host. Other reasons for substantially 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.
[0054] Nucleotide sequences encoding NCP may be joined to a variety
of other nucleotide sequences by means of well established
recombinant DNA techniques (Sambrook J et al (1989) Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor N.Y.; or Ausubel F M et al (1989) Current Protocols
in Molecular Biology, John Wiley & Sons, 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. Vectors of interest include vectors for
replication, expression, probe generation, sequencing, and the
like. In general, vectors of interest may contain an origin of
replication functional in at least one organism, convenient
restriction endonuclease sensitive sites, and selectable markers
for one or more host cell systems.
[0055] PCR as described in U.S. Pat. Nos. 4,683,195; 4,800,195; and
4,965,188 provides additional uses for oligonucleotides based upon
the ncp nucleotide sequence. Such oligomers are generally
chemically synthesized, 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 diagnostic use. The same two
oligomers, nested sets of oligomers, or even a degenerate pool of
oligomers may be employed under less stringent conditions for
identification and/or quantitation of closely related DNA or RNA
sequences.
[0056] Full length genes may be cloned utilizing 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 adjacent to a known locus. First, genomic DNA is amplified
in the presence of 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 internal to the first one. Products of each round of PCR are
transcribed with an appropriate RNA polymerase and sequenced using
reverse transcriptase. Gobinda et al present data concerning Factor
IX for which they identified a conserved stretch of 20 nucleotides
in the 3' noncoding region of the gene.
[0057] Inverse PCR is the first method to report successful
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 used as a PCR template.
Divergent primers are designed from the known region. The multiple
rounds of restriction enzyme digestions and ligations that are
necessary prior to PCR make the procedure slow and expensive
(Gobinda et al, supra).
[0058] Capture PCR (Lagerstrom M et al (1991) PCR Methods Applic
1:111-19) is a method for PCR amplification of DNA fragments
adjacent to a known sequence in human and YAC DNA. As noted by
Gobinda et al (supra), capture PCR also requires multiple
restriction enzyme digestions and ligations to place an engineered
double-stranded sequence into an unknown portion of the DNA
molecule before PCR. Although the restriction and ligation
reactions are carried out simultaneously, the requirements for
extension, immobilization and two rounds of PCR and purification
prior to sequencing render the method cumbersome and time
consuming.
[0059] Parker J D 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,
PromoterFinder.TM. a new kit available from Clontech (Palo Alto
Calif.) uses PCR and primers derived from p53 to walk in genomic
DNA. Nested primers and special PromoterFinder libraries are used
to detect upstream sequences such as promoters and regulatory
elements. This process avoids the need to screen libraries and is
useful in finding intron/exon junctions.
[0060] Another new PCR method, "Improved Method for Obtaining Full
Length cDNA Sequences" by Guegler et al, patent application Ser.
No. 08/487,112, filed Jun. 7, 1995 and hereby incorporated by
reference, employs XL-PCR.TM. (Perkin-Elmer, Foster City Calif.) 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 extended (possibly full-length) sequence within 6-10 days. This
new method replaces methods which use labeled probes to screen
plasmid libraries and allow one researcher to process only about
3-5 genes in 14-40 days.
[0061] In the first step, which can be performed in about two days,
any two of a plurality of primers are designed 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
appropriate vector. Step 5, which takes about one day, involves
transforming and growing up host bacteria. In step 6, which takes
approximately five hours, PCR is used to screen bacterial clones
for extended sequence. The final steps, which take about one day,
involve the preparation and sequencing of selected clones.
[0062] 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 preferred library may be one that has
been size-selected to include only larger cDNAs or may consist of
single or combined commercially available libraries, eg. lung,
liver, heart and brain from Gibco/BRL (Gaithersburg Md.). The cDNA
library may have been prepared with oligo d(T) or random priming.
Random primed libraries are preferred in that they will contain
more sequences which contain 5' ends of genes. A randomly primed
library may be particularly useful if an 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.
[0063] 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 Elmer (Foster City
Calif.), Beckman Instruments (Fullerton Calif.), and other
companies. Capillary sequencing employs flowable polymers for
electrophoretic separation, four different fluorescent dyes (one
for each nucleotide) which are laser activated, and detection of
the emitted wavelengths by a charge coupled devise camera.
Output/light intensity is converted to electrical signal using
appropriate software (eg. Genotyper.TM. and Sequence Navigator.TM.
from Perkin Elmer) and the entire process from loading of samples
to computer analysis and electronic data display is computer
controlled. Capillary electrophoresis 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 amounts in a particular sample. The
reproducible sequencing of up to 350 bp of M13 phage DNA in 30 min
has been reported (Ruiz-Martinez MC et al (1993) Anal Chem
65:2851-8).
