U.S. patent application number 11/598473 was filed with the patent office on 2007-03-15 for regulators of type-1 tumor necrosis factor receptor and other cytokine receptor shedding.
This patent application is currently assigned to THE GOVERNMENT OF THE USA OF AMERICA, REP. BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES. Invention is credited to Stewart Levine.
Application Number | 20070059758 11/598473 |
Document ID | / |
Family ID | 22681605 |
Filed Date | 2007-03-15 |
United States Patent
Application |
20070059758 |
Kind Code |
A1 |
Levine; Stewart |
March 15, 2007 |
Regulators of type-1 tumor necrosis factor receptor and other
cytokine receptor shedding
Abstract
The present invention provides compositions and methods for the
regulation of cytokine signaling through the Tumor Necrosis Factor
(TNF) pathway. Specifically, the invention provides a novel gene,
polypeptide and related compositions and methods for the regulation
of ectodomain shedding. In preferred embodiments, methods and
compositions for the regulation of TNF Type-1 Receptor ectodomain
shedding are provided. The present invention finds use in
therapeutics, diagnostics, and drug screening applications.
Inventors: |
Levine; Stewart; (North
Potomac, MD) |
Correspondence
Address: |
LEYDIG, VOIT & MAYER, LTD.
TWO PRUDENTIAL PLAZA, SUITE 4900
180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6731
US
|
Assignee: |
THE GOVERNMENT OF THE USA OF
AMERICA, REP. BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN
SERVICES
Rockville
MD
|
Family ID: |
22681605 |
Appl. No.: |
11/598473 |
Filed: |
November 13, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10220443 |
Dec 19, 2002 |
7135303 |
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PCT/US01/06464 |
Feb 28, 2001 |
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11598473 |
Nov 13, 2006 |
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60185586 |
Feb 28, 2000 |
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Current U.S.
Class: |
435/6.16 ;
435/320.1; 435/325; 435/69.1; 530/350; 530/388.22; 536/23.5 |
Current CPC
Class: |
A61K 39/00 20130101;
C12N 9/6421 20130101; C07H 21/04 20130101; C12Q 1/6883 20130101;
C12Q 2600/158 20130101; A61K 48/00 20130101; C07K 14/7151 20130101;
C12N 15/1133 20130101; C07K 16/40 20130101; C12Q 2600/136 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 530/350; 536/023.5; 530/388.22 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C07K 14/715 20060101 C07K014/715; C07K 16/28 20060101
C07K016/28 |
Claims
1. An isolated nucleic acid having the nucleotide sequence set
forth in SEQ ID NO:1.
2. An isolated polypeptide having the amino acid sequence set forth
in SEQ ID NO:2.
3. An isolated nucleic acid encoding the polypeptide set forth in
SEQ ID NO:2.
4. A recombinant vector comprising the nucleic acid of claim 3.
5. A host cell comprising the recombinant vector of claim 4,
wherein said host cell is selected from the group consisting of
prokaryotic host cells and eukaryotic host cells.
6. An antibody directed against at least a portion of said
polypeptide of claim 2, wherein said antibody is selected from the
group consisting of monoclonal antibodies and polyclonal
antibodies.
7. An isolated nucleic acid substantially homologous to the nucleic
acid of claim 1, wherein said nucleic acid is capable of
hybridizing under high stringency conditions to the nucleic acid of
claim 1.
8. The isolated nucleic acid of claim 7, wherein said nucleic acid
encodes a polypeptide having the ability to regulate the shedding
of the extracellular domain of at least one cytokine receptor.
9. The isolated nucleic acid of claim 8, wherein said cytokine
receptor is selected from the group consisting of type-1 tumor
necrosis factor receptor, type I interleukin-1 cytokine receptor,
type II interleukin-1 cytokine receptor, and interleukin-6 cytokine
receptor alpha-chain gp80.
10. A method for the isolated of an amplifiable nucleic acid
substantially homologous to the nucleic acid of claim 1,
comprising: a) providing: i) a sample comprising template nucleic
acid suspected of encoding a gene substantially homologous to the
nucleic acid of claim 1, and ii) at least two primers; b) annealing
said primers to said template nucleic acid; c) extending said
primers with reiterated DNA synthesis under conditions such that
said template nucleic acid is amplified to produce an amplified
product; and d) visualizing and isolating said amplified
product.
11. The amplified product of claim 10.
12. The amplified product of claim 11, wherein said amplified
product encodes a polypeptide having the ability to regulate the
shedding of the extracellular domain of at least one cytokine
receptor.
13. The amplified product of claim 12, wherein said cytokine
receptor is selected from the group consisting of type-1 tumor
necrosis factor receptor, type I interleukin-1 cytokine receptor,
type II interleukin-1 cytokine receptor, and interleukin-6 cytokine
receptor alpha-chain gp80.
14. A method for regulating the shedding of the extracellular
domain of at least one cytokine receptor, comprising the steps of:
a) providing: i) a recombinant vector comprising the isolated
nucleic acid of claim 3 in sense orientation, ii) a first tissue
containing one or more cells expressing a cytokine receptor, and
iii) a second tissue comprising one or more cells capable of
expressing the polypeptide encoded by said recombinant vectors; and
b) delivering said vector to said cells of said second tissue in
the presence of said first tissue, under conditions which result in
regulation of shedding of said cytokine receptor from cells of said
first tissue.
15. The method of claim 14, wherein said cytokine receptor is
selected from the group consisting of type-1 tumor necrosis factor
receptor, type I interleukin-1 cytokine receptor, type II
interleukin-1 cytokine receptor, and interleukin-6 cytokine
receptor alpha-chain gp80.
16. The method of claim 14, wherein said delivering said vector to
said second tissue comprises a means of intracellular delivery
selected from the group consisting of direct nucleic acid
administration, liposome administration, viral vector delivery, and
ex vivo gene delivery followed by transplantation.
17. A method for regulating the shedding of the extracellular
domain of at least one cytokine receptor, comprising the steps of:
a) providing: i) the recombinant vector comprising at least a
transcribeable portion of the isolated nucleic acid of claim 1 in
an antisense orientation, ii) a first tissue comprising one or more
cells expressing a cytokine receptor, and iii) a second tissue
comprising one or more cells expressing the endogenous polypeptide
of SEQ ID NO:2, and one or more cells capable of transcribing said
antisense nucleic acid; and b) delivering said vector to said
second tissue in the presence of said first tissue, under
conditions that result in regulation of shedding of said cytokine
receptor from the cells of said first tissue.
18. The method of claim 17, wherein said cytokine receptor is
selected from the group consisting of type-1 tumor necrosis factor
receptor, type I interleukin-1 cytokine receptor, type II
interleukin-1 cytokine receptor, and interleukin-6 cytokine
receptor alpha-chain gp80.
19. The method of claim 17, wherein said delivering said vector to
said second tissue comprises a means of intracellular delivery
selected from the group consisting of direct nucleic acid
administration, liposome administration, viral vector delivery, and
ex vivo gene delivery followed by transplantation.
20. A method for regulating the shedding of the extracellular
domain of at least one cytokine receptor, comprising the steps of:
a) providing: i) the antibody of claim 6, and ii) a tissue
comprising one or more cells expressing a cytokine receptor and the
endogenous polypeptide of SEQ ID NO:2; and b) delivering said
antibody to said tissue under conditions such that said antibody
regulates the shedding of said cytokine receptor from the surface
of said cells of said tissue.
21. The method of claim 20, wherein said cytokine receptor is
selected from the group consisting of type-1 tumor necrosis factor
receptor, type I interleukin-1 cytokine receptor, type II
interleukin-1 cytokine receptor, and interleukin-6 cytokine
receptor alpha-chain gp80.
22. The method of claim 20, wherein said means of delivery is
selected from the group consisting of oral administration,
intra-arterial injection, intravenous injection, intramuscular
injection, intraperitoneal injection, subcutaneous injection,
suppository, local surgical administration, systemic surgical
administration, catheter, and any combination of these means of
delivery.
23. A method for treating a subject, comprising the steps of: a)
providing: i) a composition selected from the group consisting of a
recombinant vector comprising an isolated nucleic acid encoding the
polypeptide set forth in SEQ ID NO:2 in the sense orientation, a
recombinant vector comprising at least a portion of an isolated
nucleic acid having the nucleotide sequence set forth in SEQ ID
NO:1 in the antisense orientation, an isolated polypeptide having
the amino acid sequence set forth in SEQ ID NO:2, and an antibody
directed to at least a portion of the polypeptide having the amino
acid set forth in SEQ ID NO:2, ii) a subject, and iii) a means of
delivery of said composition to at least one tissue of said
subject; and b) delivering said composition to said subject using
said means of delivery.
24. The method of claim 23, wherein said subject is selected from
the group consisting of a subject displaying pathology resulting
from abnormal cytokine activity, a subject suspected of displaying
pathology resulting from abnormal cytokine activity, and a subject
at risk of displaying pathology resulting from abnormal cytokine
activity.
25. The method of claim 24, wherein said cytokine activity is
mediated by a cytokine selected from the group consisting of tumor
necrosis factor a, interleukin-1 alpha, interleukin-1 beta, and
interleukin-6.
26. The method of claim 23, wherein said subject is a human.
27. The method of claim 23, wherein said means of delivery is
selected from the group consisting of oral administration,
intra-arterial injection, intravenous injection, intramuscular
injection, intraperitoneal injection, subcutaneous injection,
suppository, local surgical administration, systemic surgical
administration, catheter, and any combination of these means of
delivery.
28. The method of claim 27, wherein said means of delivery is
further selected from the group consisting of direct nucleic acid
administration, liposome administration, viral vector delivery, and
ex vivo gene delivery followed by transplantation.
29. A means for detecting an ARTS-1 mRNA in a sample, wherein said
means comprises at least a portion of the nucleic acid of claim 1
complementary to at least a portion of said ARTS-1 mRNA, and
further wherein said nucleic acid is a probe.
30. The means of claim 29, wherein said means comprises Northern
blotting.
31. The means of claim 29, wherein said sample is a tissue sample
from a subject.
32. A means for detecting an ARTS-1 polypeptide in a sample,
wherein said means comprises the antibody of claim 6.
33. The means of claim 32, wherein said means comprises Western
immunoblotting.
34. The means of claim 32, wherein said means comprises an
enzyme-linked immunosorbent assay.
35. The means of claim 32, wherein said sample is a tissue sample
from a subject.
36. A diagnostic kit comprising a means to measure ARTS-1
expression, wherein said means is selected from the group
consisting of a probe comprising at least a portion of an isolated
nucleic acid having the nucleotide sequence set forth in SEQ ID
NO:1 complementary to at least a portion of ARTS-1 mRNA and a
monoclonal or polyclonal antibody directed to at least a portion of
an isolated polypeptide having the amino acid sequence set forth in
SEQ ID NO:2.
37. A method for drug screening to identify drugs having the
ability regulate ARTS-1 expression comprising the steps of: a)
providing: i) a drug, ii) cultured cells, and iii) a means to
measure ARTS-1 expression, wherein said means is selected from the
group consisting of a probe comprising at least a portion of an
isolated nucleic acid having the nucleotide sequence set forth in
SEQ ID NO:1 complementary to at least a portion of ARTS-1 mRNA and
a monoclonal or polyclonal antibody directed to at least a portion
of an isolated polypeptide having the amino acid sequence set forth
in SEQ ID NO:2; b) exposing said cells to said drug; and c) using
said means to measure ARTS-1 expression.
38. The method of claim 37, wherein said cultured cells are human
NCI-H292 pulmonary mucoepidermoid carcinoma cells.
39. A method for drug screening to identify drugs capable of
regulating the peptidase activity of ARTS-1, comprising the steps
of: a) providing: i) purified ARTS-1 polypeptide, ii) an amino acid
p-nitroaniline, iii) a means to measure amino acid p-nitroaniline
cleavage, and iv) a drug; b) exposing said purified ARTS-1
polypeptide to said amino acid p-nitroaniline in the absence and
presence of said drug; and c) measuring amino acid p-nitroaniline
cleavage in the absence and presence of said drug.
40. The method of claim 39, wherein said purified ARTS-1
polypeptide comprises glutathione-S-transferase.
41. The method of claim 39, wherein said amino acid p-nitroaniline
is selected from the group consisting of isoleucine p-nitroanilide,
phenylalanine p-nitroanilide and glycine p-nitroanilide.
42. The method of claim 39, wherein said means to measure amino
acid p-nitroaniline cleavage comprises measuring absorbance at 380
nm.
43. A method for drug screening to identify drugs capable of
regulating the shedding of a cytokine receptor, comprising the
steps of: a) providing: i) cultured cells expressing a cytokine
receptor, ii) a means to quantitate the concentration of the
soluble form of said cytokine receptor in the supernatants of said
cultured cells, and iii) a drug; b) culturing said cells in the
absence and presence of said drug; c) quantitating the
concentration of said soluble form of said cytokine receptor in
said supernatants of said cultured cells using said means; and d)
comparing the concentrations of said soluble cytokine receptor in
said supernatants of said cell cultures in the absence and presence
of drug.
44. The method of claim 43, wherein said cytokine receptor is
selected from the group consisting of type-1 tumor necrosis factor
receptor, type II interleukin-1 cytokine receptor, and
interleukin-6 cytokine receptor alpha-chain gp80.
45. The method of claim 43, wherein said means to quantitate the
concentration of the soluble form of a cytokine receptor is an
enzyme-linked immunosorbent assay.
46. The method of claim 43, wherein said cultured cells are
cultured human NCI-H292 pulmonary mucoepidermoid carcinoma cells.
Description
[0001] This application is a divisional of copending U.S. patent
application Ser. No. 10/220,443, filed Dec. 19, 2002; which is a
national stage application of PCT/US01/06464, filed Feb. 28, 2001;
which is an application claiming the benefit under 35 USC 119(e) of
U.S. Provisional Patent Application No. 60/185,586, filed Feb. 28,
2000.
[0002] This invention was made during the course of work supported
by the United States Government under the National Institutes of
Health. As such, the United States Government may have certain
rights to this invention.
FIELD OF THE INVENTION
[0003] The present invention provides compositions and methods
related to regulation of cytokine signaling through the Tumor
Necrosis Factor (TNF) pathway. Specifically, the invention provides
novel genes, polypeptides and related compositions and methods for
the regulation of TNF Type-1 Receptor ectodomain shedding. It is
contemplated that the compositions and methods of the present
invention will also find use in the regulation of ectodomain
shedding of other cytokine receptors. It is further contemplated
that these compositions and methods will find use in therapeutics
for the treatment of diseases and disorders of the immune
system.
BACKGROUND OF THE INVENTION
[0004] Aberrant regulation of cytokine signaling results in a wide
variety of hyper-inflammatory, autoimmune and immune-deficiency
pathological conditions. Cytokines are a large and diverse group of
molecules which mediate interactions between cells, ultimately
regulating the wide variety of cells of the immune repertoire.
Cytokine signaling mediates numerous facets of normal immune system
physiology, including development, response, activation,
maintenance, memory and apoptosis (Roitt et al. (Eds.), Immunology,
Fifth Edition, Mosby International Publishers [1998]).
Tumor Necrosis Factor
[0005] Tumor necrosis factor-a (commonly written as TNF, but also
written as tumor necrosis factor or TNF.alpha.) is a
multifunctional cytokine mediating pleiotropic biological functions
in both health and disease states. TNF is secreted primarily by
monocytes and macrophages, but can also be secreted by other cell
types. The list of processes regulated by TNF is extensive, and
includes inflammation, immunoregulation, cytotoxicity and antiviral
effects (See e.g., Vilcek et al., J. Biol. Chem., 266:7313-7316
[1991]). TNF plays an integral role in destroying tumors, mediating
responses to tissue injury, and protecting hosts from infections by
various microorganisms (Vassali, Ann. Rev. Immunol., 10:411-452
[1992]). TNF also induces the transcriptional activation of
numerous genes, including NF-icB and AP-1, with the consequent
expression of pro-inflammatory and immunoregulatory genes (Rothe et
al, Cell 83:1243-124 [1995]; Varfolomeev et al, J. Exp. Med.,
183:1271-1275 [1996]; Chinnaiyan et al, J. Biol Chem.,
271:4961-4965 [1996]; Hsu et al, Immunity 4:387-396 [1996]; and Hsu
et al, Cell 84:299-308 [1996]). TNF-mediated NF-KB activation is
also an important negative feedback mechanism regulating apoptosis
(Beg and Baltimore, Science 274:782-784 [1996]; Van Antwerp et al,
Science 274:787-789 [1996]; and Wang et al, Science 274:784-787
[1996]).
TNF in Disease
[0006] TNF has also been implicated in the pathogenesis of a
variety of diseases and disorders. It is theorized that these
pathologies result from the aberrant regulation of TNF activity, in
which the pathologies manifest as a result of excessive or
insufficient TNF activity. Among the activities for which TNF is
most noted are its pro-inflammatory actions, sometimes termed the
"acute phase immune response." Unfortunately, if not properly
regulated, these proinflammatory responses can result in tissue
injury and chronic inflammatory diseases, such as rheumatoid
arthritis, inflammatory bowel disease, septic shock, cachexia,
autoimmune disorders, graft-versus-host disease and insulin
resistance (Piguet et al, J. Exp. Med., 166:1280 [1987];
Pujol-Borrell et al, Nature 326:304-306 [1987]; Tracey et al,
Nature 330:662-664 [1987]; Oliff, Cell 54:141-142 [1988]; Vilcek et
al, J. Biol Chem., 266:7313-7316 [1991]; and Eigler et al, Immunol
Today 18:487-92 [1997]). Excessive TNF activity results in the
detrimental effects of an exaggerated immune response demonstrated
in some of these diseases, exemplified by overstimulation of
interleukin-6 and granulocyte/macrophage-colony stimulating factor
(GM-CSF) secretion, enhanced cytotoxicity of polymorphonuclear
neutrophils, prolonged expression of cellular adhesion molecules,
induction of procoagulant activity on vascular endothelial cells,
increased adherence of neutrophils and lymphocytes, and stimulation
of the release of platelet activating factor from macrophages,
neutrophils and vascular endothelial cells (Vassali, Ann. Rev.
Immunol., 10:411-452 [1992]; Vilcek et al, J. Biol. Chem.,
266:7313-7316 [1992]; and Barbara et al, Immunol. and Cell Biol,
74:434-443 [1996]).
[0007] Recent evidence also implicates TNF activity in the
pathogenesis of many infections. TNF is thought to play a central
role in the pathophysiological consequences of Gram-negative sepsis
and endotoxic shock, including fever, malaise, anorexia, and
cachexia (Beutler et al, Nature 316:552-554 [1985]; Bauss et al,
Infect. Immun., 55:1622-1625 [1987]; Tracey et al, Nature
330:662-664 [1987]; and Vassali, Ann. Rev. Immunol, 10:411-452
[1992]). Because TNF can mimic many of the biological effects of
endotoxin, it is theorized that TNF is a central mediator
responsible for the clinical manifestations of endotoxin-related
and other critical illnesses (Waage et al, Lancet 1:355-357 [1987];
Cerami et al, Immunol Today 9:28 [1988]; Mitchie et al, N. Eng. J.
Med., 318:1481-1486 [1988]; Revhaug et al, Arch. Surg., 123:162-170
[1988]; and Michie et al., Ann. Surg., 209:19-24 [1989]).
Tumor Necrosis Factor Receptor
[0008] The numerous biological effects of TNF are now know to be
mediated by two transmembrane receptors, the 55 kilodalton Type I
receptor (also written as "CD120a," and referred to herein as
"TNFR1") and the 75 kilodalton Type II receptor (also written as
"CD120b," and referred to herein as TNFR2). Although both TNFR1 and
TNFR2 demonstrate strong affinity for TNF.alpha., these two
receptors demonstrate no apparent homology in their cytoplasmic
(i.e., intracellular) domains. This fact is consistent with the
observation that these two receptors transduce different signals to
the nucleus via distinct signaling intermediates (Lewis et al.,
Proc. Natl Acad Sci. USA 88:2830-2834 [1991]; Tartaglia and
Goeddel, Immunol. Today 13:151-153 [1992]; and Barbara et al,
Imunol Cell Biol, 74:434-443 [1996]).
[0009] Soluble TNF inhibitors have been identified in normal human
urine, as well as in sera and other body fluids of patients with
infectious, neoplastic and immunologic disorders. This observation
ultimately led to the revelation that these soluble TNF inhibitors
were actually the extracellular domains of TNF receptors derived by
proteolytic cleavage of the transmembrane forms (Engelmann et al,
J. Biol Chem., 264:11974-11980 [1989]; Olsson et al, Eur. J.
Haematol, 42:270-275 [1989]; Seckinger et al, J. Biol Chem.,
264:11966-11973 [1989]; Engelmann et al, J. Biol Chem.,
265:1531-1536 [1990]; and Aderka et al, J. Exp. Med., 175:323-329
[1992]). In the case of the TNFR1, this proteolytic activity
results in the cleavage and shedding of the extracellular
N-terminal domain (also called the ectodomain). These free, soluble
TNFR1 ectodomains ("sTNFR1s") have an affinity for TNF that is
similar to that of intact membrane receptors. Due to this affinity,
the free receptors are able to bind and sequester TNF, thereby
inhibiting the biological action of TNF. Furthermore, the
generation of sTNFR1 is also likely to suppress TNF signaling by
reducing the number of functional TNF receptors acting at the cell
membrane. The sTNFR1 ectodomains are also theorized to serve a more
complex buffering function in the regulation of TNF activity
(Aderka et al, J. Exp. Med., 175:323-329 [1992]; and Werb and Yan,
Science 282:1279-1280 [1998]). The complexity of TNF signaling is
further illustrated by the observation that many of the stimuli
that result in TNF release also result in the release of the
soluble TNF receptor, suggesting that these soluble TNF inhibitors
may serve as part of a regulated feedback mechanism to control TNF
activity (Adreke et al., J. Exp. Med., 175:323-329 [1992]; and
Porteu and Nathan, /. Exp. Med., 172:599-607 [1990]).
[0010] The importance of TNFR1 in the regulation of TNF activity in
host defense, immunoregulation and development has been further
demonstrated in studies utilizing TNFR1 knockout mice. Mice
deficient in TNFR1 show a variety of phenotypes, including
phenotypes which mimic human immune disorders (Pfeffer et al., Cell
73:457-467 [1993]; Rothe, Nature 364:798-802 [1993]; Le Hir et al,
J. Exp. Med., 183:2367-2372 [1996]; Matsumoto et al, Science
271:1289-1291[1996]; Mori et al. J. Immunol 157:3178-3182 [1996];
Speiser et al., J. Immunol, 158:5185-5190 [1997]; Tkachuk et al, J.
Exp. Med., 187:469-477 [1998]; and Kagi et al, J. Immunol,
162:45984605 [1999]).
[0011] The key role of TNFR1 shedding in the regulation of TNF
bioactivity is highlighted by the association of germline mutations
in TNFR1 extracellular domains with impaired TNFR1 shedding and
autoinflammatory disease characterized by autosomal dominant
periodic fever syndromes (McDermott et al, Cell 97:133-144
[1999]).