[0064] 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
stringency of the hybridization conditions will determine whether
the probe 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 the detection of related cysteine protease encoding
sequences, they should preferably contain at least 50% of the
nucleotides of the sequences presented herein. Hybridization probes
of the subject invention may be derived from the nucleotide
sequences of the SEQ ID NO: 1 and 5-24 or from surrounding genomic
sequences comprising untranslated regions such as promoters,
enhancers and introns. Such hybridization probes may be labeled
with appropriate reporter molecules.
[0065] Means for producing specific hybridization probes for
cysteine proteases include oligolabeling, nick translation,
end-labeling or PCR amplification using a labeled nucleotide.
[0066] 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 synthesize RNA
probes in vitro by addition of an appropriate RNA polymerase such
as T7, T3 or SP6 and labeled nucleotides. A number of companies
(such as Pharmacia Biotech, Piscataway N.J.; Promega, Madison Wis.;
U.S Biochemical Corp, Cleveland, Ohio; etc.) supply commercial kits
and protocols for these procedures.
[0067] It is also possible to produce a DNA sequence, or portions
thereof, entirely by synthetic chemistry. Sometimes the source of
information for producing this sequence comes from the known
homologous sequence from closely related organisms. 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 chemistry may
be used to introduce specific mutations into the nucleotide
sequence. Alternatively, a portion of sequence in which a mutation
is desired can be synthesized and recombined with a portion of an
existing genomic or recombinant sequence.
[0068] The ncp nucleotide sequences can be used individually in a
diagnostic test or assay to detect disorder or disease processes
associated with 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 compatible fluid is rinsed off, the reporter
molecule is quantitated and compared with a standard for that
fluid, cell or tissue. If ncp expression is significantly different
from the standard, the assay indicates the presence of disorder or
disease. The form of such qualitative or quantitative methods may
include northern analysis, dot blot or other membrane-based
technologies, dip stick, pin or chip technologies, PCR, ELISAs or
other multiple sample format technologies.
[0069] This same assay, combining a sample with the nucleotide
sequence, is applicable in evaluating the efficacy of a particular
therapeutic treatment regime. It may be used in animal studies, in
clinical trials, or in monitoring the treatment of an individual
patient. First, standard expression must be established for use as
a basis of comparison. Second, samples from the animals 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 therapeutic agent is administered, and
a treatment profile is generated. The assay is evaluated to
determine whether the profile progresses toward or returns to the
standard pattern. Successive treatment profiles may be used to show
the efficacy of treatment over a period of several days or several
months.
[0070] The nucleotide sequence for ncp can also be used to generate
probes for mapping the native genomic 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 of Basic Techniques, Pergamon Press, New York
City), flow-sorted chromosomal preparations, or artificial
chromosome constructions such as yeast artificial chromosomes
(YACs), bacterial artificial chromosomes (BACs), bacterial P1
constructions or single chromosome cDNA libraries.
[0071] In situ hybridization of chromosomal preparations and
physical mapping techniques such as linkage analysis using
established chromosomal markers are invaluable in extending genetic
maps. Examples of genetic maps can be found in Science (1994;
265:1981f). Often the placement of a gene on the chromosome of
another mammalian species may reveal associated markers even if the
number or arm of a particular human chromosome is not known. New
sequences can be assigned to chromosomal arms, or parts thereof, by
physical 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
11q22-23 (Gatti et al (1988) Nature 336:577-580), any sequences
mapping to that area may represent associated or regulatory genes
for further investigation. The nucleotide sequence of the subject
invention may also be used to detect differences in the chromosomal
location due to translocation, inversion, etc. between normal and
carrier or affected individuals.
[0072] 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 Expression Technology Methods and
Enzymology, Vol 185, Academic Press, San Diego Calif.) is one among
many publications which teach expression 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.
[0073] Cells transformed with ncp nucleotide sequence may be
cultured under conditions suitable for the expression and recovery
of peptide from cell culture. The peptide produced by a recombinant
cell may be secreted or may be contained 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 ligating 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 D J et al (1993) DNA Cell Biol 12:441-53).
[0074] Direct peptide synthesis using solid-phase techniques
(Stewart et al (1969) Solid-Phase Peptide Synthesis, W H Freeman
Co, San Francisco Calif.; 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 manufacturer. 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.
[0075] Although an amino acid sequence or oligopeptide used for
antibody induction does not require biological activity, it must be
immunogenic. NCP used to induce specific antibodies may have an
amino acid sequence consisting of at least five amino acids and
preferably at least 10 amino 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 domain.
[0076] Antibodies specific for NCP may be produced by inoculation
of an appropriate 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. Antibody production includes not
only the stimulation of an immune response by injection into
animals, but also analogous processes such as the production of
synthetic antibodies, the screening of recombinant immunoglobulin
libraries for specific-binding molecules (Orlandi R et al (1989)
PNAS 86:3833-3837, or Huse W D et al (1989) Science 256:1275-1281),
or the in vitro stimulation of lymphocyte 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
adapted to produce molecules which specifically bind NCP.
Antibodies or other appropriate molecules generated against a
specific immunogenic peptide fragment or oligopeptide can be used
in Western analysis, enzyme-linked immunosorbent assays (ELISA) or
similar tests to establish the presence of or to quantitate amounts
of NCP active in normal, diseased, CT therapeutically treated cells
or tissues.
[0077] The examples 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 invention.
EXAMPLES
[0078] I Adrenal Gland cDNA Library Construction
[0079] Although both the adrenal gland and human umbilical vein
endothelial cell cDNA libraries were employed to clone the full
length gene; for purposes of example, the adrenal gland cDNA
library construction will be described.
[0080] The adrenal gland cDNA library was constructed from a pooled
sample of five, whole, normal adrenal glands from Caucasian males
and females who ranged in age from 10 to 46 years. The poly A.sup.+
RNA was obtained from Clontech Laboratories Inc (Catalogue #6571-2;
Palo Alto Calif.)
[0081] Stratagene (La Jolla Calif.) made the cDNA library using
this poly A.sup.+ RNA. The cDNA synthesis was primed using both
oligo d(T) and random hexamers, and the two cDNA libraries were
treated separately. Synthetic adapter oligonucleotides were ligated
onto the ends of the cDNAs enabling their insertion into the
Uni-ZAP.TM. vector system (Stratagene).
[0082] The pBluescript.TM. phagemid (Stratagene) cDNA clones were
obtained by the in vivo excision process, and phagemids from the
two cDNA libraries were combined into a single library by mixing
equal numbers of bacteriophage. The latter were used to transform
E. coli host strain XL1-Blue.TM. (Stratagene). Enzymes from both
pBluescript and a cotransformed f1 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
reinfect fresh host cells (SOLR.TM., Stratagene). Presence of the
.beta.-lactamase gene on the phagemid allowed transformed bacteria
to grow on medium containing ampicillin.
[0083] II Isolation of cDNA Clones
[0084] Phagemid DNAs containing the cDNA insert may be purified
using the QIAWELL-8.TM. Plasmid purification system from QIAGEN
(Chatsworth Calif.). This high-throughput method isolates highly
purified phagemid DNA from lysed bacterial cells using QIAGEN
anion-exchange resin particles and EMPORE.TM. membrane technology
from 3M (Minneapolis Minn.) in a multiwell format. The DNA was
eluted and prepared for DNA sequencing and other analytical
manipulations.
[0085] III Sequencing of cDNA Clones
[0086] The cDNA inserts from random isolates of the adrenal gland
library were sequenced in part. Methods for DNA sequencing are well
known in the art and employ such enzymes as the Klenow fragment of
DNA polymerase I, SEQUENASE.RTM. (US Biochemical Corp) or Taq
polymerase. Methods to extend the DNA from an oligonucleotide
primer annealed to the DNA template of interest have been developed
for both single- and double-stranded templates. Chain termination
reaction products were separated using electrophoresis and detected
via their incorporated, labeled precursors. Recent improvements in
mechanized reaction preparation, sequencing and analysis have
permitted expansion in the number of sequences that can be
determined per day. Preferably, the process is automated with
machines such as the Applied Biosystems Catalyst 800 and 373 DNA
sequencers.
[0087] The quality of any particular cDNA library may be determined
by performing a pilot scale analysis of the cDNAs and checking for
percentages of clones containing vector, lambda or E. coli DNA,
mitochondrial or repetitive DNA, and clones with exact or
homologous matches to public databases. The number of unique
sequences, those having no known match in any available database,
are then recorded.
[0088] IV Homology Searching of cDNA Clones and Their Deduced
Proteins
[0089] Each sequence so obtained was compared to sequences in
GenBank using a search algorithm developed by Applied Biosystems
and incorporated into the INHERIT.TM. 670 Sequence Analysis System.