Other Mediators of Acute Phase Response
[0012] In addition to TNF, other cytokines have been implicated in
the induction of the pro-inflammatory response (i.e., the acute
phase immune response). These cytokines which demonstrate
overlapping activities with TNF include the interleukins (e.g.,
IL-1 and IL-6) (Suffredini et al, J. Clin. Immunol, 19:203-214
[1999]).
[0013] IL-1 (consisting of both a and p forms) is an important
proinflammatory cytokine which regulates the expression of a wide
variety of target genes and proteins in nearly every cell type
(Dinarello, Blood 77:1627-1652 [1991]; and Dinarello, The Cytokine
Handbook (ed. Angus W. Thomson), 3.sup.rd edition, Academic Press,
San Diego, p. 35-72 [1998]). The spectrum of IL-1-mediated biologic
effects includes inflammatory, metabolic, physiologic,
hematopoietic, and immunologic functions. IL-1 is thought to play a
role in the pathogenesis of several disease states, including
septic shock, rheumatoid arthritis, inflammatory bowel disease,
myelogenous leukemia, diabetes mellitus, and atherosclerosis
(Dinarello et al, N. Engl. J. Med., 328:106-113 [1993]).
[0014] IL-6 is also a multifunctional cytokine with pleiotropic
pro-inflammatory effects (DiCosmo, et al, J. Clin. Invest,
94:2028-2035 [1994]; and Kishimoto et al, Blood 86:1243-1254
[1995]). For example, IL-6 plays an important role in regulating B
cell immunoglobulin production, T-cell activation, growth and
differentiation, hematopoiesis, hepatic acute phase reactions and
osteoclast development (Hirano, The Cytokine Handbook (ed. Angus W.
Thomson), 3.sup.rd edition, Academic Press, San Diego, p. 197-228
[1998]). Dysregulated production of IL-6 may contribute to the
pathogenesis of a variety of inflammatory, neoplastic and
autoimmune disorders, such as plasma cell neoplasia and Castleman's
disease (Yoshizaki, et al, Blood 74:1360-1367 [1989]; and Hirano,
Int. J. Cell Cloning 9:166-184 [1991]).
[0015] The signal transduction pathways utilized by TNF, IL-1 and
IL-6 also show shared signaling intermediates. For example, both
TNF and IL-1 can activate both NF-KB and AP-1, which are important
pro-inflammatory transcription factors (Ashkenazi et al, Science
281:1305-1308 [1998]; and Dinarello, The Cytokine Handbook (ed.
Angus W. Thomson), 3.sup.rd edition, Academic Press, San Diego, p.
35-72 [1998]). Similarly, IL-6 signaling uses components of the
JAK-STAT pathway, which has also been reported to be induced by TNF
(Guo et al, J. Immunol, 160:2742-2750 [1998]; and Hirano, The
Cytokine Handbook (ed. Angus W. Thomson), 3rd edition, Academic
Press, San Diego, p. 197-228 [1998]).
[0016] The cognate receptors for the IL-1 and IL-6 cytokines are
known. There are two IL-1 receptor forms, type I and type II. There
is a single IL-6 receptor, consisting of gp80 alpha chain and gp!30
beta chain subunits, where ligand binding is mediated by the alpha
subunit. The IL-1 and IL-6 receptors are also present as soluble
forms analogous to the soluble form of TNFR1. Furthermore, it has
been suggested that these receptors play a role in the regulation
of IL-1 and IL-6 activity and pro-inflammatory response (Dower et
al., J. Immunol, 142:4314 [1989]; Novick et al, J. Exp. Med.,
170:1409 [1989]; Eastgate et al., FBBS Lett., 260:213 [1990]; Giri
et al., J. Biol Chem., 265:17416 [1990]; Symons et al., Cytokine
2:190 [1990]; Symons et al, FEES Lett., 272:133 [1990]; Symons et
al, J. Exp. Med., 174:1251-1254 [1991]; Mullberg et al, Biochem.
Biophys. Res. Commun., 189:794 [1992]; Mullberg et al, Eur. J.
Immunol, 23:473 [1993]; Svenson et al, Cytokine 5:427 [1993]; and
Arend et al, J. Immunol, 153:4766-4774 [1994]).
[0017] Analogy between regulation of TNF and other cytokines is
further illustrated by studies utilizing peptide-hydroxamate
metalloprotease inhibitors. Specifically, the protease inhibitors
TAPI (TNF-a protease inhibitor) and RU36156 have been reported to
inhibit the proteolytic cleavage and shedding of both TNFR1 and
IL-6R (Mullberg et al, J. Immunol, 155:5198-5205 [1995]; and
Gallea-Robache et al., Cytokine 9:340-346 [1997]).
[0018] As discussed above, in view of the importance of TNF, IL-1
and IL-6 in both health and disease states, there exists a need for
methods and compositions for the regulation of TNF, IL-1 and IL-6
cytokine activity. These methods and compositions will find use as
therapeutic agents for the treatment of disease states.
SUMMARY OF THE INVENTION
[0019] The present invention provides compositions and methods
related to regulation of cytokine signaling through the Tumor
Necrosis Factor (TNF) pathway, as well as other signaling pathways
controlled by other cytokines, including IL-1 and IL-6. It is
contemplated that these compositions and methods will find use in
therapeutics for the treatment of diseases and disorders of the
immune system.
[0020] The present invention also provides novel polypeptides and a
nucleic acid sequences. In particular, the present invention
provides isolated nucleic acids comprising the nucleotide sequence
set forth in SEQ ID NO:1, which encodes a polypeptide referred to
as "ARTS-"! (i.e., aminopeptidase regulator of type I, 55 kDa tumor
necrosis factor receptor ectodomain shedding) which has the ability
to promote the shedding of the extracellular domain of Type I Tumor
Necrosis Factor Receptor (TNFR1). Thus, the present invention also
provides the isolated polypeptides comprising the amino acid
sequence set forth in SEQ ID NO:2, as well as isolated nucleic
acids encoding the polypeptide set forth in SEQ ID NO:2.
[0021] The present invention further provides recombinant vectors
comprising the ARTS-1 gene and host cells comprising these vectors.
In one embodiment, the host cell is eukaryotic, while in an
alternative embodiment, the host cell is prokaryotic. The present
invention also provides antibodies raised against at least a
portion of the ARTS-1 polypeptide of SEQ ID NO:2. In some
embodiments, the antibodies are monoclonal, while in alternative
embodiments, the antibodies are polyclonal.
[0022] In other embodiments, the present invention provides
isolated nucleic acids that are substantially homologous to the
nucleic acid of SEQ ID NO:1, wherein the nucleic acid is capable of
hybridizing under high stringency conditions to the nucleic acid of
SEQ ID NO:1. In a preferred embodiment, the nucleic acid
substantially homologous to the ARTS-1 gene encodes a polypeptide
having the ability to regulate the shedding of the extracellular
domain of at least one cytokine receptor. In particularly preferred
embodiments, the cytokine receptor is selected from the group
consisting of type-1 tumor necrosis factor receptor, type I
interleukin-1 cytokine receptor, type II interleukin-1 cytokine
receptor, and interleukin-6 cytokine receptor alpha-chain gp80. In
some embodiments, the nucleic acid substantially homologous to the
nucleic acid of the ARTS-1 gene is identified using PCR methods. In
alternative embodiments, the nucleic acid substantially homologous
to the ARTS-1 gene is identified using hybridization screening
methods.
[0023] The present invention further provides methods for the
isolation of amplifiable nucleic acid substantially homologous to
the nucleic acid of SEQ ID NO:1 comprising: providing a sample
comprising template nucleic acid suspected of encoding a gene
substantially homologous to the nucleic acid of SEQ ID NO:1; and at
least two primers; annealing the primers to the template nucleic
acid; extending the primers (e.g., with reiterated DNA synthesis)
under conditions such that the template nucleic acid is amplified,
to produce an amplified product; and visualizing the amplified
product. In some preferred embodiments, the amplified product is
isolated. The present invention further provides the product of
these amplification methods. In preferred embodiments, the
amplified product encodes a polypeptide having the ability to
regulate the shedding of the extracellular domain of at least one
cytokine receptor. In particularly preferred embodiments, the
cytokine receptor is selected from the group consisting of type-1
tumor necrosis factor receptor, type I interleukin-1 cytokine
receptor, type II interleukin-1 cytokine receptor, and
interleukin-6 cytokine receptor alpha-chain gp80.
[0024] The present invention also provides methods for the use of
these compositions to regulate the shedding of sTNFR1. It is
contemplated that methods which regulate the shedding of the sTNFR1
also regulate the activity of TNR In a most preferred embodiment,
the invention provides methods for use of these compositions in
therapeutic applications in the treatment of immune system diseases
and disorders resulting from aberrant cytokine activity.
[0025] The present invention provides methods for regulating the
shedding of the extracellular domain of at least one cytokine
receptor, comprising the steps of: providing a recombinant vector
comprising SEQ ID NO:1 in the sense orientation, a first tissue
containing one or more cells expressing at least one cytokine
receptor, and a second tissue comprising one or more cells capable
of expressing the polypeptide encoded by the recombinant vector;
delivering the vector to the cells of the second tissue in the
presence of the first tissue, under conditions which result in
regulation of shedding of the cytokine receptor(s) from cells of
the first tissue. In some preferred embodiments, the cytokine
receptor is selected from the group consisting of type-1 tumor
necrosis factor receptor, type I interleukin-1 cytokine receptor,
type II interleukin-1 cytokine receptor, and interleukin-6 cytokine
receptor alpha-chain gp80. In alternative preferred embodiments,
the delivery of the vector to the second tissue comprises a means
of intracellular delivery selected from the group consisting of
direct nucleic acid administration, liposome administration, viral
vector delivery, and ex vivo gene delivery followed by
transplantation.
[0026] In other embodiments, the present invention provides
compositions and methods suitable for regulating TNFR1 ectodomain
shedding by overexpressing or suppressing the activity of the
ARTS-1 polypeptide. In some of these embodiments, TNFR1 ectodomain
shedding is regulated by the intracellular delivery of a vector
which results in overexpression of the ARTS-1 polypeptide (e.g.,
SEQ ID NO:2). In another embodiment, TNFR1 ectodomain shedding is
regulated by delivering purified ARTS-1 polypeptide (e.g., SEQ ID
NO:2) to tissues.
[0027] The present invention also provides methods for regulating
the shedding of the extracellular domain of at least one cytokine
receptor, comprising the steps of: providing a recombinant vector
comprising at least a transcribeable portion of the nucleic acid of
SEQ ID NO:1 in an antisense orientation, a first tissue comprising
one or more cells expressing at least one cytokine receptor, and a
second tissue comprising one or more cells expressing the
endogenous polypeptide of SEQ ID NO:2, and one or more cells
capable of transcribing the antisense nucleic acid; and delivering
the vector to the second tissue in the presence of the first
tissue, under conditions that result in regulation of shedding of
the cytokine receptor(s) from the cells of the first tissue. In
preferred embodiments, the cytokine receptor is selected from the
group consisting of type-1 tumor necrosis factor receptor, type I
interleukin-1 cytokine receptor, type II interleukin-1 cytokine
receptor, and interleukin-6 cytokine receptor alpha-chain gp80. In
alternative preferred embodiments, the delivery of the vector to
the second tissue comprises a means of intracellular delivery
selected from the group consisting of direct nucleic acid
administration, liposome administration, viral vector delivery, and
ex vivo gene delivery followed by transplantation.
[0028] The present invention further provides methods for
regulating the shedding of the extracellular domain of at least one
cytokine receptor, comprising the steps of: providing a polypeptide
having the amino acid sequence set forth in SEQ ID NO:2, and a
tissue comprising one or more cells expressing at least one
cytokine receptor on their plasma membrane extracellular surface;
and delivering the polypeptide to the tissue under conditions such
that the polypeptide regulates the shedding of the cytokine
receptor(s) from the surface of the cells of the tissue. In some
preferred embodiments, the cytokine receptor is selected from the
group consisting of type-1 tumor necrosis factor receptor, type I
interleukin-1 cytokine receptor, type II interleukin-1 cytokine
receptor, and interleukin-6 cytokine receptor alpha-chain gp80. In
alternative preferred embodiments, the delivery of the polypeptide
to the tissue comprises a means of delivery selected from the group
consisting of oral administration, intra-arterial injection,
intravenous injection, intramuscular injection, intraperitoneal
injection, subcutaneous injection, suppository, local surgical
administration, systemic surgical administration, catheter, and any
combination of these means of delivery.
[0029] The present invention further provides methods for
regulating the shedding of the extracellular domain of at least one
cytokine receptor, providing an antibody raised against the ARTS-1
polypeptide, and a tissue comprising one or more cells expressing
at least one cytokine receptor and the endogenous polypeptide of
SEQ ID NO:2; and delivering the antibody to the tissue under
conditions such that the antibody regulates the shedding of the
cytokine receptor(s) from the surface of the cells of the tissue.
In some preferred embodiments, the cytokine receptor is selected
from the group consisting of type-1 tumor necrosis factor receptor,
type I interleukin-1 cytokine receptor, type II interleukin-1
cytokine receptor, and interleukin-6 cytokine receptor alpha-chain
gp80. In alternative preferred embodiments, the means of delivery
is selected from the group consisting of oral administration,
intra-arterial injection, intravenous injection, intramuscular
injection, intraperitoneal injection, subcutaneous injection,
suppository, local surgical administration, systemic surgical
administration, catheter, and any combination of these means of
delivery.
[0030] In view of the overlapping activities between TNF and other
proinflammatory cytokines, the present invention also provides
compositions and methods suitable for regulating the shedding of
other cytokine receptors in addition to TNFR1, including, but not
limited to IL-1 and IL-6 cytokine receptors.
[0031] In some particularly preferred embodiments, the present
invention provides compositions and methods are provided to treat
subjects displaying pathology, as well as subjects suspected of
displaying or at risk of displaying pathology resulting from
abnormal cytokine activity. The compositions provided for use in
the most preferred embodiment include vectors capable of expressing
the ARTS-1 polypeptide, vectors capable of transcribing at least a
portion of the ARTS-1 gene in an antisense orientation, the ARTS-1
polypeptide set forth in SEQ ID NO:2, and antibodies raised against
at least a portion of the ARTS-1 polypeptide.
[0032] The present invention also provides methods for treating a
subject, comprising the steps of: providing a composition selected
from the group consisting of a recombinant vector comprising at
least a portion of SEQ ID NO:1 in the sense orientation, a
recombinant vector comprising at least a portion of SEQ ID NO:1 in
the antisense orientation, at least a portion of the ARTS-1
polypeptide, at least a portion of SEQ ID NO:2, and antibody
directed against at least a portion of the ARTS-1 polypeptide, as
well as a subject, and a means of delivery of the composition to at
least one tissue of the subject; and delivering the composition to
the subject using the means of delivery. In preferred embodiments,
the subject is selected from the group consisting of a subject
displaying pathology resulting from abnormal cytokine activity, a
subject suspected of displaying pathology resulting from abnormal
cytokine activity, and a subject at risk of displaying pathology
resulting from abnormal cytokine activity. In some preferred
embodiments, the cytokine activity is mediated by a cytokine
selected from the group consisting of tumor necrosis factor a,
interleukin-1 alpha, interleukin-1 beta, and interleukin-6. In some
particularly preferred embodiments, the subject is a human. In
alternative preferred embodiments, the means of delivery is
selected from the group consisting of oral administration,
intra-arterial injection, intravenous injection, intramuscular
injection, intraperitoneal injection, subcutaneous injection,
suppository, local surgical administration, systemic surgical
administration, catheter, and any combination of these means of
delivery. In further preferred embodiments, the means of delivery
is further selected from the group consisting of direct nucleic
acid administration, liposome administration, viral vector
delivery, and ex vivo gene delivery followed by
transplantation.
[0033] In other embodiments, the present invention provides ARTS-1
markers, including those selected from the group consisting of the
ARTS-1 mRNA transcript and the ARTS-1 polypeptide. Furthermore, the
invention also provides compositions and means for detecting the
ARTS-1 mRNA and polypeptide markers. In preferred embodiments,
these compositions are selected from the group consisting of
nucleic acid complementary to the ARTS-1 mRNA, and antibodies
specific for at least a portion of the ARTS-1 polypeptide.
[0034] The present invention also provides means for detecting an
ARTS-1 mRNA in a sample, wherein the means comprises at least a
portion of the nucleic acid of SEQ ID NO:1 complementary to at
least a portion of ARTS-1 mRNA, and further wherein the nucleic
acid is a probe. In alternative embodiments, the means comprises
Northern blotting. In preferred embodiments, the sample is a tissue
sample from a subject. In additional preferred embodiments, the
methods provide means for detecting an ARTS-1 polypeptide in a
sample, wherein the means comprises an antibody directed against at
least a portion of the ARTS-1 polypeptide. In some preferred
embodiments, the means comprises Western immunoblotting, while in
alternative preferred embodiments, the means comprises an
enzyme-linked immunosorbent assay. In alternative preferred
embodiments, the sample is a tissue sample from a subject.
[0035] The present invention also provides diagnostic kits
comprising a means to measure ARTS-1 expression, wherein the means
comprises at least a portion of SEQ ID NO:1 that is complementary
to at least a portion of ARTS-1 mRNA, and further wherein the
nucleic acid is a probe. In alternative embodiments, the diagnostic
kits of the present invention provides means for detecting an
ARTS-1 polypeptide in a sample, wherein the means comprises
antibody directed against at least a portion of the ARTS-1
polypeptide. In some preferred embodiments, the means comprises
Western immunoblotting, while in alternative preferred embodiments,
the means comprises an enzyme-linked immunosorbent assay.
[0036] The present invention further provides compositions and
methods for the screening for drugs with the ability to regulate
ARTS-1 peptidase activity and cytokine receptor shedding regulatory
activity. In some embodiments, the present invention provides
methods for drug screening to identify drugs having the ability to
regulate ARTS-1 expression comprising the steps of: providing a
drug, cultured cells, and a means to measure ARTS-1 expression,
wherein the means comprises at least a portion of SEQ ID NO:1 that
is complementary to at least a portion of ARTS-1 mRNA, and further
wherein the nucleic acid is a probe; exposing the cells to the
drug; and using the means to measure ARTS-1 expression. In
alternative embodiments, the diagnostic kits of the present
invention provide means for detecting an ARTS-1 polypeptide in a
sample, wherein the means comprises an antibody directed against at
least a portion of the ARTS-1 polypeptide. In some preferred
embodiments, the cultured cells are human NCI-H292 pulmonary
mucoepidermoid carcinoma cells.
[0037] The present invention further provides methods for drug
screening to identify drugs capable of regulating the peptidase
activity of ARTS-1, comprising the steps of: providing purified
ARTS-1 polypeptide, an amino acid p-nitroaniline, a means to
measure amino acid p-nitroaniline cleavage, and a drug; exposing
the purified ARTS-1 polypeptide to the amino acid p-nitroaniline in
the absence and presence of the drug; and measuring amino acid
p-nitroaniline cleavage in the absence and presence of the drug. In
some preferred embodiments, the purified ARTS-1 polypeptide
comprises glutathione-S-transferase. In alternative preferred
embodiments, the amino acid p-nitroaniline is selected from the
group consisting of isoleucine p-nitroanilide, phenylalanine
p-nitroanilide and glycine p-nitroanilide. In still further
preferred embodiments, the means to measure amino acid
p-nitroaniline cleavage comprises measuring absorbance at 380
nm.
[0038] The present invention also provides methods for drug
screening to identify drugs capable of regulating the shedding of a
cytokine receptor, comprising the steps of: providing cultured
cells expressing at least one cytokine receptor, a means to
quantitate the concentration of the soluble form of the cytokine
receptor(s) in the supernatants of the cultured cells, and a drug;
culturing the cells in the absence and presence of the drug;
quantitating the concentration of the soluble form of the cytokine
receptor(s) in the supernatants of the cultured cells; and
comparing the concentrations of the soluble cytokine receptor(s) in
the supernatants of the cell cultures in the absence and presence
of drug. In some preferred embodiments, the cytokine receptor is
selected from the group consisting of type-1 tumor necrosis factor
receptor, type II interleukin-1 cytokine receptor, and
interleukin-6 cytokine receptor alpha-chain gp80. In alternative
preferred embodiments, the means to quantitate the concentration of
the soluble form of a cytokine receptor comprises an enzyme-linked
immunosorbent assay. In further embodiments, the cultured cells are
cultured human NCI-H292 pulmonary mucoepidermoid carcinoma
cells.
DESCRIPTION OF THE FIGURES
[0039] FIG. 1 presents the ARTS-1 transcription unit and open
reading frame translation.
[0040] FIG. 2 provides a schematic representation of the ARTS-1
protein, indicating domains of homology with the aminopeptidase
family of gluzincin zinc metalloproteases.
[0041] FIG. 3 provides a Northern blot analysis of multiple human
tissues using a .sup.32P-labelled ARTS-1 cDNA probe. Panel A shows
the blot probed with the ARTS-1 cDNA probe. Panel B shows the same
blot following stripping and rehybridization to a probe specific
for the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene
as a reference for RNA loading normalization.
[0042] FIG. 4 provides Western immunoblots using pre-immune and
polyclonal anti-ARTS-1 antisera in conjunction with crude whole
cell homogenates, and membrane and cytosolic fractions made from
cultured NCI-H292 cells. Panel A provides a Western immunoblot
using the immune sera. Panel B provides a Western immunoblot using
the pre-immune sera. Panels C and D provide results from Western
immunoblot competition experiments using bovine serum albumin or
ARTS-1 cognate peptide, respectively.
[0043] FIG. 5 provides Western immunoblots using polyclonal
anti-ARTS-1 antisera and membrane and cytosolic fractions made from
primary cells and cell lines. Panel A provides a Western immunoblot
using human bronchial brush cells collected from human subjects.
Panel B provides a Western immunoblot using the NCI-H292, BEAS-2B,
BET-1A and A549 cultured cell lines. Panel C provides a Western
immunoblot using primary cultures of normal human bronchial
epithelial cells (NHBE), human umbilical vein endothelial cells
(HUVEC) and human fibroblasts.
[0044] FIG. 6 provides results of an analysis of recombinant
GST-ARTS-1 fusion protein purification and analysis of the
aminopeptidase activity of this protein following purification.
Panel A provides a Coomassie stained gel of protein samples
obtained during the production and purification of the GST-ARTS-1
fusion protein. Panel B provides an FPLC elution profile of the
purified GST-ARTS-1 fusion protein. Panel C provides the results of
an assay of phenylalanine p-nitroaniline substrate aminopeptidase
activity of the FPLC eluted fractions.
[0045] FIG. 7 provides a Western immunoblot using anti-ARTS-1
polyclonal antiserum as the primary antibody. Samples analyzed in
the blot are from membrane protein fractions derived from stably
transfected NCI-H292 cells which were untransfected (WT), control
transfected (Mock), ARTS-1 sense-overexpressing (ARTS-1) or ARTS-1
anti-sense expressing (AS). Two independent clones each from the
ARTS-1 and AS cell lines were analyzed.
[0046] FIG. 8 provides results of an ELISA to determine the levels
of sTNFR1 resulting from TNFR1 ectodomain shedding in cell culture
supernatants from cultures of the same cell lines as indicated for
FIG. 7.