In this algorithm, Pattern Specification Language (TRW Inc, Los
Angeles Calif.) was used to determine regions of homology. The
three parameters that determine how the sequence comparisons run
were window size, window offset, and error tolerance. Using a
combination of these three parameters, the DNA database was
searched for sequences containing regions of homology to the query
sequence, and the appropriate sequences were scored with an initial
value. Subsequently, these homologous regions were examined using
dot matrix homology plots to distinguish regions of homology from
chance matches. Smith-Waterman alignments were used to display the
results of the homology search.
[0090] Peptide and protein sequence homologies were ascertained
using the INHERIT.TM. 670 Sequence Analysis System in a way similar
to that used in DNA sequence homologies. Pattern Specification
Language and parameter windows were used to search protein
databases for sequences containing regions of homology which were
scored with an initial value. Dot-matrix homology plots were
examined to distinguish regions of significant homology from chance
matches.
[0091] Alternatively, BLAST, which stands for Basic Local Alignment
Search Tool, is used to search for local sequence alignments
(Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S F et al
(1990) J Mol Biol 215:403-10). BLAST produces alignments of both
nucleotide and amino acid sequences to determine sequence
similarity. Because of the local nature of the alignments, BLAST is
especially useful in determining exact matches or in identifying
homologs. While it is useful for matches which do not contain gaps,
it is inappropriate for performing motif-style searching. The
fundamental unit of BLAST algorithm output is the High-scoring
Segment Pair (HSP).
[0092] An HSP consists of two sequence fragments of arbitrary but
equal lengths whose alignment is locally maximal and for which the
alignment score meets or exceeds a threshold or cutoff score set by
the user. The BLAST approach is to look for HSPs between a query
sequence and a database sequence, to evaluate the statistical
significance of any matches found, and to report only those matches
which satisfy the user-selected threshold of significance. The
parameter E establishes the statistically significant threshold for
reporting database sequence matches. E is interpreted as the upper
bound of the expected frequency of chance occurrence of an HSP (or
set of HSPs) within the context of the entire database search. Any
database sequence whose match satisfies E is reported in the
program output.
[0093] Using the criteria described above, the following partial
novel cysteine protease molecules were identified and are disclosed
herein: Incyte Clone 1098 (SEQ ID NO 3) from the U937 (a
histiocytic lymphoma cell line) library, Incyte Clone 75848 (SEQ ID
NO 5) from the THP-1 (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 library, 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 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 library--were identified using the criteria above.
The full length nucleic and amino acid sequences for this novel
human cysteine protease are shown in FIGS. 2A, 2B, 2C and 2D. FIGS.
3A and 3B show the alignment between the translated amino acid
sequence for ncp and the closest related cysteine protease,
hemoglobinase from Schistosoma 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.
[0094] V Extension of cDNAs to Full Length
[0095] The Incyte clones presented here can be and were used to
design oligonucleotide primers for extension of the cDNAs to full
length. Primers are designed based on known sequence; one primer is
synthesized to initiate extension in the antisense direction (XLR)
and the other to extend sequence in the sense direction (XLF). The
primers allow the sequence to be extended "outward" generating
amplicons containing new, unknown nucleotide sequence for the gene
of interest. The primers may be designed using Oligo 4.0 (National
Biosciences Inc, Plymouth Minn.), or another appropriate 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 temperatures about
68.degree.-72.degree. C. Any stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was
avoided.
[0096] 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 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 remaining portion of the cysteine protease
sequence.
[0097] 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 primer
and the recommended concentrations of all other components of the
kit, PCR is performed using the Peltier Thermal Cycler (PTC200; MJ
Research, Watertown Mass.) and the following parameters:
[0098] Step 1 94.degree. C. for 1 min (initial denaturation)
[0099] Step 2 65.degree. C. for 1 min
[0100] Step 3 68.degree. C. for 6 min
[0101] Step 4 94.degree. C. for 15 sec
[0102] Step 5 65.degree. C. for 1 min
[0103] Step 6 68.degree. C. for 7 min
[0104] Step 7 Repeat step 4-6 for 15 additional cycles
[0105] Step 8 94.degree. C. for 15 sec
[0106] Step 9 65.degree. C. for 1 min
[0107] Step 10 68.degree. C. for 7:15 min
[0108] Step 11 Repeat step 8-10 for 12 cycles
[0109] Step 12 72.degree. C. for 8 min
[0110] Step 13 4.degree. C. (and holding)
[0111] A 5-10 .mu.l aliquot of the reaction mixture is analyzed by
electrophoresis on a low concentration (about 0.6-0.8%) agarose
mini-gel to determine which reactions were successful in extending
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 involves using a
commercial gel extraction method such as QIAQuick.TM. (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.
[0112] After ethanol precipitation, the products are redissolved in
13 .mu.l of ligation buffer. Then, 1 .mu.l T4-DNA ligase (15 units)
and 1 .mu.l T4 polynucleotide kinase are added, and the mixture is
incubated at room temperature for 2-3 hours or overnight at
16.degree. C. Competent E. coli cells (in 40 .mu.l of appropriate
media) are transformed with 3 .mu.l of ligation mixture and
cultured in 80 .mu.l of SOC medium (Sambrook J et al, supra). After
incubation for one hour at 37.degree. C., the whole transformation
mixture is plated on Luria Bertani (LB)-agar (Sambrook J et al,
supra) containing 2x Carb. The following day, 12 colonies are
randomly picked from each plate and cultured in 150 .mu.l of liquid
LB/2x Carb medium placed in an individual well of an appropriate,
commercially-available, sterile 96-well microtiter plate. The
following day, 5 .mu.l of each overnight culture is transferred
into a non-sterile 96-well plate and after dilution 1:10 with
water, 5 .mu.l of each sample is transferred into a PCR array.
[0113] For PCR amplification, 18 .mu.l of concentrated PCR reaction
mix (3.3x) containing 4 units of rTth DNA polymerase, a vector
primer and one or both of the gene specific primers used for the
extension reaction are added to each well. Amplification is
performed using the following conditions:
[0114] Step 1 94.degree. C. for 60 sec
[0115] Step 2 94.degree. C. for 20 sec
[0116] Step 3 55.degree. C. for 30 sec
[0117] Step 4 72.degree. C. for 90 sec
[0118] Step 5 Repeat steps 2-4 for an additional 29 cycles
[0119] Step 6 72.degree. C. for 180 sec
[0120] Step 7 4.degree. C. (and holding)
[0121] Aliquots of the PCR reactions are run on agarose gels
together with molecular weight markers. The sizes of the PCR
products are compared to the original partial cDNAs, and
appropriate clones are selected, ligated into plasmid and
sequenced.
[0122] VI Diagnostic Assay Using NCP Specific Oligomers
[0123] In those cases where a specific condition (see definitions
supra) is suspected to involve altered quantities of ncp, oligomers
may be designed to establish the presence and/or quantity OF mRNA
expressed in a biological sample. There are several methods
currently being used to quantitate the expression of a particular
molecule. Most of these methods use radiolabeled (Melby P C et al
1993 J Immunol Methods 159:235-44) or biotinylated (Duplaa C et al
199.3 Anal Biochem 229-36) nucleotides, coamplification of a
control nucleic acid, and standard curves onto which the
experimental results are interpolated. Quantitation may be speeded
up by running the assay in an ELISA format where the
oligomer-of-interest is presented in various dilutions and a
calorimetric response gives rapid quantitation. For example, NCP
deficiency may result in an abundance of the proinflammatory
interleukin molecules, much swelling and discomfort. In like
manner, overexpression may cause apoptosis 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 to monitor progress of the patient as
his/her physiological situation moves toward the normal range
during therapy.
[0124] VII Sense or Antisense Molecules
[0125] 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 H F Lodish 1993) Mol Cell Biol
13:98-104) or antisense (Eguchi et al (1991) Annu Rev Biochem
60:631-652) technologies for the investigation or alteration of
gene expression. To inhibit in vivo or in vitro ncp expression, an
oligonucleotide based on the coding sequence of a fragment of an
ncp designed 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 expression.
Furthermore, antisense molecules can be designed to inhibit
promoter binding in the upstream nontranslated leader or at various
sites along the ncp coding region. Alternatively, antisense
molecules may be designed to inhibit translation of an mRNA into
polypeptide by preparing an oligomer or fragment which will bind in
the region spanning approximately -10 to +10 nucleotides at the 5'
end of the coding sequence. These technologies are now well known
in the art.
[0126] In addition to using fragments constructed to interrupt
transcription of the open reading frame, modifications of gene
expression can be obtained by designing antisense sequences to
enhancers, introns, or even 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 compromises the ability of the double helix to open
sufficiently for the binding of polymerases, transcription factors,
or regulatory molecules.
[0127] Any of these types of antisense molecules may be placed in
expression vectors and used to transform preferred cells or
tissues. This may include introduction of the expression vector
into a synovial cavity for transient or short term therapy.