[0047] FIG. 9 provides a graph depicting the ability of ARTS-1
overexpression to potentiate the cleavage and shedding of TNFR
ectodomain from the surface of NCI-H292 cells in response to PMA
stimulation, as determined by an ELISA measuring sTNFR1 in the cell
culture supernatants.
[0048] FIG. 10 provides a histogram showing the results of an ELISA
analysis. The ELISA determined the levels of sTNFR1 (a measure of
TNFR1 ectodomain shedding) in cell culture supernatants for stably
transfected NCI-H292 cell lines expressing various ARTS-1
mutants.
[0049] FIG. 11 provides a Western immunoblot using an anti-TNFR1
antibody as the primary antibody to detect membrane bound TNFR1.
Samples analyzed in the blot include membrane protein fractions
derived from stably transfected NCI-H292 cells which were
untransfected (WT), control transfected (Mock), ARTS-1
sense-overexpressing (ARTS-1), or ARTS-1 anti-sense expressing
(AS). Two independent clones of each cell line were analyzed.
[0050] FIG. 12 provides two Western immunoblots following two in
vivo immunoprecipitation experiments using membrane protein
fractions isolated from cultured NCI-H292 cells. In the top panel,
an anti-TNFR1 antibody was used in the immunoprecipitation step
(indicated as "IP"), and anti-ARTS-1 antiserum was used as the
primary antibody in the immunoblotting (indicated as "IB").
Conversely, in the lower panel, the anti-ARTS-1 antiserum was used
in the immunoprecipitation, while the anti-TNFR1 antibody was used
as the primary antibody in the immunoblotting.
[0051] FIG. 13 provides a Western immunoblot following an in vivo
immunoprecipitation experiment using an anti-TNFR1 monoclonal
antibody for the immunoprecipitation and anti-ARTS-1 antiserum as
the primary antibody in the blot. The immunoprecipitations used
cell membrane protein fractions derived from stably transfected
NCI-H292 cells overexpressing ARTS-1 (ARTS-1), expressing an
anti-sense ARTS-1 message (AS), as well as control-transfected
(Mock) and non-transfected (WT) cell lines.
[0052] FIG. 14 provides results of two drug screening assays. Panel
A provides a Western immunoblot using anti-ARTS-1 polyclonal
antiserum as the primary antibody, tested with membrane protein
fractions from NCI-H292 cells following exposure to 4b-phorbol
12-myristate 13-acetate (PMA) (over a time course). Panel B
provides the results of an ELISA to determine the levels of sTNFR1
in cell culture supernatants from NCI-H292 cell cultures following
exposure PMA, over time.
DEFINITIONS
[0053] To facilitate an understanding of the present invention, a
number of terms and phrases are defined or clarified below:
[0054] The terms "peptide," "polypeptide" and "protein" all refer
to a primary sequence of ammo acids that are joined by covalent
"peptide linkages." In general, a peptide consists of a few amino
acids, typically from 2-25 amino acids, and is shorter than a
protein. Polypeptides may encompass either peptides or proteins.
Where "amino acid sequence" is recited herein to refer to an amino
acid sequence of a naturally occurring protein molecule, "amino
acid sequence" and like terms, such as "polypeptide" or "protein"
are not meant to limit the amino acid sequence to the complete,
native amino acid sequence associated with the recited protein
molecule.
[0055] As used herein, the term "nucleic acid" refers to any
sequence of the bases adenine, thymine, cytosine and guanine, and
various analogs of these bases. A nucleic acid is characterized by
a specific nucleotide sequence (i.e., the sequence of the bases and
base analogs in the molecule). A "nucleic acid" is not limited to
DNA or RNA, and is not limited in any way by the size of the
molecule. A nucleic acid may be double stranded or single stranded.
The term "nucleic acid" encompasses sequences that include any of
the bases adenine, thymine, guanine and cytosine, as well as known
analogs of these bases, including but not limited to,
4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,
pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil,
5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
[0056] As used herein, the term "oligonucleotide," refers to a
short length of single-stranded polynucleotide chain.
Oligonucleotides are typically less than 100 residues long (e.g.,
between 15 and 50), however, as used herein, the term is also
intended to encompass longer polynucleotide chains.
Oligonucleotides are often referred to by their length. For example
a 24 residue oligonucleotide is referred to as a "24-mer."
Oligonucleotides can form secondary and tertiary structures by
self-hybridizing or by hybridizing to other polynucleotides. Such
structures can include, but are not limited to, duplexes, hairpins,
cruciforms, bends, and triplexes.
[0057] As used herein, "recombinant nucleic acid," "recombinant
gene" or "recombinant DNA molecule" indicate that the nucleotide
sequence or arrangement of its parts is not a native configuration,
and has been manipulated by molecular biological techniques. The
term implies that the DNA molecule is comprised of segments of DNA
that have been artificially joined together. Protocols and reagents
to manipulate nucleic acids are common and routine in the art (See
e.g., Maniatis et a/.(eds.), Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, NY, [1982]; Sambrook
et al. (eds.), Molecular Cloning: A Laboratory Manual, Second
Edition, Volumes 1-3, Cold Spring Harbor Laboratory Press, NY,
[1989]; and Ausubel et al. (eds.), Current Protocols in Molecular
Biology, Vol. 1-4, John Wiley & Sons, Inc., New York
[1994]).
[0058] Similarly, a "recombinant protein" or "recombinant
polypeptide" refers to a protein molecule that is expressed from a
recombinant DNA molecule. Use of these terms indicates that the
primary amino acid sequence, arrangement of its domains or nucleic
acid elements which control its expression are not native, and have
been manipulated by molecular biology techniques. As indicated
above, techniques to manipulate recombinant proteins are also
common and routine in the art.
[0059] The terms "exogenous" and "heterologous" are sometimes used
interchangeably with "recombinant." An "exogenous nucleic acid,"
"exogenous gene" and "exogenous protein" indicate a nucleic acid,
gene or protein, respectively, that has come from a source other
than its native source, and has been artificially supplied to the
biological system. In contrast, the terms "endogenous protein,"
"native protein," "endogenous gene," and "native gene" refer to a
protein or gene that is native to the biological system, species or
chromosome under study. A "native" or "endogenous" polypeptide does
not contain amino acid residues encoded by recombinant vector
sequences; that is, the native protein contains only those amino
acids found in the polypeptide or protein as it occurs in nature. A
"native" polypeptide may be produced by recombinant means or may be
isolated from a naturally occurring source. Similarly, a "native"
or "endogenous" gene is a gene that does not contain nucleic acid
elements encoded by sources other than the chromosome on which it
is normally found in nature.
[0060] As used herein, the term "portion" when in reference to a
protein (as in "a portion of a given protein") refers to fragments
of that protein. The fragments may range in size from four amino
acid residues to the entire amino acid sequence minus one amino
acid.
[0061] Nucleic acid molecules (e.g., DNA or RNA) are said to have
"5' ends" and "3' ends" because mononucleotides are reacted to make
oligonucleotides or polynucleotides in a manner such that the 5'
phosphate of one mononucleotide pentose ring is attached to the 3'
oxygen of its neighbor in one direction via a phosphodiester
linkage. Therefore, an end of an oligonucleotides or
polynucleotide, referred to as the "5' end" if its 5' phosphate is
not linked to the 3' oxygen of a mononucleotide pentose ring and as
the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a
subsequent mononucleotide pentose ring. As used herein, a nucleic
acid sequence, even if internal to a larger oligonucleotide or
polynucleotide, also may be said to have 5' and 3' ends. In either
a linear or circular DNA molecule, discrete elements are referred
to as being "upstream" or 5' of the "downstream" or 3' elements.
This terminology reflects the fact that transcription proceeds in a
5' to 3' fashion along the DNA strand. The promoter and enhancer
elements that direct transcription of a linked gene are generally
located 5' or upstream of the coding region. However, enhancer
elements can exert their effect even when located 3' of the
promoter element or the coding region. Transcription termination
and polyadenylation signals are located 3' or downstream of the
coding region.
[0062] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence comprised of parts, that when appropriately combined in
either a native or recombinant manner, provide some product or
function. Genes may or may not comprise coding sequences necessary
for the production of a polypeptide. Examples of genes which do not
encode polypeptide sequences include ribosomal RNA genes (rRNA) and
transfer RNA (tRNA) genes. Genes can encode a polypeptide or any
portion of a polypeptide within the gene's "coding region" or "open
reading frame." The polypeptide produced by the open reading frame
of a gene may or may not display functional activity or properties
of the full-length polypeptide (e.g., enzymatic activity, ligand
binding, signal transduction, etc.).
[0063] In addition to the coding region of the nucleic acid, the
term "gene" also encompasses the transcribed nucleotide sequences
of the full-length mRNA adjacent to the 5' and 3' ends of the
coding region. These noncoding regions are variable in size, and
typically extend for distances up to or exceeding 1 kb on both the
5' and 3' ends of the coding region. The sequences that are located
5' and 3' of the coding region and are contained on the mRNA are
referred to as 5' and 3' untranslated sequences (5' UT and 3' UT).
Both the 5' and 3' UT may serve regulatory roles, including
translation initiation, post-transcriptional cleavage and
polyadenylation. The term "gene" encompasses mRNA, cDNA and genomic
forms of a gene.
[0064] It is contemplated that the genomic form or genomic clone of
a gene may contain the sequences of the transcribed mRNA, as well
as other non-coding sequences which lie outside of the mRNA. The
regulatory regions which lie outside the mRNA transcription unit
are sometimes called "5' or 3' flanking sequences." A functional
genomic form of a gene must contain regulatory elements necessary
for the regulation of transcription. The term "promoter/enhancer
region" is usually used to describe this DNA region, typically but
not necessarily 5' of the site of transcription initiation,
sufficient to confer appropriate transcriptional regulation. The
word "promoter" alone is sometimes used synonymously with
"promoter/enhancer." A promoter may be constitutively active, or
alternatively, conditionally active, where transcription is
initiated only under certain physiological conditions or in the
presence of certain drugs. The 3' flanking region may contain
additional sequences which regulate transcription, especially the
termination of transcription. "Introns" or "intervening regions" or
"intervening sequences" are segrnents of a gene which are contained
in the primary transcript (i.e., hetero-nuclear RNA, or hnRNA), but
are spliced out to yield the processed mRNA form. Introns may
contain transcriptional regulatory elements such as enhancers. The
mRNA produced from the genomic copy of a gene is translated in the
presence of ribosomes to yield the primary amino acid sequence of
the polypeptide.
[0065] As used herein, the term "regulatory element" refers to a
genetic element which controls some aspect of the expression of
nucleic acid sequences. For example, a promoter is a regulatory
element that enables the initiation of transcription of an operably
linked coding region. Other regulatory elements are splicing
signals, polyadenylation signals, termination signals, etc.
[0066] Transcriptional control signals in eukaryotes comprise
"promoter" and "enhancer" elements. Promoters and enhancers consist
of short arrays of DNA sequences that interact specifically with
cellular proteins involved in transcription (Maniatis et al.,
Science 236:1237 [1987]). Promoter and enhancer elements have been
isolated from a variety of eukaryotic sources including genes in
yeast, insect and mammalian cells, as well as viruses. Analogous
control elements (i.e., promoters and enhancers) are also found in
prokaryotes. The selection of a particular promoter and enhancer to
be operably linked in a recombinant gene depends on what cell type
is to be used to express the protein of interest. Some eukaryotic
promoters and enhancers have a broad host range while others are
functional only in a limited subset of cell types (for review see,
Voss et al, Trends Biochem. Sci., 11:287 [1986 and Maniatis et al,
Science 236:1237 [1987]). For example, the SV40 early gene enhancer
is very active in a wide variety of mammalian cell types (Dijkema
et al, EMBO J., 4:761 [1985]). Two other examples of
promoter/enhancer elements active in a broad range of mammalian
cell types are those from the human elongation factor la gene
(Uetsuki et al, J. Biol Chem., 264:5791 [1989]; Kim et al, Gene
91:217 [1990]; Mizushima and Nagata, Nuc. Acids. Res., 18:5322
[1990]), the long terminal repeats of the Rous sarcoma virus
(Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777 [1982]), and
human cytomegalovirus (Boshart et al, Cell 41:521 [1985]). Some
promoter elements serve to direct gene expression in a
tissue-specific manner.
[0067] As used herein, the term "promoter/enhancer" denotes a
segment of DNA which contains sequences capable of providing both
promoter and enhancer functions (i.e., the functions provided by a
promoter element and an enhancer element). For example, the long
terminal repeats of retroviruses contain both promoter and enhancer
functions. The promoter/enhancer may be "endogenous," or
"exogenous," or "heterologous." An "endogenous" promoter/enhancer
is one which is naturally linked with a given gene in the genome.
An "exogenous" or "heterologous" promoter/enhancer is one placed in
juxtaposition to a gene by means of genetic manipulation (i.e.,
molecular biological techniques such as cloning and recombination)
such that transcription of the gene is controlled by the linked
promoter/enhancer.
[0068] The presence of "splicing signals" on an expression vector
often results in higher levels of expression of the recombinant
transcript. Splicing signals mediate the removal of introns from
the primary RNA transcript and consist of a splice donor and
acceptor site (See e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, New York [1989], pp. 16.7-16.8). A commonly used splice
donor and acceptor site is the splice junction from the 16S RNA of
SV40.
[0069] Efficient expression of recombinant DNA sequences in
eukaryotic cells requires the presence of signals directing the
efficient termination and polyadenylation of the resulting
transcript. Transcription termination signals are generally found
downstream of the polyadenylation signal and are a few hundred
nucleotides in length. The term "poly A site" or "poly A sequence"
as used herein denotes a nucleic acid sequence that directs both
the termination and polyadenylation of the nascent RNA transcript.
Efficient polyadenylation of the recombinant transcript is
desirable as transcripts lacking a poly A tail are unstable and are
rapidly degraded. The poly A signal utilized in an expression
vector may be "heterologous" or "endogenous." An endogenous poly A
signal is one that is found naturally at the 3' end of the coding
region of a given gene in the genome. A heterologous poly A signal
is one that is isolated from one gene and placed 3' of another
gene. A commonly used heterologous poly A signal is the SV40 poly A
signal. The SV40 poly A signal is contained on a 237 bp BamHI/Bcll
restriction fragment and directs both termination and
polyadenylation (See e.g., Sambrook et al, Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, New York [1989], pp. 16.6-16.7).
[0070] The terms "in operable combination," "in operable order,"
"operably linked" and similar phrases when used in reference to
nucleic acid herein are used to refer to the linkage of nucleic
acid sequences in such a manner that a nucleic acid molecule
capable of directing the transcription of a given gene and/or the
synthesis of a desired protein molecule is produced. The term also
refers to the linkage of amino acid sequences in such a manner so
that a functional protein is produced.
[0071] As used herein, the terms "an oligonucleotide having a
nucleotide sequence encoding a gene," "polynucleotide having a
nucleotide sequence encoding a gene," and similar phrases are meant
to indicate a nucleic acid sequence comprising the coding region of
a gene (i.e., the nucleic acid sequence which encodes a gene
product). The coding region may be present in either a cDNA,
genomic DNA or RNA form. When present in a DNA form, the
oligonucleotide, polynucleotide or nucleic acid may be
single-stranded (i.e., the sense strand or the antisense strand) or
double-stranded. Suitable control elements such as
enhancers/promoters, splice junctions, polyadenylation signals,
etc. may be placed in close proximity to the coding region of the
gene if needed to permit proper initiation of transcription and/or
correct processing of the primary RNA transcript. Alternatively,
the coding region utilized in the expression vectors of the present
invention may contain endogenous enhancers/promoters, splice
junctions, intervening sequences, polyadenylation signals, etc. or
a combination of both endogenous and exogenous control
elements.
[0072] As used herein, the terms "nucleic acid molecule encoding,"
"DNA sequence encoding," and "DNA encoding" and similar phrases
refer to the order or sequence of deoxyribonucleotides along a
strand of deoxyribonucleic acid. The order of these
deoxyribonucleotides determines the order of amino acids along the
polypeptide (e.g., protein) chain. The DNA sequence thus codes for
the amino acid sequence.
[0073] As used herein, the term "gene expression" refers to the
process of converting genetic information encoded in a gene into
RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of
the gene (i.e., via the enzymatic action of an RNA polymerase), and
for protein encoding genes, into protein through "translation" of
the mRNA. Gene expression can be regulated at many stages.
"Up-regulation" or "activation" refers to regulation that increases
the production of gene expression products (i.e., RNA or protein),
while "down-regulation" or "repression" refers to regulation that
decreases mRNA or protein production. Molecules (e.g.,
transcription factors) that are involved in up-regulation or
down-regulation are often called "activators" and "repressers,"
respectively.
[0074] As used herein, the terms "complementary" or
"complementarity" are used in reference to polynucleotides (i.e., a
sequence of nucleotides) related by the rules of antiparallel
base-pairing. For example, the sequence 5'-C-T-A-G-T-3' is
complementary to the sequence 5'-A-C-T-A-G-3'. Complementarity may
be "partial," in which only some of the nucleic acids' bases are
matched according to antiparallel base pairing rules.
[0075] Also, there may be "complete" or "total" complementarity
between two nucleic acids. The degree of complementarity between
nucleic acid strands has significant effects on the efficiency and
strength of hybridization between nucleic acid strands. This is of
particular importance in polymerase chain reaction (PCR)
amplification reactions, as well as detection methods that depend
upon binding between nucleic acids. As used herein, the terms
"antiparallel complementarity" and "complementarity" are
synonymous.
[0076] As used herein, the term "antisense" is used in reference to
any nucleic acid which is antiparallel to and complementary to
another nucleic acid. Antisense DNA or RNA may be produced by any
method. For example, a cDNA or a portion of a cDNA may be subcloned
into an expression vector containing a promoter which permits
transcription either in vitro or in vivo. The cDNA or a portion of
the cDNA is subcloned in such a way that it is in the reverse
orientation relative to the direction of transcription of the cDNA
in its native chromosome. Transcription of this antisense cDNA
produces an RNA transcript that is complementary and antiparallel
to the native mRNA. The mechanism by which an antisense nucleic
acid produces effects in a biological system is unclear, however,
likely involves the formation of a duplex with its complementary
nucleic acid within either the nucleus or cytoplasm of a cell.
These duplexes are theorized to block transcription of the native
mRNA or prevent its translation. Using antisense techniques, an
"artificial knockout" mutant may be reproduced in an animal or
animal cell line. The term "antisense strand" is used in reference
to a nucleic acid strand that is complementary to the "sense"
strand. The designation (-) (i.e., "negative") is sometimes used in
reference to the antisense strand, with the designation (+) (i.e.,
"positive") sometimes used in reference to the sense strand.
[0077] The following definitions are the commonly accepted
definitions of the terms "identity," "similarity" and "homology."
Percent identity is a measure of strict amino acid conservation.
Percent similarity is a measure of amino acid conservation which
incorporates both strictly conserved amino acids, as well as
"conservative" amino acid substitutions, where one amino acid is
substituted for a different amino acid having similar chemical
properties (i.e. a "conservative" substitution). The term
"homology" can pertain to either proteins or nucleic acids. Two
proteins can be described as "homologous" or "non-homologous," but
the degree of amino acid conservation is quantitated by percent
identity and percent similarity. Nucleic acid conservation is
measured by the strict conservation of the bases adenine, thymine,
guanine and cytosine in the primary nucleotide sequence. When
describing nucleic acid conservation, conservation of the nucleic
acid primary sequence is sometimes expressed as percent homology.
In the same nucleic acid, one region may show a high percentage of
nucleotide sequence conservation, while a different region can show
no or poor conservation. Nucleotide sequence conservation can not
be inferred from an amino acid similarity score. Two proteins may
show domains that in one region are homologous, while other regions
of the same protein are clearly non-homologous.
[0078] As used herein, the term "hybridization" is used in
reference to the pairing of complementary nucleic acids.
Hybridization can be demonstrated using a variety of hybridization
assays (Southern blot, Northern Blot, slot blot, phage plaque
hybridization, and other techniques). These protocols are common in
the art (See e.g., Sambrook et al. (eds.), Molecular Cloning: A
Laboratory Manual, Second Edition, Volumes 1-3, Cold Spring Harbor
Laboratory Press, NY, [ 1989]; Ausubel et al. (eds.), Current
Protocols in Molecular Biology, Vol. 1-4, John Wiley & Sons,
Inc., New York [1994]). Hybridization is the process of one nucleic
acid pairing with an antiparallel counterpart which may or may not
have 100% complementarity. Two nucleic acids which contain 100%
antiparallel complementarity will show strong hybridization. Two
antiparallel nucleic acids which contain no antiparallel
complementarity (generally considered to be less than 30%) will not
hybridize. Two nucleic acids which contain between 31-99%
complementarity will show an intermediate level of hybridization. A
single molecule that contains pairing of complementary nucleic
acids within its structure is said to be "self-hybridized."
[0079] As used herein, the term "stringency" is used in reference
to the conditions of temperature, ionic strength, and the presence
of other compounds such as organic solvents, under which nucleic
acids hybridize. "Low or weak stringency" conditions are reaction
conditions which favor the complementary base pairing and annealing
of two nucleic acids. "High stringency" conditions are those
conditions which are less optimal for complementary base pairing
and annealing. The art knows well that numerous variables affect
the strength of hybridization, including the length and nature of
the probe and target (DNA, RNA, base composition, present in
solution or immobilized, the degree of complementary between the
nucleic acids, the T.sub.m of the formed hybrid, and the G:C ratio
within the nucleic acids). Conditions may be manipulated to define
low or high stringency conditions: factors such as the
concentration of salts and other components in the hybridization
solution (e.g., the presence or absence of formamide, dextran
sulfate, polyethylene glycol) as well as temperature of the
hybridization and/or wash steps. Conditions of "low" or "high"
stringency are specific for the particular hybridization technique
used.
[0080] During hybridization of two nucleic acids under high
stringency conditions, complementary base pairing will occur only
between nucleic acid fragments that have a high frequency of
complementary base sequences. Thus, conditions of "weak" or "low"
stringency are often required with nucleic acids that are derived
from organisms that are genetically diverse, as the frequency of
complementary sequences is usually less. As used herein, two
nucleic acids which are able to hybridize under high stringency
conditions are considered "substantially homologous."
[0081] Whether sequences are "substantially homologous" may be
verified using hybridization competition assays. For example, a
"substantially homologous" nucleotide sequence is one that at least
partially inhibits a completely complementary probe sequence from
hybridizing to a target nucleic acid under conditions of low
stringency. This is not to say that conditions of low stringency
are such that non-specific binding is permitted; low stringency
conditions require that the binding of two sequences to one another
be a specific (i.e., selective) interaction. The absence of
non-specific binding may be verified by the use of a second target
that lacks even a partial degree of complementarity (e.g., less
than about 30% identity); in the absence of non-specific binding
the probe will not hybridize to the second non-complementary
target. When used in reference to a double-stranded nucleic acid
sequence such as a cDNA or genomic clone, the term "substantially
homologous" refers to any probe that can hybridize to either or
both strands of the double-stranded nucleic acid sequence under
conditions of high stringency.
[0082] A gene may produce multiple RNA species that are generated
by differential splicing of the primary RNA transcript. cDNAs that
are splice variants of the same gene contain regions of nucleotide
sequence identity (100% homo logy), representing the presence of
the same exon or portion of the same exon on both cDNAs, as well as
regions of complete non-identity. Because the two cDNAs contain
regions of sequence identity they will both hybridize to a probe
derived from the entire gene or portions of the gene containing
sequences found on both cDNAs. As used herein, the two splice
variants are therefore substantially homologous to such a probe and
to each other.
[0083] As used herein, the term "T.sub.m" is used in reference to
the "melting temperature." The melting temperature is the
temperature at which a population of double-stranded nucleic acid
molecules becomes half dissociated into single strands. The
equation for calculating the T.sub.m of nucleic acids is well known
in the art. As indicated by standard references, a simple estimate
of the T.sub.m value may be calculated by the equation:
T.sub.m=81.5+0.41(% G+C), when a nucleic acid is in aqueous
solution at 1 M NaCl (See e.g., Anderson and Young, Quantitative
Filter Hybridization, in Nucleic Acid Hybridization [1985]). Other
references include more sophisticated computations that take
structural as well as sequence characteristics into account for the
calculation of T.sub.m.
[0084] As used herein, the term "amplifiable nucleic acid" is used
in reference to nucleic acids which may be amplified by any
amplification method. It is contemplated that "amplifiable nucleic
acid" will usually comprise "template." As used herein, the term
"template" refers to nucleic acid originating from a sample that is
to be used as a substrate for the generation of the amplified
nucleic acid.
[0085] As used herein, the term "primer" refers to an
oligonucleotide, typically but not necessarily produced
synthetically, that is capable of acting as a point of initiation
of nucleic acid synthesis when placed under conditions in which
synthesis of a primer extension product that is complementary to a
nucleic acid strand is induced, (i.e., in the presence of
nucleotides, an inducing agent such as DNA polymerase, and at a
suitable temperature and pH). The primer is preferably single
stranded for maximum efficiency in amplification, but may
alternatively be double stranded. If double stranded, the primer is
first treated to separate its strands before being used to prepare
extension products. Preferably, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
inducing agent. The exact lengths of the primers will depend on
many factors, including temperature, source of primer and the use
of the method.
[0086] As used herein, the term "probe" refers to an
oligonucleotide (i.e., a sequence of nucleotides), whether produced
as a purified restriction digest or produced by synthetic means,
recombinantly or by amplification, that is capable of hybridizing
to another oligonucleotide of interest. A probe may be
single-stranded or double-stranded. Probes are useful in the
detection, identification and isolation of particular gene
sequences. It is contemplated that in preferred embodiments, any
probe used in the present invention will be labelled with any
"reporter molecule," so that is detectable in any detection system,
including, but not limited to enzyme (e.g., ELISA, as well as
immunohistochemical assays), fluorescent, radioactive, and
luminescent systems. It is not intended that the present invention
be limited to any particular detection system or label.
[0087] As used herein, the term "polymerase chain reaction" ("PCR")
refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195,
4,683,202 and 4,965,188, each of which is hereby incorporated by
reference, which describe a method for increasing the concentration
of a segment of a target sequence in a mixture of genomic DNA
without cloning or purification. This process for amplifying the
target sequence consists of introducing a large excess of two
oligonucleotide primers to the DNA mixture containing the desired
target sequence, followed by a precise sequence of thermal cycling
in the presence of a thermostable DNA polymerase. The two primers
are complementary to their respective strands of the double
stranded target sequence. To effect amplification, the mixture is
denatured and the primers then annealed to their complementary
sequences within the target molecule. Following annealing, the
primers are extended with a polymerase so as to form a new pair of
complementary strands. The steps of denaturation, primer annealing
and polymerase extension can be repeated many times (i.e.,
denaturation, annealing and extension constitute one "cycle"; there
can be numerous "cycles") to obtain a high concentration of an
amplified segment of the desired target sequence. The length of the
amplified segment of the desired target sequence is determined by
the relative positions of the primers with respect to each other,
and therefore, this length is a controllable parameter. By virtue
of the repeating aspect of the process, the method is referred to
as the "polymerase chain reaction" (hereinafter "PCR"). Because the
desired amplified segments of the target sequence become the
predominant sequences (in terms of concentration) in the mixture,
they are said to be "PCR amplified".
[0088] With PCR, it is possible to amplify a single copy of a
specific target sequence in genomic DNA to a level detectable by
several different methodologies (e.g., hybridization with a
labelled probe; incorporation of biotinylated primers followed by
avidin-enzyme conjugate detection; incorporation of
.sup.32P-labelled deoxynucleotide triphosphates, such as dCTP or
dATP, into the amplified segment). In addition to genomic DNA, any
oligonucleotide or polynucleotide sequence can be amplified with
the appropriate set of primer molecules. In particular, the
amplified segments created by the PCR process are, themselves,
efficient templates for subsequent PCR amplifications.
[0089] J. As used herein, the terms "PCR product," "PCR fragment,"
and "amplification product" refer to the resultant mixture of
compounds after two or more cycles of the PCR steps of
denaturation, annealing and extension are complete. These terms
encompass the case where there has been amplification of one or
more segments of one or more target sequences.
[0090] As used herein, the term "amplification reagents" refers to
those reagents (e.g., deoxyribonucleotide triphosphates, buffer,
etc.), needed for amplification except for primers, nucleic acid
template and the amplification enzyme. Typically, amplification
reagents along with other reaction components are placed and
contained in a reaction vessel (test tube, microwell, etc.).
[0091] As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to bacterial enzymes, each of which cut
double-stranded DNA at or near a specific nucleotide sequence.
[0092] The term "isolated" when used in relation to a nucleic acid,
as in "an isolated nucleic acid," "an isolated oligonucleotide" or
"isolated polynucleotide" refers to a nucleic acid sequence that is
identified and separated from at least one contaminant nucleic acid
with which it is ordinarily associated in its natural source.
Isolated nucleic acid is present in a form or setting that is
different from the form or setting of that nucleic acid found in
nature. In contrast, non-isolated nucleic acids are found in the
state in which they exist in nature. For example, a given DNA
sequence (e.g., a gene) is found on the host cell chromosome in
proximity to neighboring genes; RNA sequences, such as a specific
mRNA sequence encoding a specific protein, are found in the cell in
a mixture with numerous other mRNAs that encode a multitude of
proteins. However, isolated nucleic acid encoding a given
polypeptide includes, by way of example, such nucleic acid in cells
ordinarily expressing the given protein where the nucleic acid is
in a chromosomal location different from that of natural cells, or
is otherwise flanked by a different nucleic acid sequence than that
found in nature. The isolated nucleic acid, oligonucleotide, or
polynucleotide may be present in single-stranded or double-stranded
form. When an isolated nucleic acid, oligonucleotide or
polynucleotide is to be utilized to express a protein, the
oligonucleotide or polynucleotide will contain at a minimum the
sense or coding strand (i.e., the oligonucleotide or polynucleotide
may be single-stranded), but may contain both the sense and
anti-sense strands (i.e., the oligonucleotide or polynucleotide may
be double-stranded).
[0093] As used herein, the term "purified" or "to purify" refers to
the removal of contaminants from a sample. For example, antibodies
are purified by removal of contaminating non-immunoglobulin
proteins; they are also purified by the removal of immunoglobulin
that does not bind to the target molecule. The removal of
non-immunoglobulin proteins and/or the removal of immunoglobulins
that do not bind to the target molecule results in an increase in
the percent of target-reactive immunoglobulins in the sample. In
another example, recombinant polypeptides are expressed in
bacterial host cells and the polypeptides are purified by the
removal of host cell proteins; the percent of recombinant
polypeptides is thereby increased in the sample.
[0094] As used herein, the term "vector" is used in reference to
nucleic acid molecules that transfer DNA segment(s) from one cell
to another. The term "vehicle" is sometimes used interchangeably
with "vector." A vector "backbone" comprises those parts of the
vector which mediate its maintenance and enable its intended use
(e.g., the vector backbone may contain sequences necessary for
replication, genes imparting drug or antibiotic resistance, a
multiple cloning site, and possibly operably linked
promoter/enhancer elements which enable the expression of a cloned
nucleic acid). The cloned nucleic acid (e.g., such as a cDNA coding
sequence, or an amplified PCR product) is inserted into the vector
backbone using common molecular biology techniques. Vectors are
often derived from plasmids, bacteriophages, or plant or animal
viruses. A "cloning vector" or "shuttle vector" or "subcloning
vector" contain operably linked parts which facilitate subcloning
steps (e.g., a multiple cloning site containing multiple
restriction endonuclease sites). A "recombinant vector" indicates
that the nucleotide sequence or arrangement of its parts is not a
native configuration, and has been manipulated by molecular
biological techniques. The term implies that the vector is
comprised of segments of DNA that have been artificially
joined.
[0095] The term "expression vector" as used herein refers to a
recombinant DNA molecule containing a desired coding sequence and
operably linked nucleic acid sequences necessary for the expression
of the operably linked coding sequence in a particular host
organism (e.g., a bacterial expression vector, a yeast expression
vector or a mammalian expression vector). Nucleic acid sequences
necessary for expression in prokaryotes typically include a
promoter, an operator (optional), and a ribosome binding site,
often along with other sequences. Eukaryotic cells utilize
promoters, enhancers, and termination and polyadenylation signals
and other sequences which are different from those used by
prokaryotes.
[0096] Eukaryotic expression vectors may also contain "viral
replicons" or "viral origins of replication." Viral replicons are
viral DNA sequences that allow for the extrachromosomal replication
of a vector in a host cell expressing the appropriate replication
factors. Some vectors replicate their nucleic acid to high copy
numbers (e.g., vectors that contain either the SV40 or polyoma
virus origin of replication replicate to high "copy number" (up to
10.sup.4 copies/cell) in cells that express the appropriate viral T
antigen). Other vectors replicate their nucleic acid in low copy
numbers (e.g., vectors that contain the replicons from bovine
papillomavirus or Epstein-Barr virus replicate extrachromosomally
at "low copy number" (.about.100 copies/cell)). The viral origins
of replication listed above are not limiting, as the art is aware
of other origins of replication that are commonly used in
eukaryotic expression vectors.
[0097] The term "transgene" as used herein refers to a foreign gene
that is placed into an organism by, for example, introducing the
foreign gene into newly fertilized eggs or early embryos. The term
"foreign gene" refers to any nucleic acid (e.g., gene sequence)
that is introduced into the genome of an animal by experimental
manipulations and may include gene sequences found in that animal
so long as the introduced gene does not reside in the same location
as does the naturally-occurring gene.
[0098] Embryonal cells at various developmental stages can be used
to introduce transgenes for the production of transgenic, non-human
animals. Different methods are used depending on the stage of
development of the embryonic cell. The zygote is the best target
for micro-injection. In the mouse, the male pronucleus reaches the
size of approximately 20 micrometers in diameter which allows
reproducible injection of 1-2 picoliters (pi) of DNA solution. The
use of zygotes as a target for gene transfer has a major advantage
in that in most cases the injected DNA will be incorporated into
the host genome before the first cleavage (Brinster et al, Proc.
Natl. Acad Sci. USA 82:4438-4442 [1985]). As a consequence, all
cells of the transgenic non-human animal will carry the
incorporated transgene. This will, in general, also be reflected in
the efficient transmission of the transgene to offspring of the
founder since 50% of the germ cells will harbor the transgene.
Micro-injection of zygotes is the preferred method for
incorporating transgenes in practicing the invention. U.S. Pat. No.
4,873,191, herein incorporated by reference, describes a method for
the micro-injection of zygotes.
[0099] Retroviral infection can also be used to introduce
transgenes into a non-human animal. The developing embryo can be
cultured in vitro to the blastocyst stage. During this time, the
blastomeres can be targets for retroviral infection (Jaenisch,
Proc. Natl. Acad. Sci. USA 73:1260-1264 [1976]). Efficient
infection of the blastomeres is obtained by enzymatic treatment to
remove the zona pellucida (See e.g., Hogan et al., in Manipulating
the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. [1986]). The viral vector system used to introduce the
transgene is typically a replication-defective retro virus carrying
the transgene (Jahner et al, Proc. Natl. Acad Sci. USA 82:6927-693
[1985]). Transfection is easily and efficiently obtained by
culturing the blastomeres on a monolayer of virus-producing cells
(van der Putten, Proc. Natl. Acad Sci. USA 82(18):6148-52 [1985];
and Stewart, et al., EMBO J. 6:383-388 [1987]). Alternatively,
infection can be performed at a later stage. Virus or
virus-producing cells can be injected into the blastocoele (Jahner
et al Nature 298:623-628 [1982]). Most of the founders will be
mosaic for the transgene since incorporation occurs only in a
subset of cells that form the transgenic animal. Further, the
founder may contain various retroviral insertions of the transgene
at different positions in the genome that generally will segregate
in the offspring. In addition, it is also possible to introduce
transgenes into the germline, albeit with low efficiency, by
intrauterine retroviral infection of the midgestation embryo
(Jahner et al, Nature 298:623-628 [1982]). Additional means of
using retroviruses or retroviral vectors to create transgenic
animals known to the art include, but are not limited to, the
micro-injection of retroviral particles or mitomycin C-treated
cells producing retrovirus into the perivitelline space of
fertilized eggs or early embryos (PCT International Application WO
90/08832 [1990], and Haskell and Bowen, Mol. Reprod. Dev., 40:386
[1995]).
[0100] A third type of target cell for transgene introduction is
the embryonal stem (ES) cell. ES cells are obtained by culturing
pre-implantation embryos in vitro under appropriate conditions
(Evans et al, Nature 292:154-156 [1981]; Bradley et al, Nature
309:255-258 [1984]; Gossler et al, Proc. Acad. Sci. USA
83:9065-9069 [1986]; and Robertson et al, Nature 322:445-448
[1986]). Transgenes can be efficiently introduced into the ES cells
by DNA transfection using a variety of methods known to the art
including calcium phosphate co-precipitation, protoplast or
spheroplast fusion, lipofection and DEAE-dextran-mediated
transfection. Transgenes may also be introduced into ES cells by
retro virus-mediated transduction or by micro-injection. Such
transfected ES cells can thereafter colonize an embryo following
their introduction into the blastocoel of a blastocyst-stage embryo
and contribute to the germ line of the resulting chimeric animal
(for review, see Jaenisch, Science 240:1468-1474 [1988]). Prior to
the introduction of transfected ES cells into the blastocoel, the
transfected ES cells may be subjected to various selection
protocols to enrich for ES cells that have integrated the
transgene, assuming that the transgene provides a means for such
selection. Alternatively, PCR may be used to screen for ES cells
that have integrated the transgene. This technique obviates the
need for growth of the transfected ES cells under appropriate
selective conditions prior to transfer into the blastocoel.
[0101] The terms "overexpression" and "overexpressing" and
grammatical equivalents are used in reference to levels of mRNA or
protein where the level of expression of the mRNA or protein is
higher than that typically observed in a given tissue in a control
or non-transgenic animal. Levels of mRNA or protein are measured
using any of a number of techniques known to those skilled in the
art. For example, mRNA levels may be assayed using (but not limited
to) a Northern blot analysis. Appropriate controls are included on
the Northern blot to control for differences in the amount of RNA
loaded from each tissue analyzed (e.g., the amount of 28S rRNA, an
abundant RNA transcript present at essentially the same amount in
all tissues, present in each sample can be used as a means of
normalizing or standardizing the mRNA-specific signal observed on
Northern blots). The amount of mRNA present in the band
corresponding in size to the correctly spliced transgene RNA is
quantified; other minor species of RNA which hybridize to the
transgene probe are aot considered in the quantification of the
expression of the transgenic inRNA.
[0102] The term "transfection" as used herein refers to the
introduction of foreign DNA into cells. Transfection may be
accomplished by a variety of means known to the art including
calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated
transfection, polybrene-mediated transfection, electroporalion,
microinjection, liposome fusion, lipofection, protoplast fusion,
retroviral infection, and biolistics. Mammalian
cell.cndot.transfection-techniques are common in the art, and are
described in many sources (See, e.g., Ausubel et rt/.-(eds.),
Current Protocols in Molecular Biology, Vol. 1-4, John Wiley &
Sons, Inc., New York [1994]).
[0103] The term "stable transfcctioa" or "stably triuisfected"
refers to ihejntrocluction and integration of foreign DNA into the
genome -of. the transfected.cell. The term "stable transfectant"
refers to a cell which contains stably integrated foreign DNA
within its own gertomic DNA.
[0104] The term "transient transfection" or "transiently
transfected" refers to the introduction of foreign DNA into a cell
where the foreign DNA fails to integrate into the genome of the
trans fected cell. The foreign DNA persists in the nucleus of the
transfected ceii for several days. During this time the foreign DNA
is subject to the regulatory controls that govern the expression of
endogenous genes in the chromosomes. The term "transient
transfectant" refers to ceils which have taken up foreign DNA but
have failed to integrate this DNA.
[0105] The term "calcium phosphate co-precipitation" refers to a
technique for the introduction of nucleic acids into;i eukaryotic
cell, and. most typically .mammalian cells. The uptake of nucleic
acids by cells is enhanced-when the nucleic acid is presented as a
calcium phosphate-nucleic acid co-precipitate. Various
modifications of the original technique of Graham and van der-Eb
(Graham and van der Eb, Virol., 52:456 [1973]) are "known in which
the conditions for the transfection of a particular cell type has
been optimized. The art is well aware of these various methods.
[0106] The term "transformation" has multiple meanings, depending
on its usage. In one sense, the term.sup.|;transformation" is i-sed
to describe the process of introduction of foreign DNA into
prokaryotic cells (i.e., bacterial cells), and most frequently E.
coli strains. Bacterial cell transformation may be accomplished by
a variety of means well known to the art, including the preparation
of "competent" bacteria by the use of calcium chloride, magnesium
chloride or rubidium chloride, and electroporation. When a plasmid
is used as the transformation vector, the plasmid typically
contains a gene conferring drug resistance, such as the genes
encoding ampicillin, tetracycline or kanamycin resistance.
Bacterial transformation techniques are common in the art, and are
described in many sources (e.g., Cohen et al., Proc. Natl. Acad.
Sci. USA 69: 2110-2114 [1972]; Hanahan, J. Mol. Biol, 166:557-580
[1983]; Sambrook et al. (eds.), Molecular Cloning: A Laboratory
Manual, Second Edition, Volumes 1-3, Cold Spring Harbor Laboratory
Press, NY, [1989]; Ausubel et al. (eds.), Current Protocols in
Molecular Biology, Vol. 1-4, John Wiley & Sons, Inc., New York
[1994]).
[0107] "Transformation" also describes the physiological process by
which a normal eukaryotic cell acquires the phenotypic qualities of
a malignant cell. Such properties can include the ability to grow
in soft agar, the ability to grow in nutrient poor conditions,
rapid proliferation, and the loss of contact inhibition. A
eukaryotic cell which is "transformed" displays the properties of
malignant cells. A eukaryotic cell may acquire its transformed
phenotype in vivo, or be artificially transformed in culture.
[0108] As used herein, the term "selectable marker" refers to the
use of a gene that encodes an enzymatic activity that confers the
ability to grow in medium lacking what would otherwise be an
essential nutrient (e.g., the HISS gene in yeast cells); in
addition, a selectable marker may confer resistance to an
antibiotic or drug upon the cell in which the selectable marker is
expressed. Furthermore, selectable markers may be "dominant."
Dominant selectable markers encode an enzymatic activity that can
be detected in any eukaryotic cell line. Examples of dominant
selectable markers include the bacterial aminoglycoside 3'
phosphotransferase gene (i.e., the neo gene) that confers
resistance to the drug G-418 in mammalian cells, as well as the
bacterial hygromycin G phosphotransferase (hyg) gene that confers
resistance to the antibiotic hygromycin, and the bacterial
xanthine-guanine phosphoribosyl transferase gene (i.e., the gpt
gene) that confers the ability to grow in the presence of
mycophenolic acid. The use of non-dominant selectable markers must
be in conjunction with a cell line that lacks the relevant enzyme
activity. Examples of non-dominant selectable markers include the
thymidine kinase (tk) gene (used in conjunction with tk.about. cell
lines), the CAD gene (used in conjunction with CAD-deficient cells)
and the mammalian hypoxanthine-guanine phosphoribosyl transferase
(hprj) gene (used in conjunction with hprt' cell lines). A review
of the use of selectable markers in mammalian cell lines is
provided in Sambrook et al., Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor Laboratory Press, New
York (1989), at pp.16.9-16.15.
[0109] As used herein in the context of protein purification, the
terms "source culture," "starting culture," "starting material" or
"culture" or the like can include any of the following materials:
culture supernatant, cultured eukaryotic or prokaryotic cells
(e.g., animal cells or bacteria), crushed eukaryotic or prokaryotic
cells, tissue removed from an organism, or the product of an in
vitro translation or in vitro coupled transcription/translation
reaction. The cells of such a culture may or may not contain
recombinant nucleic acid.
[0110] As used herein, the term "cell culture" refers to any in
vitro culture of cells. Included within this term are continuous
cell lines (e.g., with an immortal phenotype), primary cell
cultures, finite cell lines (e.g., non-transformed cells), and any
other cell population maintained in vitro.
[0111] The term "test compound" refers to any chemical entity,
pharmaceutical, drug, composition, and the like that can be used to
treat or prevent a disease, illness, sickness, or disorder of
bodily function. Test compounds comprise both known and potential
therapeutic compounds. A "known therapeutic compound" refers to a
therapeutic compound that has been previously described.
Particularly preferred known therapeutics are those that have been
shown (e.g., through animal trials or prior experience with
administration to humans) to be effective in treatment or
prevention of pathology.
[0112] As used herein, a "drug" can be any molecule of any
composition, including protein, peptide, nucleic acid, organic
molecule, inorganic molecule, or combinations of molecules,
biological or non-biological, which are capable of producing a
physiological response. As used herein, a "drug" provides at least
one beneficial response in the cure, mitigation, treatment or
prevention of a disease or disorder. A compound is considered a
"drug candidate" if it is not yet known if that compound will
provide at least one beneficial response in the cure, mitigation,
treatment or prevention of a disease, disorder or condition. A
"drug library" is a collection of molecules, where it may or may
not be known if one or multiple drugs in the library have
therapeutic value.
[0113] As used herein, the terms "host," "expression host," and
"transformant" refer to organisms and/or cells which harbor an
exogenous DNA sequence (e.g., via transfection), an expression
vector or vehicle, as well as organisms and/or cells that are
suitable for use in expressing a recombinant gene or protein. It is
not intended that the present invention be limited to any
particular type of cell or organism. Indeed, it is contemplated
that any suitable organism and/or cell will find use in the present
invention as a host.
[0114] As used herein, the term "subject" refers to any animal
being examined, studied or treated. It is not intended that the
present invention be limited t6 any particular type of subject. It
is contemplated that multiple organisms will find use in the
present invention as subjects. In some embodiments, humans are the
preferred subject.