Expression of the antisense sequence would continue to flood the
cell with inhibitory molecules until all copies of the vector were
disabled by endogenous nucleases. Such transient expression may
last for a month or more with a non replicating vector and three
months or more if appropriate replication elements are used in the
transformation or expression system.
[0128] Stable transformation of appropriate dividing cells with a
vector containing the antisense molecule can produce a transgenic
cell line, tissue or organism (see, for example, Trends in
Biotechnol 11:155-215 (1993) and U.S Pat. No. 4,736,866, Apr. 12,
1988). Those cells which assimilate or replicate enough copies of
the vector to allow stable integration will also produce enough
antisense molecules to compromise or entirely eliminate 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 morphology, etc. at the cellular,
tissue or organismal level.
[0129] VIII Expression of NCP
[0130] Expression of the NCP may be accomplished by subcloning the
cDNAs into appropriate vectors and transfecting the vectors into
host cells. In this case, the cloning vector previously used for
the generation of the tissue library also provides for direct
expression of the ncp sequence in E. coli. Upstream of the cloning
site, this vector contains a promoter for .beta.-galactosidase,
followed by sequence containing the amino-terminal Met and the
subsequent 7 residues of .beta.-galactosidase. Immediately
following these eight residues is a bacteriophage promoter useful
for transcription and a linker containing a number of unique
restriction sites.
[0131] Induction of an isolated, transfected bacterial strain with
IPTG using standard methods will produce a fusion protein
corresponding to the first seven residues of .beta.-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 essentially random process, there is one chance in three that
the included cDNA will lie in the correct frame for proper
translation. If the cDNA is not in the proper reading frame, it can
be obtained by deletion or insertion of the appropriate number of
bases by well known methods including in vitro mutagenesis,
digestion with exonuclease III or mung bean nuclease, or
oligonucleotide linker inclusion.
[0132] The cDNA can be shuttled into other vectors known to be
useful for expression of protein in specific hosts. Oligonucleotide
linkers containing cloning sites as well as a stretch of DNA
sufficient to hybridize to the end of the target cDNA (25 bases)
can be synthesized chemically by standard methods. These primers
can then used to amplify the desired gene fragments by PCR. The
resulting fragments can be digested with appropriate restriction
enzymes under standard conditions and isolated by gel
electrophoresis. Alternatively, similar gene fragments can be
produced by digestion of the cDNA with appropriate restriction
enzymes and filling in the missing gene sequence with chemically
synthesized oligonucleotides. Partial nucleotide sequence from more
than one cysteine protease homolog can be ligated together and
cloned into appropriate vectors to optimize expression.
[0133] Suitable expression hosts for such chimeric molecules
include but are not limited to mammalian cells such as Chinese
Hamster Ovary (CHO) and human 293 cells, insect cells such as Sf9
cells, yeast cells such as Saccharomyces 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
.beta.-lactamase antibiotic resistance 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 expression hosts may require RNA processing elements
such as 3' polyadenylation sequences if such are not part of the
cDNA of interest.
[0134] If native promoters are not part of the cDNA, other host
specific promoters may be specifically combined with the coding
region of ncp. They include MMTV, SV40, and metallothiomine
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 enhancers, such as
the rous sarcoma virus (RSV) enhancer, may be used in mammalian
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.
[0135] IX Isolation of Recombinant NCP
[0136] 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 chelating 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
extension/affinity purification system (Immunex Corp, Seattle
Wash.). 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 expression of NCP.
[0137] X Cysteine Protease Activity
[0138] The activity of purified or expressed NCP may be tested by
mixing a known quantity 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 appropriate period of
time. A zymogram, which consists of a nondenaturing polyacrylamide
gel soaked in the proteinaceous material onto which various
concentrations, preferably between 10 and 100 ng/.mu.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).
[0139] XI Identification of or Production of NCP Specific
Antibodies
[0140] 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 immunize 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
immunize a rabbit. For identifying mouse hybridomas, the denatured
protein can be radioiodinated 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.
[0141] In a monoclonal approach, the amino acid sequence of NCP, as
deduced from translation of the cDNA, is analyzed to determine
regions of high immunogenicity. Oligopeptides comprising
appropriate hydrophilic regions, as shown in FIGS. 3A and 3B, are
synthesized and used in suitable immunization protocols to raise
antibodies. Analysis to select appropriate epitopes is described by
Ausubel FM et al (supra). The optimal amino acid sequences for
immunization are usually at the C-terminus, the N-terminus and
those intervening, hydrophilic regions of the polypeptide which are
likely to be exposed to the external environment when the protein
is in its natural conformation.