[0115] A subject displaying pathology resulting from abnormal
cytokine activity may display symptoms that include, but are not
limited to, inflammation, cachexia, insulin resistance,
overstimulation of interleukin-6 and granulocyte/macrophage-colony
stimulating factor (GM-CSF) secretion, enhanced cytotoxicity of
polymorphonuclear neutrophils, prolonged expression of cellular
adhesion molecules, induction of procoagulant activity on vascular
endothelial cells, increased adherence of neutrophils and
lymphocytes, stimulation of the release of platelet activating
factor from macrophages, neutrophils and vascular endothelial
cells, fever, malaise, and anorexia.
[0116] As used herein, a "disease" is a disruption of normal body
function, generally where two of three criteria are met: 1) the
etiological agent is known, 2) an identifiable group of symptoms
appears, and 3) there are consistent anatomical or physiological
alterations. Examples include, but are not limited to rheumatoid
arthritis, inflammatory bowel disease and graft-versus-host
disease. A disorder is, in general, a disruption of some aspect of
normal body function (e.g., rheumatoid arthritis and inflammatory
bowel disease are immune disorders).
[0117] As used herein, the term "antigen" refers to any agent
(e.g., any substance, compound, molecule [including
macromolecules], or other moiety), that is recognized by an
antibody, while the term "immunogen" refers to any agent (e.g., any
substance, compound, molecule [including macromolecules], or other
moiety) that can elicit an immunological response in an individual.
These terms may be used to refer to an individual macromolecule or
to a homogeneous or heterogeneous population of antigenic
macromolecules. It is intended that the terms antigen and immunogen
encompass protein molecules or at least one portion of a protein
molecule, which contains one or more epitopes. In many cases,
antigens are also immunogens, thus the term "antigen" is often used
interchangeably with the term "immunogen." The substance may then
be used as an antigen in an assay to detect the presence of
appropriate antibodies in the serum of the immunized animal.
[0118] The term "antigenic determinant" as used herein refers to
that portion of an antigen that makes contact with a particular
antibody (i.e., an epitope). When a protein or fragment of a
protein is used to immunize a host animal, numerous regions of the
protein may induce the production of antibodies which bind
specifically to a given region or three-dimensional structure on
the protein; these regions or structures are referred to as
antigenic determinants. An antigenic determinant may compete with
the intact antigen (i.e., the "immunogen" used to elicit the immune
response) for binding to an antibody.
[0119] The terms "specific binding" or "specifically binding" when
used in reference to the interaction of an antibody and a protein
or peptide means that the interaction is dependent upon the
presence of a particular structure (i.e., the antigenic determinant
or epitope) on the protein; in other words the antibody is
recognizing and binding to a specific protein structure rather than
to proteins in general.
[0120] As used herein, the term "adjuvant" is defined as a
substance known to increase the immune response to other antigens
when administered with other antigens. If adjuvant is used, it is
not intended that the present invention be limited to any
particular type of adjuvant--or that the same adjuvant, once used,
be used all the time. It is contemplated that adjuvants may be used
either separately or in combination. The present invention
contemplates all types of adjuvant, including but not limited to
agar beads, aluminum hydroxide or phosphate (alum), Incomplete
Freund's adjuvant (incomplete or complete), as well as Quil A
adjuvant and Gerbu adjuvant (Accurate Chemical and Scientific
Corporation), and bacterins (i.e., killed preparations-of bacterial
cells). It is further contemplated that the vaccine comprise at
least one "excipient" (i.e., a pharmaceutically acceptable carrier
or substance) suitable for administration to a human or other
animal subject. It is intended that the term "excipient" encompass
liquids, as well as solids, and colloidal suspensions.
[0121] As used herein the term "immunogenically-effective amount"
refers to that amount of an immunogen required to invoke the
production of protective levels of antibodies in a host upon
vaccination.
[0122] The term "protective level," when used in reference to the
level of antibodies induced upon immunization of the host with an
immunogen means a level of circulating antibodies sufficient to
protect the host from challenge with a lethal dose of an organism
or other material (e.g., toxins, etc.).
[0123] The terms "self antigen" or "autoantigen" refer to an
antigen or a molecule normally expressed by an individual, but
which solicits an immune response. Under normal conditions, these
autoantigens are recognized during an immune response as self
(i.e., an antigen that is normally part of the individual), and do
not solicit an immune response. This is in contrast to antigens
which are foreign, or exogenous, and are thus not normally part of
the individual's antigenic makeup. "Self antigen" or "autoantigen"
is recognized as foreign, although the antigen is native to the
individual's physiology.
[0124] As used herein, the term "autoimmune disease" means a set of
sustained organ-specific or systemic clinical symptoms and signs
associated with altered immune homeostasis that is manifested by
qualitative and/or quantitative defects of expressed autoimmune
repertoires. Autoimmune diseases are characterized by antibody or
cytotoxic immune responses to epitopes on self antigens. The immune
system of the individual then activates an inflammatory cascade
aimed at cells and tissues presenting those specific self antigens.
The destruction of the antigen, tissue, cell type, or organ
attacked by the individual's own immune system gives rise to the
signs and symptoms of the disease. Clinically significant
autoimmune diseases include, for example, rheumatoid arthritis,
multiple sclerosis, juvenile-onset diabetes, systemic lupus
erythematosus (SLE), autoimmune uveoretinitis, autoimmune
vasculitis, bullous pemphigus, myasthenia gravis, autoimmune
thyroiditis or Hashimoto's disease, Sjogren's syndrome,
granulomatous orchitis, autoimmune oophoritis, Crohn's disease,
sarcoidosis, rheumatic carditis, ankylosing spondylitis,
glomerulonephritis, Grave's disease, and autoimmune
thrombocytopenic purpura.
[0125] The ability of a particular antigen to stimulate a
cell-mediated immunological response may be determined by a number
of assays, including but not limited to lymphoproliferation (i.e.,
lymphocyte activation) assays, CTL cytotoxic cell assays such as
chromium-release assays, or by assaying for T lymphocytes specific
for the antigen in a sensitized subject. Such assays are well known
in the art (See e.g., Erickson et al., J. Immunol., 151:4189-4199
[1993 and Doe et al., Eur. J. Immunol, 24:2369-2376 [1994]).
[0126] The term "modulate," as used herein, refers to a change in
the biological activity of a biologically active molecule.
Modulation can be an increase or a decrease in activity, a change
in binding characteristics, or any other change in the biological,
functional, or immunological properties of biologically active
molecules.
[0127] The term "agonist," as used herein, refers to a molecule
which, when interacting with an biologically active molecule,
causes a change (e.g., enhancement) in the biologically active
molecule, which modulates the activity of the biologically active
molecule. Agonists may include proteins, nucleic acids,
carbohydrates, or any other molecules which bind or interact with
biologically active molecules. For example, agonists can alter the
activity of gene transcription by interacting with KNA polymerase
directly or through a transcription factor.
[0128] The terms "antagonist" or "inhibitor," as used herein, refer
to a molecule which, when interacting with a biologically active
molecule, blocks or modulates the biological activity of the
biologically active molecule. Antagonists and inhibitors may
include proteins, nucleic acids, carbohydrates, or any other
molecules that bind or interact with biologically active molecules.
Inhibitors and antagonists can effect the biology of cells,
tissues, organs or entire organisms.
[0129] The terms "Western blot," "Western immunoblot" "immunoblot"
and "Western" refer to the immunological analysis of protein(s),
polypeptides or peptides that have been immobilized onto a membrane
support. The proteins are first resolved by polyacrylamide gel
electrophoresis (i.e., SDS-PAGE) to separate the proteins, followed
by transfer of the protein from the gel to a solid support, such as
nitrocellulose or a nylon membrane. The immobilized proteins are
then exposed to an antibody having reactivity towards an antigen of
interest. In preferred embodiments, the binding of the antibody
(i.e., the primary antibody) is detected by use of a secondary
antibody which specifically binds the primary antibody. The
secondary antibody is typically conjugated to an enzyme which
permits visualization of the antigen-antibody complex by the
production of a colored reaction product or catalyzes a luminescent
enzymatic reaction (e.g., the ECL reagent, Amersham).
[0130] As used herein, the term "ELISA" refers to enzyme-linked
immunosorbent assay (or EIA). Numerous ELISA methods and
applications are known in the art, and are described in many
references (See e.g., Crowther, "Enzyme-Linked Immunosorbent Assay
(ELISA)," in Molecular Biomethods Handbook, Rapley et al. [eds.],
pp. 595-617, Humana Press, Inc., Totowa, N.J. [1998]; See also,
Harlow and Lane (eds.), Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory Press [1988]; and Ausubel et al. (eds.),
Current Protocols in Molecular Biology, Ch. 11, John Wiley &
Sons, Inc., New York [1994], for general descriptions of ELISA
methodology). In addition, there are numerous commercially
available ELISA test systems, equipment and individual
reagents.
[0131] One ELISA method is a "direct ELISA," where an antigen
(e.g., sTNFR1 or ARTS-1) in a sample is detected. In one embodiment
of the direct ELISA, a sample containing antigen is exposed to a
solid (i.e., stationary or immobilized) support (e.g., a microtiter
plate well). The antigen within the sample becomes immobilized to
the stationary phase, and is detected directly using an
enzyme-conjugated antibody specific for the antigen.
[0132] In an alternative embodiment, an antibody specific for an
antigen is detected in a sample. In this embodiment, a sample
containing an antibody (e.g., anti-ARTS-1 antiserum) is immobilized
to a solid support (e.g., a microtiter plate well). The
antigen-specific antibody is subsequently detected using purified
antigen and an enzyme-conjugated antibody specific for the
antigen.
[0133] In further alternative embodiments, an "indirect ELISA" is
used to detect antibody or antigen in samples. In one embodiment,
an antigen (or antibody) is immobilized to a solid support (e.g., a
microtiter plate well) as in the direct ELISA, but is detected
indirectly by first adding an antigen-specific antibody (or
antigen), then followed by the addition of a detection antibody
specific for the antibody that specifically binds the antigen, also
known as "species-specific" antibodies (e.g., a goat anti-rabbit
antibody), which are available from various manufacturers known to
those in the art (e.g., Santa Cruz Biotechnology; Zymed and
Pharmingen/Transduction Laboratories).
[0134] In other embodiments, a "sandwich ELISA" is used, where the
antigen is immobilized on a solid support (e.g., a microtiter
plate) via an antibody (i.e., a capture antibody) that is
immobilized on the solid support and is able to bind the antigen of
interest. Following the affixing of a suitable capture antibody to
the immobilized phase, a sample is then added to the microtiter
plate well, followed by washing. If the antigen of interest is
present in the sample, it is bound to the capture antibody present
on the support. In some embodiments, a sandwich ELISA is a "direct
sandwich" ELISA, where the captured antigen is detected directly by
using an enzyme-conjugated antibody directed against the antigen.
Alternatively, in other embodiments, a sandwich ELISA is an
"indirect sandwich" ELISA, where the captured antigen is detected
indirectly by using an antibody directed against the antigen, which
is then detected by another enzyme-conjugated antibody which binds
the antigen-specific antibody, thus forming an
antibody-antigen-antibody-antibody complex. Suitable reporter
reagents are then added to detect the third antibody.
Alternatively, in some embodiments, any number of additional
antibodies are added as necessary, in order to detect the
antigen-antibody complex. In some preferred embodiments, these
additional antibodies are labelled or tagged, so as to permit their
visualization and/or quantitation.
[0135] As used herein, the term "capture antibody" refers to an
antibody that is used in a sandwich ELISA to bind (i.e., capture)
an antigen in a sample prior to detection of the antigen. In one
embodiment of the present invention, biotinylated capture
antibodies are used in the present invention in conjunction with
avidin-coated solid support. Another antibody (i.e., the detection
antibody) is then used to bind and detect the antigen-antibody
complex, in effect forming a "sandwich" comprised of
antibody-antigen-antibody (i.e., a sandwich ELISA).
[0136] As used herein, a "detection antibody" is an antibody which
carries a means for visualization or quantitation. Typically,
detection antibodies are conjugated enzyme moieties that yield a
colored, fluorescent, or luminescent reaction product following the
addition of a suitable substrate. Conjugated enzymes commonly used
with detection antibodies in the ELISA include, but are not limited
to horseradish peroxidase, urease, alkaline phosphatase,
glucoamylase and p-galactosidase. In some embodiments, the
detection antibody is directed against the antigen of interest,
while in other embodiments, the detection antibody is not directed
against the antigen of interest. Thus, in some embodiments, the
detection antibody is directed against another antibody. In some
embodiments, the detection antibody is an anti-species antibody.
Alternatively, the detection antibody is prepared with a label
(e.g., biotin, a fluorescent marker, or a radioisotope), and is
detected and/or quantitated using this label.
[0137] As used herein, the terms "reporter reagent," "reporter
molecule," "detection substrate" and "detection reagent" are used
in reference to reagents which permit the detection and/or
quantitation of an antibody bound to an antigen. For example, in
some embodiments, the reporter reagent is a calorimetric substrate
for an enzyme that has been conjugated to an antibody. Addition of
a suitable substrate to the antibody-enzyme conjugate results in
the production of a colorimetric, fluorimetric, or luminescent
signal (e.g., following the binding of the conjugated antibody to
the antigen of interest). Other reporter reagents include, but are
not limited to, radioactive compounds. This definition also
encompasses the use of biotin and avidin-based compounds (e.g.,
including but not limited to neutravidin and streptavidin) as part
of the detection system.
[0138] As used herein, the term "signal" is used generally in
reference to any detectable process that indicates that a reaction
has occurred, for example, binding of antibody to antigen. It is
contemplated that signals in the form of radioactivity,
fluorimetric or colorimetric products/reagents will all find use
with the present invention. In various embodiments of the present
invention, the signal is assessed qualitatively, while in
alternative embodiments, the signal is assessed quantitatively.
[0139] As used herein, the term "amplifier" is used in reference to
a system which enhances the signal in a detection method, such as
an ELISA (e.g., an alkaline phosphatase amplifier system used in an
ELISA).
[0140] As used herein, the term "solid support" is used in
reference to any solid or stationary material to which reagents
such as antibodies, antigens, and other test components are
attached. For example, in preferred ELISA methods, the wells of
microtiter plates provide solid supports. Other examples of solid
supports include microscope slides, coverslips, beads, particles,
cell culture flasks, as well as any other suitable items.
[0141] As used herein, the term "kit" is used in reference to a
combination of reagents and other materials which facilitate sample
analysis. In various embodiments of the present invention, a kit
can include antibodies (e.g., a suitable capture antibody, reporter
antibody, primary antibody, secondary antibody, detection antibody)
purified control antigen, detection reagents, amplifier system, and
nucleic acid probes. Furthermore, in other embodiments, the kit
includes, but is not limited to, components such as apparatus for
sample collection, sample tubes, holders, trays, racks, dishes,
plates, instructions on the use of the kit, solutions or other
chemical reagents, and samples to be used for standardization,
normalization, and/or control samples. It is contemplated that kits
of the present invention can also include apparatus and reagents
for electrophoresis and blotting.
[0142] As used herein, the term "in vitro" refers to an artificial
environment and to processes or reactions that occur within an
artificial environment. In vitro environments consist of, but are
not limited to, controlled laboratory conditions. The term "in
vivo" refers to the natural environment (e.g., an animal or a cell)
and to processes or reactions that occur within that natural
environment.
[0143] As used herein, the terms "local" or "localized" and the
like refer to confinement to a small area, a single tissue, a
single organ (e.g., a lung) or other defined structure.
[0144] As used herein, the term "localized delivery" is delivery of
an agent (e.g., a gene therapy agent or a drug) to a small area, a
single tissue, a single organ or other specific structure. For
example, localized delivery of a gene therapy agent to a single
site (e.g., the liver) in a subject is typically achieved by
injection into that site.
[0145] As used herein, the term "systemic" refers to multiple
sites, tissues or organs in an organism, or to the entire organism.
Use of the word "systemic" generally indicates involvement of the
circulatory or lymphatic systems.
[0146] As used herein, the term "systemic delivery" (in contrast to
localized delivery) is delivery of an agent (e.g., a drug) to
multiple sites, tissues or organs in an organism, or to the entire
organism via the circulatory system following an intravenous
injection, or via gastrointestinal absorption of an orally
administered agent.
[0147] As used herein, the term "surgical delivery" refers to the
delivery of an agent (e.g., a gene therapy agent) by surgical means
(i.e., by operation or some other invasive manipulation). Thus, in
some embodiments, surgical techniques provide means for localized
delivery of an agent.
DESCRIPTION OF THE INVENTION
[0148] The present invention provides compositions and methods
related to regulation of cytokine signaling through the Tumor
Necrosis Factor (TNF) pathway. Specifically, the present invention
provides a novel polypeptide, and a gene which encodes the
polypeptide, as set forth in FIG. 1 and SEQ ID NOS:1 and 2, which
has the ability to promote the shedding of the extracellular domain
of Type I Tumor Necrosis Factor Receptor (TNFR1). This polypeptide
and gene are called ARTS-1, for aminopeptidase regulator of type I,
55 kDa tumor necrosis factor receptor ectodomain shedding. It is
contemplated that methods which regulate the shedding of the sTNFR1
also regulate the activity of TNF. It is further contemplated that
the ARTS-1 gene, as well as genes substantially homologous to the
ARTS-1 gene (and the gene product) regulate ectodomain shedding of
other cytokine receptors, including IL-1 and IL-6. It is also
contemplated that the compositions and methods provided by the
present invention will find use in therapeutics for the treatment
of diseases and disorders resulting from aberrant TNF activity.
TNFR1 Shedding
[0149] The complete mechanisms underlying TNFR1 ectodomain shedding
are unknown, but are thought to be mediated via proteolytic
cleavage of the spacer region located between the transmembrane and
TNF-ligand binding domain (Brakebusch et al., J. Biol. Chem.,
269(51):32488-96 [1994]). Based upon amino acid sequencing of
sTNFR1 isolated from urine, the major cleavage site has been
identified as occurring between Asn-180 and Val-181, with a minor
site located between Lys-182 and Gly-183 (Nophar et al, EMBO J.,
9:3269-3278 [1990]; Wallach et al, In Tumor Necrosis Factor:
Structure-Function Relationship and Clinical Application (Osawa and
Baonavida, eds.) Vol III, 47-57, S. Karger, Basel, Switzerland
[1991]; and Brakebusch et al, J. Biol. Chem., 269(51):32488-96
[1994]). However, an understanding of the mechanism(s) is not
necessary in order to use the present invention.
[0150] Again, although an understanding of the mechanism(s) is not
necessary in order to use the present invention, a complete
understanding of the mechanism regulating TNFR1 shedding requires
the identification and characterization of the interactions between
regulatory proteins, such as ARTS-1, TNFR1, and TNFR1 receptor
sheddases, which appear to belong to the
metalloprotease-disintegrin (ADAM) family of zinc metalloproteases
(Blobel et al, Cell 90:589-592 [1997]). For example, TNF-a
converting enzyme (TACE, AD AM 17) has been reported to mediate the
ectodomain shedding of TNF-a, tranforming growth factor-a,
L-selectin and TNFR2 (Black et al., Nature 385:729-733 [1997]; Moss
et al, Nature 385:733-736 [1997]; and Peschon et al, Science
282:1281-1284 [1998]). Similarly, TACE has been implicated in the
regulated cc-secretase cleavage of the amyloid precursor protein
and ectodomain shedding of erbB4/HER4 (Buxbaum et al, J. Biol
Chem., 273:27765-27767 [1998]; Rio et al, J. Biol. Chem.,
275:10379-10387 [2000]). Recent data suggest that TACE may also
mediate TNFR1 ectodomain shedding based upon the demonstration of
increased TNFR1 shedding in reconstituted TACE-deficient cell lines
(Reddy et al, J. Biol. Chem., 275:14608-14614 [2000]). Indeed, TACE
has been implicated as having sheddase activity toward both TNFR1
and IL-1R type II (Reddy et al, supra).
[0151] Studies have also been undertaken to identify enzymes
capable of cleaving (i.e., shedding) the leucocyte selectin
(L-selectin) receptor, a peripheral lymph node homing receptor. The
enzymes which shed the L-selectin receptor have been theorized to
be metalloproteases (Preece et al, J. Biol. Chem., 271:11634-11640
[1996]; Peschon et al, Science 282:1281-1284 [1998]; and Borland et
al., J. Biol. Chem., 274:2810-2815 [1999]).
[0152] Neither the sheddase nor the mechanism regulating the
shedding of sTNFR1 are known, although attempts have been made to
identify sTNFR1 shedding activities in crude preparations. However,
the results indicate metal requirements for these activities,
thereby tentatively categorizing the enzymes as metalloproteases
(Bjonberg et al., Scand J. Immunol, 42:418-424 [1995]; Mullberg et
al, J. Immunol, 155:5198-5205 [1995]; Katsura et al, Biochem.
Biophys. Res. Comm., 222:298-302 [1996]; Williams et al, J. Clin.
Invest., 97(12):2833-2841 [1996]; and Gallea-Robache et al,
Cytokine 9:340-346 [1997]). However, the molecular identities of
these metalloprotease enzymes remain unknown.
[0153] Bjornberg et al (Bjornberg et al, Scand. J. Immunol,
42:418-424 [1995]) indicate that in assays to identify and
characterize enzymes involved in TNFR1 processing, inhibitors of
aminopeptidases had negligible effects on release of sTNFR1. These
authors indicate that aminopeptidases are not involved in release
of sTNFR1.
[0154] The mechanisms which regulate TNF activity, TNFR activation,
TNFR1 signaling, sTNFR1 activity and sTNFR1 shedding are poorly
understood. However, an understanding of these mechanisms is not
necessary to practice the present invention. Indeed, the present
invention is not limited to any particular mechanism or
mechanisms.
IL-IRII and IL-6R Shedding
[0155] As discussed above, the cognate receptors for the cytokines
IL-1 and IL-6 also exhibit soluble forms akin to the soluble form
of TNFR1. Furthermore, it has been suggested that these soluble
receptor forms play a role in the regulation of IL-1 and IL-6
activity and pro-inflammatory response. However, the proteins
responsible for the shedding of ectodomains of the receptors for
IL-1 and IL-6 remain unidentified. Nonetheless, it is contemplated
that a protein which regulates the shedding of TNFR1 ectodomain
(e.g., a protein of the present invention) will also regulate the
shedding of ectodomains of other cytokine receptors, including
IL-1RII and IL-6R. ARTS-1 binds the soluble form of the IL-6
receptor and promotes the shedding of the IL-6 receptor. ARTS-1
also binds to the soluble form of the type II IL-1 receptors and
promotes the shedding of the type II IL-1 receptors.
Anti-TNF Therapeutic Strategies
[0156] The recognition of TNF as an important inflammatory mediator
in both health and disease has fostered the development of a
variety of therapeutic strategies directed at inhibiting TNF
bioactivity.
[0157] The benefits of inhibiting TNF activity during inflammatory
reactions have been demonstrated using neutralizing monoclonal
antibodies to TNF (Tracey et al., Nature 330:662-664 [1987];
Hinshaw et al, Circ. Shock 30:279-292 [1990]; Opal et al., J.
Infect. Dis., 161:1148-1152 [1990]; Silva et al, J. Infect. Sis.,
162:421-427 [1990]; Emerson et al, Circ. Shock 38:75-84 [1992];
Fieldler et al, J. Lab. Clin. Med., 120:574-588 [1992]; Jesmok et
al, Am. J. Pathol, 141:1197-1207 [1992]; Walsh et al, Arch. Surg.,
127:138-144 [1992]; and Williams et al, Proc. Natl Acad. Sci. USA
89:9784-9788 [1992]). Unfortunately, the development of widespread
clinical application of neutralizing monoclonal antibodies directed
against human TNF is hampered by the potential for immune rejection
of the mouse anti-TNF antibodies in human hosts. The development of
an immune response to non-human anti-TNF antibodies administered to
human subjects may decrease the duration of therapeutic efficacy
and also result in adverse events related to the formation of
immune complexes or the development of hypersensitivity (Kempeni,
Ann. Rheum. Dis., 58(S1):173-181 [1999]).
[0158] A chimeric monoclonal antibody consisting of the variable
region of a murine anti-TNF monoclonal antibody fused to the
constant region of IgGlk has also been studied in clinical trials
for the treatment of Crohn's disease (i.e., inflammatory bowel
disease) (Knight et al, Mol Immunol, 30(16):1443-53 [1993]; Targan
et al, N. Engl. J. Med., 337:1029-1035 [1997]; and U.S. Pat. No.
5,656,272 to Le et al, hereby incorporated by reference). However,
this anti-TNF antibody is not optimal, due to the likely
development of human anti-chimeric antibodies directed against the
non-human elements or the artificially fused sequences within the
chimeric anti-TNF antibody. These deleterious antibodies are likely
to reduce the half-life and therapeutic efficacy of the chimeric
antibody, and possibly result in the formation of unwanted immune
complexes or the development of hypersensitivity (Targan et al, N.
Engl. J. Med., 337:1029-1035 [1997]; Harriman et al., Ann. Rheum.
Dis., 58:161-164 [1999]; and Kempeni, J. Ann. Rheum. Dis.,
58:173-181 [1999]).
[0159] Anti-TNF therapies utilizing TNF receptor fragments and
chimeric, soluble fusion proteins consisting of TNF receptor and
IgG Fc, have been reported to be efficacious for patients with
rheumatoid arthritis and inflammatory bowel disease (Moreland et
al., New Engl. J. Mod, 337:141-147 [1997]; and U.S. Pat. No.
5,605,690 to Jacobs et al. (hereby incorporated by reference)).
However, the therapeutic efficacy of these anti-TNF therapeutic
strategies utilizing soluble forms of the TNF receptor or chimeric
antibodies are also likely to be limited by the development of an
immune response to the artificially fused human sequences (Kempeni,
Ann. Rheum. Dis., 58(S1):173-181 [1999]).
[0160] Current methods of controlling TNF activity in patients also
include the use of non-specific inhibitors of cytokine gene
transcription such as corticosteroids (e.g., dexamethasone and
cyclosporin-A). These methods have significant and well known toxic
side effects which result in significant discomfort and potentially
life-threatening susceptibility to infection, as well as tissue and
organ damage. Toxicities associated with cyclosporin-A include
nephrotoxicity, hypertension, hepatic toxicity, neurotoxicity,
hirsutism, gingival hyperplasia and gastrointestinal toxicity
(Goodman & Oilman's The Pharmacological Basis of Therapeutics,
9.sup.th edition, McGraw-Hill, NY, [1996], p. 1299). Toxicity
associated with corticosteroids include adrenal suppression,
hyperglycemia, hypertension, edema, hypokalemic alkalosis,
myopathy, peptic ulcer disease, osteoporosis, aseptic (ischemic or
avascular) bone necrosis, mental status changes, glaucoma,
cataracts and hyperlipidemia (Goodman & Oilman's The
Pharmacological Basis of Therapeutics, 9.sup.th edition,
McGraw-Hill, NY, 1996, p.1475).
[0161] Induction of sTNFR1 shedding from cell culture systems can
be induced by a variety of physiological and non-physiological
mediators, such as, IFN-y, IL-1p, IL-6, formyl-Met-Leu-Phe (fMLP),
lipopolysaccharide, 4b-phorbol 12-myristate 13-acetate (PMA),
calcium ionophore, staurosporine, and sodium salicylate (Porteu et
al, J. Biol. Chem., 266:18846-18853 [1991]; Nicod.ef al, Ann.
NYAcad. Sci., 28:323-333 [1994]; Tilg et al,. Blood 83:113-118
[1994]; Zhang et al, J. Biol. Chem., 269:10270-10279 [1994];
Mullberg et al, J. Immunol, 155:5198-5205 [1995]; Levine et al, Am.
J. Respir. Cell Mol Biol, 14:254-261 [1996]; and Madge et al, J.
Biol Chem., 274:13643-13649 [1999]). However, these agents are poor
candidates for development as drugs to regulate TNF activity in
humans for a variety of reasons, most notably for the fact that
most of these agents are toxic and not suitable for human
therapeutic use. In addition, most of these agents are known to
have or are likely to have detrimental or unwanted side effects
resulting from their disruption of normal body processes in
addition to their effects on TNF signaling.
[0162] Other strategies for the regulation of TNF activity have
also been attempted or proposed. For example, U.S. Pat. Nos.
5,519,000 and 5,641,751 to Heavner et al. (hereby incorporated by
reference) describe short (4-25 amino acid) peptides which bind TNF
and inhibit TNF activity. U.S. Pat. Nos. 5,665,859 and 5,766,917 to
Wallach et al. (hereby incorporated by reference) describe methods
to isolate a TNFR1 protease or other polypeptides which influence
TNFR1 shedding. U.S. Pat. No. 5,945,397 to Smith et al (hereby
incorporated by reference) describes human and mouse soluble TNF
receptors and mutant variations of these receptors. U.S. Pat. No.
5,919,452 to Le et al. (hereby incorporated by reference) describes
the use of anti-TNF antibodies, anti-TNF peptides, and soluble TNFR
to treat TNF mediated pathologies.
[0163] However, until the development of the present invention, a
nucleic acid in the form of a cDNA and a polypeptide encoded by the
cDNA which has the ability to regulate the shedding of the TNFR1
ectodomain from the extracellular surface of a cell plasma membrane
to yield a free, soluble form of the receptor, sTNFR1 had not been
described. Indeed, database searches revealed that this gene and
polypeptide had not heretofore been identified. These searches also
revealed amino acid sequence motifs indicative of peptidase
activity. Specifically, this gene has been named aminopeptidase
regulator of type I, 55 kDa tumor necrosis factor receptor
shedding, or "ARTS-1." Methods for the use of the ARTS-1 gene,
polypeptide and other related compositions are provided by the
present invention. In addition, methods for the identification of
genes and polypeptides substantially homologous to ARTS-1 gene and
polypeptide are also provided by the present invention. However, an
understanding of the mechanisms of ARTS-1 activity is not necessary
to practice the present invention.
[0164] It is contemplated that the protein (or proteins), and their
corresponding genes, which regulate the cleavage of TNFR1 from the
cell surface are likely to have important roles in regulating TNF
activity in vivo in health and disease states. It is contemplated
that cleavage of the TNFR1 ectodomain from the cell surface limits
TNF activity by providing a pool of free receptors capable of
binding and sequestering TNF, as well as by removing functional
TNFR1 from the cell surface. It is further contemplated that
occupation of the TNF bindings site on the free sTNFR1 ectodomain
does not activate the intracellular components involved in TNF
signaling. It is further contemplated that the genes and proteins
which regulate the shedding of soluble TNFR1 ectodomain will find
significant use in diagnostics and therapeutic regimens, as well as
in the research setting. Furthermore, it is contemplated that genes
and proteins which regulate the shedding of TNFR1 also regulate the
shedding of other cytokine receptors important in inflammatory
diseases and disorders (e.g., IL-1 and IL-6 receptors).
[0165] The present invention provides compositions and methods to
identify genes and proteins which regulate the cleavage and
shedding of the TNFR1 ectodomain. In addition, the present
invention provides compositions suitable for the production of
monoclonal and/or polyclonal antibodies directed against the
proteins involved in the shedding of cytokine receptors (e.g.,
TNFR1). The present invention also provides compositions and
methods suitable for the development of diagnostic tools and assay
systems for the assessment of the factors involved in regulation of
soluble TNFR1 as an indicator of health and/or disease.
[0166] The present invention also provides compositions and methods
suitable for the development of more effective therapies for
treating diseases and disorders resulting from aberrant TNF
activity (e.g., for downregulation or upregulation of TNF
activity), and consequently provide therapeutic advantages for
treatment of diseases or disorders resulting from inflammatory
response or immune-deficiency.
[0167] In addition, the present invention provides compositions and
methods to develop additional means for suppressing damaging
TNF-mediated proinflammatory diseases or disorders without the
toxic side effects associated with existing techniques for immune
suppression. The compositions and methods provided by the present
invention address the need to identify genes and proteins which
regulate TNF activity, and consequently, have therapeutic value in
the treatment of immune disorders.
[0168] The remainder of the Description of the Invention is divided
into the following sections: [0169] I. Cloning Of Genes Encoding
Tnfr1 Ectodomain Binding Polypeptides [0170] II. Preparation of
Recombinant Vectors and Transformants [0171] III. Analysis of
ARTS-1 mRNA Expression [0172] IV. Antibodies Directed Against the
ARTS-1 Polypeptide [0173] V. Detection of ARTS-1 Polypeptide in
Cultured Cell Lines and Primary Cells [0174] VI. GST-ARTS-1
Polypeptide Expression and Purification [0175] VII. Analysis of
ARTS-1 Polypeptide Aminopeptidase Activity [0176] VIII. Analysis of
ARTS-1 TNFR1 Ectodomain Sheddase Regulatory Activity [0177] IX.
Analysis of TNFR1 Ectodomain Sheddase Regulatory Activity of ARTS-1
Catalytic Mutants [0178] X. Analysis of ARTS-1/TNFR1 Interaction in
vivo [0179] XI. Therapeutic Agents to Treat Immune Diseases and
Disorders [0180] XII. Diagnostic Agents for Immune Diseases and
Disorders [0181] XIII. Identification of Genes Substantially
Homologous to ARTS-1 [0182] XIV. Methods for Drug Screening I.
Cloning Of Genes Encoding Tnfr1 Ectodomain Binding Polypeptides
[0183] A) Yeast Two-Hybrid Screening
[0184] It was contemplated that polypeptides which form a physical
interaction with the ectodomain of TNFR1 would be potential
candidates for polypeptides which regulate the cleavage and
shedding of the TNFR1 ectodomain. A yeast two-hybrid screen was
performed in order to identify such TNFR1 interacting polypeptides
using a TNFR1 bait and human lung cDNA two-hybrid prey library.
Yeast two-hybrid screening is a common technique in the molecular
biology arts (Ausubel et al. (eds.), Current Protocols in Molecular
Biology, p. 20.0.1-20.1.40, John Wiley & Sons, Inc., New York
[1994]). In the development of the present invention, the
protocols, yeast strains and reagents used in these experiments
were those supplied or recommended by the manufacturer (Matchmaker
System 2; Yeastmaker Transformation System; Clontech), except where
explicitly stated otherwise. The methods and compositions provided
and used during the development of the present invention are
provided in more detail below and in the Examples.
[0185] Conventional methods known to one of skill in the art may be
used to prepare mRNA to construct a yeast-two hybrid library. In
general, the source of the mRNA (e.g., tissue or cultured cells)
are treated with a guanidine reagent, phenol reagent or the like to
obtain the total RNA. Subsequently, poly(A+)RNA (i.e., mRNA) is
obtained therefrom by an affinity column method using oligo
dT-cellulose or poly U-Sepharose carried on Sepharose 2B. Further,
the resultant poly(A+) RNA can be further fractionated by sucrose
gradient centrifugation or the like.
[0186] Single-stranded cDNA is synthesized using the thus obtained
mRNA as a template, an oligo(dT) primer and a reverse
transcriptase. Then, a double-stranded cDNA is synthesized from the
resultant single-stranded cDNA. The resultant double-stranded cDNA
is integrated (i.e., ligated) into an appropriate cloning vector.
In the case of this yeast two-hybrid library, the resulting
double-stranded cDNAs are cloned into the pGADIO plasmid
(Clontech), a recombinant vector which contains operably linked
parts which enable the transcription and translation of a chimeric
gene consisting of the transcriptional activation domain of the
yeast GAL4 transcription factor and the subcloned cDNA. The pGADIO
vector contains additional operably linked parts which enable its
selection, replication and propagation in yeast strains as well as
E. coli.
[0187] As indicated above, the yeast two-hybrid screen was
conducted using the manufacturer's recommended protocols. A bait
vector was constructed using the pAS2-1 plasmid (Clontech), to
produce a chimeric fusion gene containing nucleic acid sequences
encoding the DNA binding domain of GAL4 and the extracellular
domain of the human TNFR1 receptor, corresponding to amino acids
26-216. Following sequential transformation of the chimeric pAS2-1
bait and pGADIO prey vector library into yeast strain Y190, the
library was screened for clones positive for growth on histidine
(HIS) deficient media, followed by identification of those
HIS.sup.+ clones which were also positive in a p-galactosidase
filter lift assay. Thirty three clones positive for the ability to
grown on HIS deficient substrate and P-galactosidase activity were
identified, and each of those clones was sequenced. It is noted
that numerous protocols and reagents for DNA sequeneing are
commercially available. It is contemplated that any suitable method
known in the art may readily be used in place of the protocol
described here as well as other aspects of the production of the
compositions of the present invention.
[0188] One positive clone, L26C-53A, was selected for further
study. This clone contained a 2355 bp insert containing a 631 amino
acid open reading frame with a consensus zinc metalloprotease
catalytic motif. The nucleotide sequence of this clone corresponds
to bases 1044 to 3082 of the nucleic acid of FIG. 1 (SEQ ID NO:1).
This gene was named aminopeptidase regulator of type I, 55 kDa
tumor necrosis factor receptor ectodomain shedding, or
"ARTS-1."
[0189] The yeast two-hybrid screening described herein is not meant
to limit the present invention to the particular reagents and
methods described. Indeed, numerous other vectors and reagents may
be used to produce the ARTS-1 gene provided by the present
invention. For example, a different two-hybrid prey library may be
substituted for the human lung cDNA library to yield the ARTS-1
gene. Similarly, pAS2-1 and pGADIO vector variants may be used, for
example those which have different multiple cloning sites to
facilitate subcloning of the cDNA insert. Vector variants which use
the LexA operator components in place of the GAL4 system may also
be used in place of those described here. Also, variants of these
vectors with or without an operably joined HA antigenic tag may be
used in order to facilitate immunodetection of the fusion protein.
Similarly, Saccharomyces cerevisiae strains other than Y190 may be
used as the doubly transformed host strain for the library
screening (e.g., S. cerevisiae strain CG-1945). In addition,
selection conditions may be varied, for example, by changing the
concentration of 3-AT (3-amino-1,2,4-triazole) to counterselect
leaky HIS.sup.+ clones. Many of these alternative variables and
reagents are described and are commercially available (e.g., from
companies such as Stratagene and Clontech). Thus, it is intended
that the compositions of the present invention may be produced
using any suitable method known in the art.
[0190] B) Phage Plaque Hybridization/cDNA Cloning
[0191] The DNA sequence identified in the yeast interaction screen
appeared not to be a full length cDNA. This, the complete ARTS-1
cDNA was isolated by screening a phage library using a
.sup.32P-labelled DNA probe derived from clone L26C-53A. The
library used in this cDNA screening was constructed using poly
(A.sup.+) mRNA from the human NCI-H292 pulmonary mucoepidermoid
carcinoma cell line (ATCC, CRL 1848) which had been stimulated with
1 jaM PMA (phorbol 12-myristate, 13-acetate, Sigma). This cell line
has been demonstrated to shed sTNFR1 in response to PMA stimulation
(Levine et al., Am. J. Respir. Cell Mol Biol, 14:254-261 [1996]).
The library was constructed using the uni-ZAP XR phage vector
(Stratagene).
[0192] Bacteriophage from the library were plated in a lawn of XL
1-Blue E. coli (Stratagene) at a density of 50,000 pfu per 150 mm
plate and incubated overnight at 37.degree. C. Plaques were
transferred to Hybond N+ filters (Amersham/Pharmacia) and
denatured. Filters were then neutralized and UV cross-linked.
Filters were washed in pre-hybridization solution, then hybridized
overnight at 42.degree. C. with a .sup.32P-labelled L26C-53A insert
generated by random primed labelling. Filters were washed, and were
then exposed to x-ray film overnight and positive plaques were
selected. Positive plaques were subjected to two additional rounds
of plaque hybridization prior to sequencing. Positive plaques were
recovered via in vivo excision utilizing the ExAssist helper phage
(Stratagene).
[0193] It is not intended that the present invention be limited to
any particular cDNA library, reagent or method for the production
of the nucleic acid encoding the ARTS-1 polypeptide of the present
invention. Thus, it is contemplated that any suitable library will
find use in isolating the ARTS-1 cDNA. For example, such libraries
may include libraries made from mRNA derived from human chronic
myelogenous leukemia cell line K-562, lymphoblastic leukemia cell
line MOLT-4 or lung carcinoma A549 cell line. Similarly, multiple
techniques for the radiolabelling and purification of nucleic acid
probes as well as phage plaque hybridization protocols are well
known in the art (Ausubel et al. (eds.), Current Protocols in
Molecular Biology, Vol. 1-4, John Wiley & Sons, Inc., New York
[1994]). The probe synthesis and hybridization methods and
conditions described here are not meant to limit the scope of the
present invention. Variations on these protocols include
alternative labelling methods (e.g., 5' end labelling, overhang
fill-in labelling and random primed synthesis labelling).
Alternative detection systems can also be used, including .sup.33P
probe labelling and non-radioactive probe visualization methods
such as chemiluminescence.
[0194] Following three rounds of screening, four hybridizing phage
clones were identified from the library, amplified, then sequenced.
These four clones all overlapped the L26C-53A sequence, as
expected. However, none encoded a full-length cDNA, nor did they
collectively span the entire gene. One phage clone (bp 1-1777)
contained the putative 5' UTR and three phage clones contained the
putative 3' UTR and the poly(A) tail (bp 2181-4845). cDNA sequence
encoding the portion of the gene lying between the 5' and 3'
terminal clones was amplified via PCR from the same human lung cDNA
library using primers corresponding to 5' and 3' sequence obtained
from the phage screening. The cDNA segment amplified with these
primers was subcloned and both strands were sequenced to verify PCR
fidelity.
[0195] As discussed in Section A above, it is not intended that the
present invention be limited to any particular reagents, methods,
and/or compositions for the production of the nucleic acids and
polypeptides of the present invention. Thus, it is contemplated
that any suitable methods for cDNA cloning and/or phage plaque
hybridization will find use in the production of the compositions
of the present invention. For example, one of numerous commercially
available DNA polymerases may be used in the PCR reaction,
including Tag (Stratagene, Promega), Pfu (Promega) or Sequenase
(Amersham). Furthermore, it is contemplated that alternative PCR
reaction conditions (i.e., temperatures and time intervals) will
also be successful in amplifying the ARTS-1 nucleic acid of the
present invention. Similarly, numerous vectors and reagents are
equally suitable for subcloning and sequencing reactions.
[0196] C) Analysis of the ARTS-1 cDNA and Predicted Polypeptide
[0197] Inspection of the full length cDNA obtained as described
above revealed a 4845 nucleotide transcript, containing a 2823 bp
open reading frame and 5' and 3' untranslated regions as shown in
FIG. 1. In this Figure, a consensus polyadenylation site located at
nucleotides 4795 to 4800 is indicated with a double underline and
lies 18 nucleotides upstream of a 27 nucleotide poly(A) tail. Two
mRNA destabilization motifs are also identified at nucleotides 3929
and 4457 using bold and underline.
[0198] The open reading frame (ORF) encodes a 941 amino acid
polypeptide. The first ATG codon lying in-frame relative to the
largest open reading frame is located at nucleotide 88, and a TAA
stop codon in the same reading frame is located at nucleotide 2911.
Asparagine residues comprising five potential N-glycosylation sites
are indicated with circles. A putative transmembrane domain,
extending from amino acids 5 to 28, is also indicated in the Figure
with a single underline. In order to determine this sequence,
sequence analysis, including Kyte-Doolittle hydropathy prediction
(Kyte and Doolittle, J. Mol. Biol., 157:105-132 [1982]) was
performed using MacVector 7.0 software (Oxford Molecular). The
location of the putative hydrophobic transmembrane cc-helical
domain was predicted utilizing several web-based analysis programs
(MEMSAT2 (McGuffin et al, Bioinform., 16:404-405 [2000]; Sosui
(Hirokawa et al, Bioinform., 14:378-379 [1998]; TMAP (Persson and
Argos, J. Mol. Biol, 237:182-192 [1994]); TMpred (Hofmann and
Stoffel, Biol. Chem. Hoppe-Seyler 374:166 [1993]; and TopPred2 (von
Heijne,/. Mol. Biol, 225:487-494 [1992]). In sum, ARTS-1 is
predicted to be a type II integral membrane protein with a single
hydrophobic transmembrane oc-helical domain, located between amino
acids 5 and 28 (See, FIGS. 1 and 2), and a very short hydrophobic
intracellular ammo-terminal domain (See, McGuffin et al,
Bioinform., 16:404-405 [2000]; Hirokawa et al, Bioinform.,
14:378-379 [1998]; Persson and Argos, J. Mol. Biol, 237:182-192
[1994]); Hofmann and Stoffel, Biol. Chem. Hoppe-Seyler 374:166
[1993]; and von Heijne,/. Mol. Biol., 225:487-494 [1992]).
[0199] Subdomains of homology indicate that ARTS-1 is a member of
the aminopeptidase family of gluzincin zinc metalloproteases. This
family of proteins share motifs indicating similar biochemical
activity, although the family members are extremely diverse in
overall structure in biological function (Zinc Metalloproteases in
Health and Disease, Taylor & Francis, London, England [1996],
Hooper, p.1-21, and Wang and Cooper, p.131-151). FIG. 1 also
indicates the ARTS-1 sequences which adhere to the consensus zinc
metalloprotease catalytic motif for the aminopeptidase family,
HEXXH(Y).sub.18E (SEQ ID NO: 10). Within this motif, it is
theorized that the two histidine residues (H) and the second
glutamic acid residue (E) represent the zinc binding domain while
the first glutamic acid mediates the catalytic activity. In the
ARTS-1 polypeptide, this consensus motif is observed at
T.sup.350VAHELAHQWFG (SEQ ID NO:8) and L.sup.372WLNEGFA (SEQ ID
NO:9) (boxed in FIG. 1).
[0200] Furthermore, a schematic representation of the ARTS-1
protein, indicating domains of homology with the aminopeptidase
family of gluzincin zinc metalloproteases is provided in FIG. 2. A
zinc metalloprotease consensus catalytic motifHEXXH(Y).sub.18E (SEQ
ID NO: 10), a short intracytoplasmic tail, a transmembrane domain
and a large 375 amino acid domain of homology are also depicted in
this Figure.