[0142] Typically, selected peptides, about 15 residues in length,
are synthesized using an Applied Biosystems Peptide Synthesizer
Model 431A using fmoc-chemistry and coupled to keyhole limpet
hemocyanin (KLH, Sigma) by reaction with
M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel F M et
al, supra). If necessary, a cysteine may be introduced at the
N-terminus of the peptide to permit coupling to KLH. Rabbits are
immunized with the peptide-KLH complex in complete Freund's
adjuvant. The resulting antisera are tested for antipeptide
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.
[0143] Hybridomas may also be prepared 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
of plates (FAST; Becton-Dickinson, Palo Alto, Calif.) 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 incubation the wells are exposed to labeled NCP, 1 mg/ml.
Clones producing antibodies will bind a quantity of labeled NCP
which is detectable above background. Such clones are expanded and
subjected to 2 cycles of cloning at limiting 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 least 10.sup.8/M, preferably
10.sup.9 to 10.sup.10 or stronger, will typically be made by
standard procedures as described in Harlow and Lane (1988)
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
Cold Spring Harbor N.Y.; and in Goding (1986) Monoclonal
Antibodies: Principles and Practice, Academic Press, New York City,
both incorporated herein by reference.
[0144] XII Diagnostic Test Using NCP Specific Antibodies
[0145] Particular NCP antibodies are useful for the diagnosis of
prepathologic conditions, and chronic or acute diseases which are
characterized by differences in the amount or distribution of NCP.
To date, NCP has been found in many libraries where it is
predominantly associated with organ function, inflammation or
defense.
[0146] Diagnostic tests for NCP include methods utilizing the
antibody and a label to detect NCP in human body fluids, tissues or
extracts of such tissues. The polypeptides and 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. Suitable reporter molecules or labels include
those radionuclides, enzymes, fluorescent, chemiluminescent, or
chromogenic agents previously mentioned as well as substrates,
cofactors, inhibitors, magnetic particles and the like. Patents
teaching the use of such labels include U.S Pat. Nos. 3,817,837;
3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and
4,366,241. Also, recombinant immunoglobulins may be produced as
shown in U.S Pat. No. 4,816,567, incorporated herein by
reference.
[0147] A variety of protocols for measuring soluble or
membrane-bound NCP, using either polyclonal or monoclonal
antibodies specific for the respective protein are known in the
art. Examples include enzyme-linked immunosorbent assay (ELISA),
radioimmunoassay (RIA) and fluorescent activated cell sorting
(FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
NCP is preferred, but a competitive binding assay may be employed.
These assays are described, among other places, in Maddox, D E et
al (1983, J Exp Med 158:1211).
[0148] XIII Purification of Native NCP Using Specific
Antibodies
[0149] 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.
[0150] Polyclonal immunoglobulins are prepared from immune sera
either by precipitation with ammonium sulfate or by purification on
immobilized Protein A (Pharmacia Biotech). Likewise, monoclonal
antibodies are prepared from mouse ascites fluid by ammonium
sulfate precipitation or chromatography on immobilized Protein A.
Partially purified immunoglobulin is covalently attached to a
chromatographic resin such as CnBr-activated Sepharose (Pharmacia
Biotech). The antibody is coupled to the resin, the resin is
blocked, and the derivative resin is washed according to the
manufacturer's instructions.
[0151] Such immunoaffinity columns may be utilized in the
purification of NCP by preparing a fraction from cells containing
NCP in a soluble form. This preparation 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 art.
Alternatively, soluble NCP containing a signal sequence may be
secreted in useful quantity into the medium in which the cells are
grown.
[0152] A soluble NCP-containing preparation is passed over the
immunoaffinity 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 conditions that disrupt antibody/NCP binding (eg, a
buffer of pH 2-3 or a high concentration of a chaotrcope such as
urea or thiocyanate ion), and NCP is collected.
[0153] XIV Drug Screening
[0154] This invention is particularly useful for screening
therapeutic compounds by using binding fragments of NCP in any of a
variety of drug screening techniques. The peptide fragment employed
in such a test may either be free in solution, affixed to a solid
support, borne on a cell surface or located intracellularly. One
may measure, for example, the formation of complexes between NCP
and the agent being tested. Alternatively, one can examine the
diminution in complex formation between NCP and a receptor caused
by the agent being tested.
[0155] Methods of screening for drugs or any other agents which can
affect macrophage activation comprise contacting such an agent with
NCP fragment and assaying for the presence of a complex between the
agent and the NCP fragment. In such assays, the NCP fragment is
typically labeled. After suitable incubation, free NCP fragment is
separated from 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.