[0201] Quantitative comparisons between ARTS-1 protein and other
members of the aminopeptidase-gluzincin zinc metalloprotease family
are shown in Tables 1 and 2 below. Table 1 provides quantitation of
percent identity and percent similarity between the full length
amino acid sequence of the ARTS-1 protein and other aminopeptidase
family members. Percent identities are shown above the shaded
diagonal and percent similarities are shown below the shaded
diagonal. Table 2 shows a similar comparison between the conserved
375 amino acid domain in the ARTS-1 protein containing the
consensus zinc binding motif HEXXH(Y).sub.18E (SEQ ID NO: 10) and
other members of the aminopeptidase family of gluzincin zinc
metalloproteases. The aminopeptidase family members included in
these Tables are human placental leucine aminopeptidase (FLAP)
(Rogi et al., J. Biol. Chem., 271:56-61 [1996]), rat
insulin-regulated aminopeptidase (IRAP) (Keller et al., J. Biol.
Chem., 270:23612-23618 [1995]), human aminopeptidase A (AMP A)
(Nanus et al, Proc. Natl. Acad. Sci. USA 90:7069-7073 [1993]; and
Li et al., Genomics 17:657-664 [1993]), human aminopeptidase N (AMP
N) (Olsen et al, FEES Lett., 238:307-314 [1988]), human puromycin
sensitive aminopeptidase (Tobler et al, J. Neurochem., 68:889-897
[1997]), rat thyrotropin-releasing hormone degrading enzyme (TRH
DE) (Schauder et al., Proc. Natl. Acad. Sci. USA 91:9534-9538
[1994]), S. cerevisiae aminopeptidase YSCII (Garcia-Alvarez et al.,
Eur. J. Biochem., 202:993-1002 [1991]), C. elegans cosmid F49E8.3
gene product (Wilson et al, Nature 368:32-38 [1994]), and
Lactococcus lactis aminopeptidase N (Tan et al, FEES Lett, 306:9-16
[1992]).
[0202] As mentioned above, and as indicated in these Tables,
although members of this aminopeptidase family share a similar
biochemical motif, there is significant sequence divergence among
family members, likely indicating distinct and specialized
biological functions. TABLE-US-00001 TABLE 1 Percentage Identity
and Similarity Between Full Length ARTS-1 Protein and Other Members
of the Aminopeptidase-Gluzincin Zinc Metalloprotease Family Human
Human Rat Human Human Rat Human C. eleganus L. lactis S. cerevisiae
Protein ARTS-1 PLAP IRAP AMP A AMP N TRH DE PSA F49E8.3 AMP N YSC
II Human 44 41 31 29 27 30 26 24 27 ARTS-1 Human 17 79 30 29 27 28
25 23 26 PLAP Rat 15 5 28 28 28 26 24 21 24 IRAP Human 20 19 18 34
29 30 25 22 28 AMP A Human 18 18 18 18 22 29 27 24 26 AMP N Rat 17
16 17 17 14 26 23 22 22 TRH DE Human 17 19 17 16 17 16 36 28 32 PSA
C. eleganus 18 17 15 18 17 17 16 28 31 F49E8.3 L. lactis 16 16 15
17 15 14 16 18 27 AMP N S. cerevisiae 16 18 16 15 16 16 16 19 18
YSC II
[0203] TABLE-US-00002 TABLE 2 Percentage Identity and Similarity
Between the Conserved Domains Containing the Zinc Binding Motif of
ARTS-1 Protein and Other Members of the Aminopeptidase-Gluzincin
Zinc Metalloprotease Family Amino Acid Human Human Rat Human Human
Rat Human C. eleganus L. lactis S. cerevisiae Protein Position
ARTS-1 PLAP IRAP AMP A AMP N TRH DE PSA F49E8.3 AMP N YSC II Human
161-535 51 53 39 43 41 44 42 36 41 ARTS-1 Human 192-540 15 90 42 43
39 44 44 36 44 PLAP Rat 273-621 12 6 41 42 39 44 45 37 43 IRAP
Human 202-549 21 18 18 44 41 48 44 36 45 AMP A Human 192-554 15 15
16 19 42 47 44 37 45 AMP N Rat 248-603 14 17 17 17 16 41 40 37 38
TRH DE Human 158-508 16 18 17 14 14 18 52 41 52 PSA C. eleganus
120-469 16 17 16 17 17 20 16 41 49 F49E8.3 L. lactis 99-445 16 20
18 18 17 15 18 17 40 AMP N S. cerevisiae 114-461 15 16 15 16 16 19
15 18 20 YSC II
II. Preparation of Recombinant Vectors and Transformants
[0204] A) Preparation of Recombinant Vectors
[0205] Numerous recombinant vectors relevant to the present
invention are contemplated. Such vectors, containing the gene or
portions of the gene of the present invention (i.e., the ARTS-1
gene) may be of any type, with the only limitation being that the
vector into which the gene of the invention has been inserted has
the capacity for replication in a host. As used in the present
invention, a host may be a bacteria (e.g., E. coli BL21 strain), a
yeast (e.g., S. cerevisiae Y190 strain), or an animal cell (e.g.,
the NCI-H292 human pulmonary mucoepidermoid carcinoma cell line).
Furthermore, the host cell may exist in an in vitro tissue culture
system, or exist within an intact organism (e.g., a mouse or a
human). Indeed, it is not intended that the host be limited to any
particular cell type, nor is it intended that the cell host be
maintained in any particular setting.
[0206] Protocols for the construction of recombinant vectors are
commonplace in the molecular biology arts, and reagents for the
manipulation of recombinant nucleic acids are readily available
from commercial sources. Routine procedures in the construction of
a recombinant vector encompassed by the present invention may
include restriction endonuclease digestion, ligation,
phosphorylatibn, dephosphorylation, blunt-ending, size separation,
annealing, eluting, staining, desalting, transformation,
inoculating, incubating, cesium banding and purification by a
variety of commercially available products. In the most simple of
embodiments, a recombinant vector of the invention may be made by
digesting the gene of the invention with an appropriate restriction
enzyme, ligating into an appropriately digested vector,
transforming the ligated vector into bacteria, selection using an
antibiotic resistance marker contained on the vector and purifying
the vector using a kit designed for rapid, small scale plasmid
purification.
[0207] A vector may be in the form of a DNA plasmid. Such plasmids
contain multiple parts operably arranged to permit replication in a
minimum of one specific host species. A plasmid (e.g., the
pGEX-6P-1 plasmid) may be restricted to replication in bacteria, or
may also contain operably linked sequences which permit the plasmid
to propagate or function in a second species in addition to its
usual bacterial host. For example, the pAS2-1 and pGADIO vectors
contain operably linked nucleic acid sequences which permit their
propagation in both bacteria and yeast (e.g., the S. cerevisiae
Y190 strain), while the pTarget vector contains operably linked
sequences which permit its propagation in a bacterial host, but
also contains sequences which allow it to direct the transcription
of a cloned gene in a mammalian cell.
[0208] A vector may also be in the form of a DNA or RNA
bacteriophage or other virus, which further may be in the form of a
DNA or RNA containing virus. Such phage or other virus may be
replication competent or replication defective. The phage or viral
nucleic acid may also be engineered to permit propagation in an
organism as a plasmid in addition to its viral life cycle. For
example, the bacteriophage Lambda (k) uni-ZAP II vector
(Stratagene) is capable of bacterial infection as a bacteriophage,
but also contains sequences which enable the excision and
propagation of a plasmid form of the vector. Such a vector is often
called a "phagemid." Further, recombinant viral vectors (e.g.,
retrovirus, adenovirus, adeno-associated virus or vaccinia virus)
or an insect virus vector (e.g., baculovirus) may also be used to
infect cells.
[0209] In particularly preferred embodiments, the gene of the
present invention is operably linked to other components of the
vector. For this purpose, the vector of the invention may contain,
if desired, cis elements (e.g., an enhancer, splicing signal,
poly(A) addition signal, selection marker, ribosome binding
sequence (i.e., Shine-Dalgarno or Kozak sequences) or the like) in
addition to a promoter/enhancer and the gene of the present
invention. As the selection marker, genes encoding resistance to
dihydrofolate reductase, ampicillin, kanamycin, neomycin, or the
like may be used.
[0210] B) Preparation of Transformants
[0211] In some embodiments of the present invention, a transformant
was obtained by introducing the recombinant vector containing the
gene of the invention into a host. The host is not particularly
limited as long as it can harbor the vector of the invention.
Specific examples of host include Escherichia bacteria such as E.
coli; yeast such as S. cerevisiae and Schizosaccharomyces pombe;
animal cells such as COS cells, CHO cells, NCI-H292 cells or
non-specified cells of an intact organism; or insect cells such as
SJ9 and Sfl\ cells. In some embodiments of the present invention,
the vector is introduced into the host under conditions such that
the polypeptide encoded by the gene of the invention is
expressed.
[0212] In some cases, when a bacterium such as E. coli is used as
the host, the recombinant vector of the invention is capable of
replication in the host and, at the same time, is capable of
expressing the gene of the invention within the bacterial host
(e.g., the pGEX-ARTS-1 vectors). Such vectors in general preferably
consist of a promoter, a ribosome binding sequence, the gene of the
present invention and a transcription termination sequence. The
vector may also contain a gene to control the promoter.
[0213] Any promoter may be used as long as it can appropriately
direct the expression of the gene of interest in the host cells,
such as a mammalian cell or E. coli. For example, an E. coli or
phage-derived promoter such as tip promoter, lac promoter, P.sub.L
promoter or P.sub.R promoter may be used. An artificially designed
and altered promoter such as tac promoter may also be used.
[0214] Any method for bacterial transformation may be used for
introducing a recombinant vector into a bacterium. Many methods are
commonly known in the art, including electroporation and the use of
bacteria made competent by calcium chloride (See e.g., Ausubel et
al. (eds.), Current Protocols in Molecular Biology, p.
1.8.1-1.8.10, John Wiley & Sons, Inc., New York [1994]).
[0215] Likewise, any method for introducing DNA into yeast may be
used to introduce a recombinant vector into a yeast. For example,
electroporation (Becker et al., Methods Enzymol, 194:182-187
[1991]), the spheroplast method (Hinnen et al., Proc. Natl. Acad.
Sci. USA 75:1929-1933 [1978]), the lithium acetate method (Ito, J.
Bacterial., 153:163-168 [1983]) or the like may be used.
[0216] It is contemplated that any suitable animal cells will find
use as hosts in the present invention (e.g., simian COS-7 or Vero
cells, Chinese hamster ovary cells (CHO cells), mouse L cells, rat
GH3 cells, human FL cells, human lymphoid cell lines, or the like).
A promoter/enhancer may be used to express the ARTS-1 gene where
the promoter is active in all or most cell types (e.g., the SRct
promoter, SV40 promoter, HSV-LTR promoter, CMV promoter or the
like). Alternatively, promoter/enhancer elements may be used which
direct expression of the ARTS-1 gene in only a subset of cells
(e.g., cells of the lymphoid system).
[0217] As with other host cells, any suitable method for
introducing the recombinant vector into animal or insect cells
(e.g., electroporation, the calcium phosphate method, lipofection
or the like) may be used.
III. Analysis of ARTS-1 mRNA Expression
[0218] Following the identification and isolation of the ARTS-1
gene, an experiment was undertaken to determine the pattern of
tissue expression of the endogenous ARTS-1 mRNA. This was
accomplished using a multi-tissue Northern blot and an ARTS-1
derived probe as shown in FIG. 3 and described in greater detail in
Example 3.
[0219] The blot used in this experiment was a commercially
available human multiple tissue poly(A+) Northern blot (Clontech).
This blot was probed according to the manufacturer's suggested
protocol using a full length .sup.32P-labelled ARTS-1 cDNA
probe.
[0220] The Northern blot analysis shown in FIG. 3, Panel A,
indicates that the human ARTS-1 transcript was expressed in
multiple tissues, including spleen, thymus, small and large
intestine, peripheral blood leukocyte, heart, placenta, lung,
skeletal muscle, kidney and pancreas. In these tissues, a single
predominant mRNA species of approximately 5.7 kB was detected. FIG.
3, Panel B, shows the same blot following stripping and
rehybridization to a probe specific for the human GAPDH transcript
as a reference for RNA loading normalization.
[0221] Furthermore, in view of numerous alternative protocols known
in the art for Northern blotting, it is not intended that the
present invention be limited to the Northern blotting protocol
provided in Example 3 or any other particular Northern blotting
method. For example, in some embodiments, RNA is isolated from
tissue samples using alternative methods (e.g., a commercial RNA
isolation kit such as Qiagen RNeasy Total RNA Mini Kit, Catalog No.
74103).
[0222] Similarly, alternative probe synthesis and labelling
techniques also find use with the present invention. For example,
any probe having a minimum complementarity of 25 base pairs to the
ARTS-1 cDNA will find use in the Northern blot methods of the
present invention. Furthermore, it is contemplated that the nucleic
acid comprising the probe will be generated by PCR, by restriction
digest, or by synthetic oligonucleotide synthesis. Alternative
nucleic acid probe labelling methods also find use with the present
invention (e.g., labelling with .sup.33P radioisotope or
non-radioactive labelling methods). In addition, alternative
Northern blotting protocols, reagents and equipment suitable for
use in the present invention are known in the art (See, e.g.,
Ausubel et al. (eds.), Current Protocols in Molecular Biology,
Vol.1, pages 4.9.1-4.9.16, John Wiley & Sons, Inc., New York
[1994]).
IV. Antibodies Directed Against the ARTS-1 Polypeptide
[0223] In the present invention, polyclonal antiserum directed
against the ARTS-1 polypeptide is provided. However, the antibody
of the invention may be prepared by various methods, as numerous
methods for the production of monoclonal and polyclonal antibodies
are well known in the art (See e.g., Sambrook, J. et al. (eds.),
Molecular Cloning, Cold Spring Harbor Laboratory Press [1989];
Harlow and Lane (eds.), Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory Press [1988]; Ausubel et al. (eds.),
Current Protocols in Molecular Biology, p. 11.4.2-11.15.4, John
Wiley & Sons, Inc., New York [1994]). As used herein, the term
"antibody" means an antibody molecule as a whole or a fragment of
an antibody (e.g., Fab or F(ab').sub.2 fragment) which can bind
specifically an antigen. The antibody provided by the invention is
described in more detail below and in Example 4. The present
invention also contemplates that these same methods will find use
in the production of antibodies specific for polypeptides encoded
by genes which are substantially homologous to the ARTS-1 gene.
[0224] In order to conduct additional studies on the ARTS-1
polypeptide, polyclonal antiserum was generated against an ARTS-1
polypeptide. A commercial service (Research Genetics) was used in
these studies. Specifically, a 17 amino acid ARTS-1 synthetic
peptide was used to immunize New Zealand white rabbits. This
peptide corresponded to amino acids 538 to 554 of the ARTS-1
polypeptide and had the sequence: TABLE-US-00003 (SEQ ID NO:7)
R.sup.538GRNVHMKQEHYMKGSD
This particular peptide was chosen based upon its antigenic
potential and its lack of homology with other protein sequences as
determined by a BLAST homology search.
[0225] Rabbits were immunized with this peptide using standard
techniques. The ARTS-1 peptide was conjugated to KLH and mixed with
an equal volume of Freund's complete adjuvant. The amount of
antigen utilized per immunization was 0.1 mg, which was injected
into three subcutaneous dorsal sites. The animals received boosts
at weeks 2, 6 and 8. Bleeds were obtained at weeks 4, 8 and 10, and
tested for the presence of anti-ARTS-1 antibody. In subsequent
experiments, the antiserum obtained from the 10 week bleed was
used.
[0226] In view of numerous alternative protocols known in the art
for the production of polyclonal antibodies, the present invention
is not meant to be limited to any particular method. For example,
the entire 941 amino acid ARTS-1 polypeptide, or any portion or
fragment thereof, may potentially be used as the immunogen, and the
immunogen may be either synthetic or native. It is not intended
that the present invention be limited to any particular ARTS-1
derived immunogen, method of immunization, immunizationschedule,
animal species, test protocol for determining antibody production
or antibody purification method.
[0227] Although the antibody provided by the invention is
polyclonal, the invention also contemplates monoclonal antibodies
directed against the ARTS-1 polypeptide. It is also contemplated
that any suitable method for the production of monoclonal
antibodies will find equal use in the present invention. In one
embodiment, the immunogen used to produce these monoclonal
antibodies comprises full-length ARTS-1 polypeptide, although it is
contemplated that any portion or fragment of the ARTS-1 polypeptide
may also find use in the present invention as an immunogen.
[0228] In these monoclonal antibody protocols, any suitable method
for recovery of antibody producing cells, cell fusion, selection
and cloning of hybridomas, recovery of the monoclonal antibodies,
and purification of the monoclonal antibodies of interest may be
used. Thus, it is not intended that the present invention be
limited to any particular monoclonal antibody production system or
method.
[0229] If desired, the polyclonal or monoclonal antibody
preparation of the invention can be purified from crude antiserum
or culture supernatant using a conventional method (e.g., Protein A
affinity, ammonium sulfate precipitation, ion exchange
chromatography, gel filtration, affinity chromatography, or any of
these methods in combination).
[0230] Once the polyclonal or monoclonal antibody is thus obtained,
in some embodiments of the present invention, the antigen is bound
to a solid support, so as to thereby prepare an affinity
chromatography column. Using this column, the antibody of the
present invention can be highly purified. Conversely, in an
alternative embodiment of the present invention, the antibody is
bound to a solid support as to thereby prepare an affinity
chromatography column. Using this column, the ARTS-1 polypeptide,
or fragments thereof, either native or recombinant, can be highly
purified from variable sources. These monoclonal and polyclonal
antibodies find numerous uses, including Western blotting,
immunoprecipitation, immunohistochemistry and clinical applications
using methods known in the art (See e.g., Harlow and Lane (eds.),
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press [1988]; Ausubel et al. (eds.), Current Protocols in Molecular
Biology, Vol. 1-4, John Wiley & Sons, Inc., New York [1994];
and Laurino et al, Ann. Clin. Lab Sci., 29(3):158-166 [1999]).
V. Detection of ARTS-1 Polypeptide in Cultured Cell Lines and
Primary Cells
[0231] Following the production of ARTS-1 polyclonal antiserum, the
expression of endogenous ARTS-1 polypeptide in cultured and primary
cells was investigated using standard Western immunoblotting
techniques well known in the art, as discussed below and described
in further detail in Example 5. Protein concentrations of the
samples were assayed and 20 micrograms of total protein were
prepared for analysis in Laemmli buffer. Samples were resolved via
6% polyacrylamide SDS-PAGE and electroblotted onto nitrocellulose.
Blots were incubated overnight in blocking buffer, then incubated
for 2 hours with ARTS-1 antiserum at a 1:20,000 dilution in
blocking buffer. Membranes were washed, then incubated with
horseradish peroxidase conjugated goat anti-rabbit IgG (Life
Technologies) diluted to 1:5,000 in blocking buffer, then washed
again. Membranes were then incubated in chemiluminescent detection
substrate and the signal detected on X-ray film.
[0232] The specificity of the resulting polyclonal antiserum was
first determined as shown in FIG. 4. Extracts analyzed in this
experiment were crude whole cell homogenates, and membrane and
cytosolic fractions all prepared from cultured NCI-H292 cells. As
shown in FIG. 4, Panel A, the anti-ARTS-1 antiserum detected a
predominant 100 kDa membrane form and a predominant 68 kDa
cytosolic form from the NCI-H292 cells, while the whole cell
extracts predictably revealed a mixture of these two forms. As
shown in FIG. 4, Panel B, the preimmune serum showed no reactivity
towards the same samples when used at the same concentration.
[0233] Specificity of the immune serum was further demonstrated in
competition experiments in FIG. 4, Panels C and D. Preincubation of
the immune serum with the RGRNVHMKQEHYMKGSD peptide (SEQ ID NO:7)
against which the polyclonal antiserum was raised resulted in
almost complete attenuation of the immune signal (Panel D). In
contrast, preincubation of the immune serum with bovine serum
albumin resulted in minimal attenuation of immune signal (Panel
C).
[0234] The expression of endogenous ARTS-1 polypeptide in primary
cells and other cell lines was further investigated using the
identical antiserum and Western immunoblot technique (See, FIG. 5).
These experiments were conducted to determine if there were
differences in the ARTS-1 polypeptide forms expressed in different
cell lines. These experiments analyzed membrane and cytosolic
fractions made from human bronchial brushing specimens, airway
epithelial cell lines BEAS-2B and BET-1A, human lung carcinoma cell
line A549, cultured NCI-H292 cells, primary cultures of normal
human bronchial epithelial cells (NHBE), human umbilical vein
endothelial cells (HUVEC) and human fibroblasts.
[0235] Similar to the NCI-H292 cell line, the BEAS-2B, BET-1A, NHBE
and A549 cell lines all revealed a 100 kDa band localized
predominantly in the membrane fraction, and a 68 kDa form expressed
to varying degrees in the cytosolic fraction. The protein samples
obtained from human bronchial brushings, as well as the HUVEC and
fibroblast primary cells revealed similar patterns, with the
exception that both the 100 and 68 kDa forms appear in the membrane
fraction. The human bronchial brush cells and occasionally the
NCI-H292 cell line also showed a larger 132 kDa form.
[0236] These multiple sized forms may be due to regulated
processing of the ARTS-1 polypeptide. Furthermore, the distinction
between the different sized ARTS-1 forms seen in the membrane
versus the cytosolic fractions may also indicate regulated
processing between the membrane and cytosol. However, it is not
necessary to understand the mechanism of ARTS-1 processing or
localization in order to practice the present invention, nor is it
intended that the present invention be so limited.
[0237] Furthermore, in view of numerous alternative protocols known
in the art for Western blotting, it is not intended that the
present invention be limited to the Western blotting protocol
provided in Example 5 or any other Western blotting method. For
example, in some embodiments, alternative secondary (i.e.,
detection) antibodies can be used. Alternative Western
immunoblotting protocols, reagents and equipment suitable for use
in the present invention are known in the art (See, e.g., Ausubel
et al. (eds.), Current Protocols in Molecular Biology, Section
10.8, "Immunoblotting and Immunodetection," John Wiley & Sons,
Inc., New York [1994]).
VI. GST-ARTS-1 Polypeptide Expression and Purification
[0238] In order to facilitate the analysis of ARTS-1 biochemical
activity, a highly purified form of the ARTS-1 polypeptide was
produced using a GST fusion protein protocol commonly used in the
art. The specifics of this protocol are provided in detail in
Example 6. It is not intended that the present invention be limited
to any particular method for GST-ARTS-1 polypeptide expression,
either with or without subsequent GST-ARTS-1 polypeptide
purification.