[0156] 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 European
Patent Application 84/03564, published on Sep. 13, 1984,
incorporated herein by reference. Briefly stated, large numbers of
different small peptide test compounds are synthesized 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 detected 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.
[0157] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding NCP specifically compete with a test compound for binding
to NCP fragments. In this manner, the antibodies can be used to
detect the presence of any peptide which shares one or more
antigenic determinants with NCP.
[0158] XV Identification of Molecules Which Interact with NCP
[0159] 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 preferred method involves
labeling the primary amino groups in NCP with .sup.125I
Bolton-Hunter reagent (Bolton, A. E. and Hunter, W M (1973) Biochem
J 133: 529). This reagent has been used to label various molecules
without concomitant loss of biological activity (Hebert C A et al
(1991) J Biol Chem 266: 18989-94; McColl S et al (1993) J Immunol
150:4550-4555). Membrane-bound molecules are incubated with the
labeled NCP molecules, washed to removed unbound molecules, and the
NCP complex is quantified. Data obtained using different
concentrations of NCP are used to calculate values for the number,
affinity, and association of NCP.
[0160] Labeled NCP fragments are also useful as a reagent for the
purification of molecules 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 column. 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 N-terminal protein sequencing or other
identification procedure. If the captured molecule has an amino
acid sequence, it can be used to design degenerate oligomers for
use in cloning the gene from an appropriate cDNA library.
[0161] In an alternate method, monoclonal antibodies raised against
NCP fragments are screened to identify those which inhibit the
binding of labeled NCP. These monoclonal antibodies are then used
in affinity purification or expression cloning of associated
molecules. Other soluble binding molecules are identified in a
similar manner. Labeled NCP is incubated with extracts or other
appropriate materials 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 exclusion
chromatography or density gradient centrifugation and are purified
by methods known in the art. The soluble binding protein(s) are
subjected to N-terminal sequencing to obtain information sufficient
for database identification, if the soluble protein is known, or
for cloning, if the soluble protein is unknown.
[0162] XVI Use and Administration of Antibodies or Inhibitors to
NCP
[0163] The antibodies and inhibitors can provide different effects
when administered therapeutically. The antibodies and inhibitors
are used to lessen or eliminate undue damage caused by disorders or
diseases associated with upregulated NCP expression. Each of these
molecules or treatments (TSTs) will be formulated in a nontoxic,
inert, pharmaceutically 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 being formulated and the
condition to be treated. Characteristics of TSTs include solubility
of the molecule, half-life, antigenicity/immunogenicit- y and the
ability of the inhibitor to reach its target(s). These and other
characteristics may aid in defining an 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.
[0164] TSTs may be delivered by known routes of administration
including but not limited to topical creams and gels; transmucosal
spray and aerosol; transdermal patch and bandage; injectable,
intravenous and lavage formulations; and orally administered
liquids and pills particularly formulated to resist stomach acid
and enzymes. The particular formulation, exact dosage, and route of
administration will be determined by the attending physician and
will vary according to each specific situation.
[0165] Such determinations are made by considering multiple
variables such as the condition to be treated, the TST to be
administered, 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 administration, drug combination,
reaction sensitivities, and tolerance/response to therapy. Long
acting TST formulations might be administered every 3 to 4 days,
every week, or once every two weeks depending on half-life and
clearance rate of the particular TST.
[0166] 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 administration. Guidance as to particular dosages and
methods of delivery is provided in the literature. See U.S. Pat.
No. 4,657,760; 5,206,344; or 5,225,212. Those skilled in the art
will employ different formulations for different TSTs.
Administration to lung cells may necessitate delivery in a manner
different from that to kidney or other cells.
[0167] It is contemplated that conditions associated with altered
NCP expression are treatable with TSTs. These conditions, which
specifically include, but are not limited to, anemia,
arteriosclerosis, asthma, bronchitis, emphysema, gingivitis,
inflammatory bowel disease, insulin-dependent diabetes mellitus,
leukemia, multiple endocrine neoplasias, osteoarthritis,
osteoporosis, pulmonary fibrosis, rheumatoid arthritis, septic
shock syndromes, and systemic lupus erythematosus may be
specifically diagnosed by the tests discussed above. In addition,
such tests may be used to monitor treatment.
[0168] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent 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 the above-described modes for carrying out
the invention which are obvious to those skilled in the field of
molecular biology or related fields are intended to be within the
scope of the following claims.
Sequence CWU 1
1
* * * * *