[0239] The use of GST fusion proteins to produce purified
polypeptides is common in the art. In these protocols, a
transcriptional and translational fusion is made between the genes
encoding glutathione-S-transferase (GST) and the gene of interest
(e.g., the ARTS-1 gene). Production of the fusion protein
containing a GST "tag" enables the effective and rapid purification
of the fusion protein by use of an affinity column comprising
immobilized glutathione. In order to produce a GST-ARTS-1
polypeptide, the cDNA sequence encoding ARTS-1 was first PCR
amplified and subcloned into the pGEX plasmid backbone (Amersham
Pharmacia) and used to transform the BL21 E. coli host strain. The
pGEX plasmid contains a multiple cloning site and expresses a
fusion protein consisting of the GST polypeptide and a subcloned
coding sequence (e.g., the ARTS-1 coding sequence) cloned into the
multiple cloning site of the plasmid. Transcription of the fusion
gene is controlled by the conditional tac promoter. In accordance
with the manufacturer's protocol, clones were cultured in the
presence of 0.6 mM isopropyl p-D-thiogalactoside (IPTG) and
subsequently lysed with a protein extraction buffer. Transformed
clones expressing GST-ARTS-1 fusion protein were selected by
Western immunoblotting utilizing an anti-GST antibody (Amersham
Pharmacia). Cells with confirmed expression of GST-ARTS-1
polypeptide were lysed and centrifuged to separate the soluble from
the insoluble fractions. The GST-ARTS-1 fusion protein was isolated
from the insoluble fraction by denaturation with 6M urea, then
desalted and renatured. The GST-ARTS-1 fusion protein was purified
utilizing a glutathione sepharose 4B affinity column using
techniques well known in the art.
[0240] To assess the purity of the eluted recombinant GST-ARTS-1
fusion protein, samples were subjected to SDS-PAGE and stained with
Coomassie brilliant blue, as shown in FIG. 6A. In this Figure,
soluble and insoluble protein fractions from BL21 E. coli
transformed with empty pGEX vector are shown in lanes 1-2, and
analogous samples from bacteria containing the pGEX-ARTS-1 vector
are shown in lanes 3-4. The GST-ARTS-1 fusion protein following
elution is shown in lane 5, as a predominant 132 kDa band,
corresponding to the predicted molecular weight of a GST-ARTS-1
fusion protein (104 kDa ARTS-1 extracellular domain plus 26 kDa GST
tag). The control purified GST tag was revealed as a predicted 26
kDa band in lane 6.
[0241] The recombinant purified GST-ARTS-1 fusion protein samples
were further subjected to FPLC analysis to assess their purity, the
results of which are shown in FIG. 6B. Elution from the FPLC column
was monitored by absorbance at a wavelength of 280 nm. This
analysis of the purified GST-ARTS-1 revealed a single major protein
elution peak at approximately 40 minutes.
[0242] Using the aminopeptidase activity assay described below,
FPLC fractions were assessed for aminopeptidase activity utilizing
a phenylalanine p-nitroaniline substrate. The results of these
experiments are shown in FIG. 6C. As indicated in this Figure, the
single major protein peak revealed by FPLC analysis coeluted with a
peak of aminopeptidase activity against a phenylalanine-pNA
substrate in pooled fractions from 38-44 minutes.
[0243] As those of skill in the art know, numerous protocols for
the purification of polypeptides are available. The use of numerous
alternative protocols for the production of isolated ARTS-1
polypeptide are easily envisioned. These alternative protocols may
be used to purify ARTS-1 polypeptides other than the GST-ARTS-1
polypeptide described by the present example. Indeed, it is not
intended that the present invention be limited to any particular
protocol.
[0244] Numerous reagents and variables may be manipulated or
substituted for those used in Example 6 to yield a substantially
similar purified ARTS-1 polypeptide. Such variables include the
choice of ARTS-1 polypeptide (e.g., either with or without a fusion
protein tag), the cells used for the production of the starting
materials, the promoters/enhancers used to drive expression of the
recombinant protein to be purified, the cellular culture and growth
conditions, harvesting methods, mode of purification, and methods
to assess polypeptide purity following purification. The detailed
protocol provided in Example 6 is not meant to limit the scope of
the present invention. Indeed, it is contemplated that any protocol
which will produce a substantially similar purified product will
find use with the present invention. Such alternative embodiments
are briefly discussed below.
[0245] Alternative protocols may be used to purify forms of ARTS-1
polypeptide other than the GST-ARTS-1 fusion polypeptide provided
by the present invention. These alternative forms may include a
maltose binding protein (MBP) ARTS-1 fusion polypeptide, a
polyhistidine (i.e., 6.times. His) tagged ARTS-1 fusion
polypeptide, a thioredoxin tagged ARTS-1 fusion polypeptide, or a
full length ARTS-1 polypeptide without any fused tag to facilitate
purification.
[0246] Alternative protocols may find use in the present invention.
For example, protocols which use different host systems as a source
for starting material (i.e., a source culture) for ARTS-1
purification may be used. Such alternative source systems include
insect cells with a baculovirus overexpression system (e.g., SJ9 or
Sfl\ cell lines), mammalian cell lines in conjunction with vectors
designed for recombinant polypeptide overexpression (expression
vectors), or mammalian cells or tissues for the purification of
ARTS-1 polypeptide expressed from its endogenous (i.e., native)
chromosomal location. The cultivation of the transformed,
transfected or infected host of the invention is carried out in a
medium and under conditions most appropriate for the growth of that
particular host cell. These media formulations and culture
conditions are well known to those in the art. For example, culture
of a mammalian cell line for the isolation of an overexpressed or
endogenous polypeptide will commonly use RPMI 1640 or DMEM media,
typically supplemented with 5-10% fetal or newborn calf serum, and
may be further supplemented with an antibiotic such as kanarnycin
or penicillin. In some protocols, calf serum may be omitted to
facilitate subsequent polypeptide purification. Typically, the
cultivation of mammalian cells is carried out in the presence 5%
CO.sub.2 at 37.degree. C.
[0247] Following the cultivation of a particular ARTS-1
polypeptide-containing host, the proteins of the culture can be
extracted by disrupting (by any suitable method) the microorganisms
or cells if the protein is produced within the host. If ARTS-1
polypeptide of the invention is secreted by the host cells, the
culture fluid is collected. The resultant culture extract or
culture supernatant is then subjected to conventional biochemical
techniques used for protein purification. As known in the art,
numerous techniques for polypeptide purification exist (e.g.,
ammonium sulfate precipitation, gel chromatography, ion exchange
chromatography, and affinity chromatography). These techniques may
be used independently or in an appropriate combination to isolate
and purify the polypeptide(s) of the present invention from a
culture.
VII. Analysis of ARTS-1 Polypeptide Aminopeptidase Activity
[0248] In light of the homology between the polypeptide predicted
from ARTS-1 gene with aminopeptidase members of the gluzincin zinc
metalloprotease family, it was determined if ARTS-1 polypeptide had
aminopeptidase activity, and furthermore, if the terminal
aminopeptidase activity was specific for a particular amino acid or
group of amino acids.
[0249] To accomplish this, commercially available amino acid
p-nitroanilide substrates were incubated with purified GST-ARTS-1
fusion protein for 1 hour under conditions of linear enzyme
activity over time. The rate of amide bond hydrolysis was
determined by measuring the absorbance of the p-nitroaniline
aminopeptidase reaction product at 380 nm. Each experimental point
was run in triplicate and each determination utilized six
concentrations of amino acid p-nitroanilide substrate. Kinetic
constants were determined by Lineweaver-Burk analysis. Correlation
coefficients for each line generated were greater than 0.997. The
results of this analysis are shown in Table 3 below. Each amino
acid p-nitroaniline substrate tested is shown in the left column,
along with its polar/non-polar/acidic/basic nature. F.sub.max is a
measure of the theoretical enzyme maximal velocity, K.sub.m is the
Michaelis-Menten constant for each ARTS-1/substrate combination,
.English Pound..sub.cat is the first order rate constant (i.e., the
turnover number) for the ARTS-1/substrate combination, and
k.sub.cJK.sub.m is the second order rate constant for the
ARTS-1/substrate combination (i.e., a measure of overall catalytic
efficiency).
[0250] As shown in Table 3, recombinant GST-ARTS-1 protein
possessed selective aminopeptidase activity against non-polar amino
acid substrates with a four-fold range of enzyme activity.
Isoleucine-pNA was found to be the most favorable amino acid
substrate based upon k.sub.cJK.sub.m determination, followed by
Phe>Gly>Cys>Leu>Met>Ala>Pro>Val. Recombinant
GST-ARTS-1 had no activity against either acidic (Asp or Glu) or
basic (Arg, His, or Lys) amino acid substrates. TABLE-US-00004
ARTS-1 Aminopeptidase Activity V.sub.max (pmol/pmol/ aa-pNA
Polarity min) K.sub.m(mM) k.sub.cat (s.sup.-1) .times. 10-.sup.2
Ile Non-polar 5.81 .+-. 0.87 1.67 .+-. 0.02 9.68 .+-. 0.15 Phe
Non-polar 5.1410.04 1.66 .+-. 0.03 8.57 .+-. 0.06 Gly Non-polar
8.67 .+-. 0.05 3.67 .+-. 0.03 14.45 .+-. 0.08 Cys Non-polar 8.95
.+-. 0.31 4.57 .+-. 0.20 14.92 .+-. 0.52 Leu Non-polar 9.45 .+-.
0.43 5.26 .+-. 0.25 15.75 .+-. 0.72 Met Non-polar 13.36 .+-. 0.75
7.71 .+-. 0.43 22.27 .+-. 1.25 Ala Non-polar 26.18 .+-. 0.24 16.84
.+-. 0.18 43.63 .+-. 0.40 Pro Non-polar 5.29 .+-. 0.08 4.68 .+-.
0.06 8.82 .+-. 0.13 Val Non-polar 5.31 .+-. 0.31 5.69 .+-. 0.26
8.85 .+-. 0.52 Asp Acidic No Activity -- No Activity Glu Acidic No
Activity -- No Activity Arg Basic No Activity -- No Activity His
Basic No Activity -- No Activity Lys Basic No Activity -- No
Activity
VIII. Analysis of ARTS-1 TNFR1 Ectodomain Sheddase Regulatory
Activity
[0251] In light of the ability of the ARTS-1 polypeptide to bind to
the TNFR1 ectodomain in the yeast two-hybrid interaction assay and
the peptidase/protease motif contained within the predicted
polypeptide, the ability of ARTS-1 to promote the shedding of the
TNFR1 ectodomain from the surface of human cells in culture was
examined. The methods used in this experiment are described in
detail in Examples 9 and 11. The results are depicted in FIGS. 7
and 8.
[0252] This experiment was done in two phases. The first phase
involved construction of stably transfected cell lines which
expressed either reduced or elevated levels of ARTS-1 polypeptide,
as detailed in Example 9. The NCI-H292 cell line was stably
transfected with one of three constructs, all based on the pTarget
vector. The pTarget vector contains elements which enable its
selection following stable integration in the NCI-H292 cell line,
and also has the ability to constitutively express a cloned insert
in mammalian cells. The pTarget vectors used in this experiment
were: [0253] 1) an empty pTarget vector, [0254] 2) pTarget vector
containing the full length ARTS-1 cDNA coding region in the sense
orientation, [0255] 3) pTarget vector containing ARTS-1 cDNA bases
61 to 213 in the anti-sense orientation (this region overlaps the
putative transcription start site and intracellular and
transmembrane domains).
[0256] Following the introduction and selection of these constructs
in the host cell lines, membrane fractions were prepared from the
lines and subject to Western immunoblotting in order to assess
ARTS-1 polypeptide expression. This analysis was conducted
according to the Western immunoblotting technique described in
Example 5 and used the anti-ARTS-1 polyclonal antiserum produced as
described in Example 4. The results of this analysis are shown in
FIG. 7. In that Figure, two independent clonal lines containing
ARTS-1 sense or antisense expressing vectors were analyzed. It was
found that integration of the empty pTarget vector (Mock) had
little effect on endogenous ARTS-1 expression compared to cells
that did not contain any stably integrated plasmid (WT). The cell
lines expressing the full length ARTS-1 cDNA in the sense
orientation (ARTS-1) showed a significant increase in ARTS-1
protein expression, while the cell lines expressing the ARTS-1
antisense sequence (AS) showed significant reduction in ARTS-1
protein expression.
[0257] The amount of TNFR1 ectodomain shedding occurring in each of
these cell lines is depicted in FIG. 8. The levels of sTNFR1
ectodomain in cell culture supernatants from these cell lines were
assayed using a commercially available sandwich-enzyme-linked
immunosorbent assay (ELISA) technique (R & D Systems) with a
lower limit of detection of 7.8 pg/ml. In FIG. 8, results are
displayed as the mean of 5 independent experiments, with
accompanying SEM (standard error of the mean). As shown in the
Figure, the cell lines showing increased ARTS-1 protein expression
(ARTS-1) also showed a significant increase in the amount of sTNFR1
present in cell culture supernatants as compared to cells
transfected with the empty pTarget vector (Mock). Conversely, cell
lines with decreased ARTS-1 protein expression (AS) showed
significantly decreased levels of sTNFR1 in cell culture
supernatants as compared to cells transfected with the empty
pTarget vector (Mock).
[0258] The degree of TNFR1 ectodomain shedding as a function of
ARTS-1 protein expression was also assessed indirectly by
determining the relative amounts of membrane-bound TNFR1 fragment
in each of the stably transfected cell lines described above using
the Western immunoblotting technique described in Example 5. Crude
membrane fractions from the stably transfected NCI-H292 cells
described in Example 9 were prepared and resolved by SDS-PAGE and
analyzed by Western immunoblotting using a murine anti-human TNFR1
monoclonal primary antibody (R & D System) which detects the
membrane fragment of the TNF receptor. This Western blot is shown
in FIG. 11. As shown in FIG. 11, cell lines over-expressing ARTS-1
(ARTS-1) demonstrated a decrease in membrane-associated TNFR1
relative to non-transfected (WT) or control transfected (Mock) cell
lines, consistent with an increase in constitutive TNFR1 ectodomain
shedding. Conversely, cell lines expressing anti-sense ARTS-1 mRNA
(AS) demonstrated an increase in membrane-associated TNFR1 relative
to non-transfected (WT) or control transfected (Mock) cell lines,
consistent with a reduction in constitutive TNFR1 ectodomain
shedding.
[0259] Results from an experiment analyzing the ability of ARTS-1
overexpression to potentiate the shedding of TNFR ectodomain from
the surface of NCI-H292 cells in response to PMA stimulation using
these same cell lines are shown in FIG. 9. Cell lines
overexpressing full length ARTS-1 mRNA were stimulated with 0.1 uM
phorbol 12-myristate 13-acetate (PMA), which has previously been
shown to upregulate sTNFR1 shedding in NCI-H292 cells (Levine et
al., Am. J. Respir. Cell Mol. Biol., 14:254-261 [1996]). As
indicated in FIG. 9, the cell line containing only the empty
pTarget vector showed only a modest increase in sTNFR1 shedding
following 24 hours of PMA treatment. However, the cell line
overexpressing the ARTS-1 cDNA showed a more dramatic increase in
sTNFR1 shedding following 24 hours of PMA treatment, increasing
from approximately 300 pg/ml to 415.3.+-.4.5 pg/ml, increasing from
485.+-.16.9 pg/ml to 914.2.+-.9.5 pg/ml
IX. Analysis of TNFR1 Ectodomain Sheddase Regulatory Activity of
ARTS-1 Catalytic Mutants
[0260] The predicted ARTS-1 polypeptide contains a
peptidase/protease consensus motif found in the aminopeptidase
family of gluzincin zinc metalloproteases. It was determined if
this peptidase/protease catalytic motif was necessary for the
ability of ARTS-1 to promote the shedding of the TNFR1 ectodomain.
To conduct this experiment, a series of mutants containing point
mutations predicted to abolish the ARTS-1 peptidase/protease
activity were constructed. The construction of cell lines
expressing these mutants was conducted as described in the section
above, with experimental details provided in Examples 10 and 11.
Results of this experiment are depicted in FIG. 10.
[0261] The experiment was done in two phases. The first phase
involved construction of stably transfected cell lines which
expressed either wild-type or mutant forms of the ARTS-1
polypeptide. The ARTS-1 mutants constructed for this experiment
were designed to eliminate metalloprotease catalytic activity by
disrupting the zinc metalloprotease consensus catalytic motif
HEXXH(Y).sub.18E (SEQ ID NO:10; consisting of a zinc binding and
catalytic site domains). In the ARTS-1 polypeptide, this motif is
located at H.sup.353ELAH(Y),.sub.8E.sup.376 (SEQ ID NO:11). Each of
the mutations made lies within this domain. These mutations are
H353P, E354V, H353P and E354V in combination, and H357V. These
mutations have been previously shown to abolish zinc binding and/or
catalytic (enzymatic) activity in proteins containing the motif
(Devault et al., FEES Lett., 23154-23158 [1988]; Devault et al., J.
Biol. Chem., 263:4033-4040 [1988]; Vallee and Auld, FEBS Lett.,
257:138-140 [1989]; Vallee and Auld, Biochemistry 29:5647-5659
[1990]; and Wang and Cooper, Proc. Natl. Acad. Set. USA
90:1222-1226 [1993]).
[0262] Six cell lines were created by stably transfecting the
NCI-H292 cells with the six constructs, all based on the pTarget
expression vector. These constructs (and resulting cell lines)
contained: [0263] 1) an empty pTarget vector, [0264] 2) the ARTS-1
cDNA (WT) coding region, [0265] 3) the ARTS-1 cDNA encoding a H353P
mutation, [0266] 4) the ARTS-1 cDNA encoding a E354V mutation,
[0267] 5) the ARTS-1 cDNA encoding a H353P and E354V double
mutation, and [0268] 6) the ARTS-1 cDNA encoding a H357V
mutation.
[0269] Following the introduction and selection of these constructs
in the host cell lines, the amount of TNFR1 ectodomain shedding
occurring in each of the lines was determined by measuring the
levels of sTNFR1 ectodomain in cell culture supernatants using a
commercially available sandwich-enzyme-linked immunosorbent assay
(ELISA) technique (R & D Systems) with a lower limit of
detection of 7.8 pg/ml. These results are depicted in FIG. 10 as
the mean of five independent experiments, as well as the SEM
(standard error of the mean). As shown in this Figure, the cell
line containing the recombinant ARTS-1 cDNA with no mutations
(ARTS-1) showed a significantly elevated level of sTNFR1 in the
culture supernatant compared to cell lines containing no integrated
DNA (WT) or containing the empty pTarget vector (MOCK).
Unexpectedly, each of the cell lines containing mutant forms of the
ARTS-1 polypeptide also showed elevated levels of sTNFR1 compared
to the control lines (i.e., WT and MOCK lines). This experiment
demonstrates an unexpected property of the present invention, as
the peptidase/protease activity of the ARTS-1 polypeptide appears
not to be required for its sTNFR1 shedding regulatory activity.
X. Analysis of ARTS-1/TNFR1 Interaction in vivo
[0270] In light of the of the identification of the ARTS-1 gene by
the yeast two-hybrid interaction screening, the physical
association of ARTS-1 and TNFR1 was verified in vivo in a mammalian
cell culture system using a co-immunoprecipitation assay.
[0271] Crude membrane fractions from cultured NCI-H292 cells were
isolated and incubated with murine anti-human TNFR1 monoclonal
antibody (R & D System) or 1 ml of anti-ARTS-1 antiserum
overnight. Following the incubation, the resulting antibody
complexes were immunoprecipitated using immobilized protein A/G
beads (Pierce), and the precipitated proteins analyzed by Western
immunoblotting.
[0272] Two different combinations of precipitation and
immunoblotting antibody were used. The results of these
immunoprecipitation experiments are shown in FIG. 12. In one
experiment (FIG. 12, top panel), the anti-TNFR1 antibody was used
in the immunoprecipitation (indicated as "IP" in the Figure), and
the anti-ARTS-1 antiserum was used as the primary antibody in the
immunoblotting (indicated as "IB" in the Figure). In a second
experiment (FIG. 12, bottom panel), the antibodies were reversed,
where the anti-ARTS-1 antiserum was used in the
immunoprecipitation, while the anti-TNFR1 antibody was used as the
primary antibody in the immunoblotting.
[0273] As shown in FIG. 12, immunoprecipitation of the NCI-H292
cell membrane proteins with an anti-TNFR1 monoclonal antibody
resulted in the coprecipitation of the I 00 kDa ARTS-1 species and,
conversely, immunoprecipitation with anti-ARTS-1 antiserum
coprecipitated the 55 kDa TNFR1. These results indicate an in vivo
protein-protein interaction between ARTS-1 and TNFR1 proteins.
[0274] Similar immunoprecipitation experiments were also performed
using the stably-transfected NCI-H292 cell lines described in
Example 9. In this experiment, the anti-TNFR1 antibody was used to
immunoprecipitate protein from the various membrane protein
fractions, and the resulting immunoprecipitate was examined by
Western immunoblotting using anti-ARTS-1 antiserum as the primary
antibody. As shown in FIG. 13, immunoprecipitation using an
anti-TNFR1 monoclonal antibody of cell membrane protein derived
from the anti-sense ARTS-1 cell line (AS) showed decreased amounts
of ARTS-1 protein as compared to control-transfected (Mock) or
non-transfected (WT) cells, consistent with decreased ARTS-1
protein expression in anti-sense ARTS-1 cells. No increase in
ARTS-1 protein levels relative to control cell lines was detected
following immunoprecipitation of ARTS-1 overexpressing cell lines
with an anti-TNFR1 monoclonal antibody, which likely reflects
increased TNFR1 shedding related to ARTS-1over-expression.
XI. Therapeutic Agents to Treat Immune Diseases and Disorders
[0275] The present invention provides at least one polypeptide
which promotes the shedding of TNFR1 (i.e., ARTS-1 polypeptide)
from the surface of cultured human cells, a gene encoding the
polypeptide, recombinant vectors comprising the gene, host cells
comprising the vectors and antibodies directed against the ARTS-1
polypeptide. It is contemplated that these compositions will find
use as therapeutic agents for the treatment of TNF-mediated immune
diseases. It is contemplated that the therapeutic agents or the
agents for gene therapy of the present invention will be
administered to a subject orally, parenterally, systemically or
locally. It is also contemplated that genes and polypeptides which
are substantially homologous to the ARTS-1 gene provided by the
present invention will also find use in the treatment of TNF
mediated diseases and disorders.
[0276] When compositions of the present invention are used as
therapeutic agents or agents for gene therapy for immune diseases,
it is not intended that the present invention be limited to a
particular disease. For example, the gene or polypeptide of the
invention may be used, alone or in combination, to treat
inflammatory diseases including, but not limited to, rheumatoid
arthritis, inflammatory bowel disease, septic shock, cachexia,
autoimmune disorders, graft-versus-host disease and insulin
resistance.
[0277] In some preferred embodiments of the present invention, when
the therapeutic agent of the invention is administered orally, the
agent may be formulated into a tablet, capsule, granule, powder,
pill, troche, liquid drops, suspension, emulsion, syrup or the
like. Alternatively, the therapeutic agent may be prepared into a
dry product which is re-dissolved just before use. In preferred
embodiments, when the therapeutic agent of the invention is
administered parenterally, the agent may be formulated for
intravenous injection, intramuscular injection, intraperitoneal
injection, subcutaneous injection, as a suppository, etc.
Injections are supplied in the form of unit dosage ampules or
multi-dosage containers. The formulations of the present invention
may be prepared by conventional methods using appropriate
excipients, fillers, binders, wetting agents, disintegrating
agents, lubricating agents, surfactants, dispersants, buffers,
preservatives, dissolution aids, antiseptics, flavoring/perfuming
agents, analgesics, stabilizers, isotonicity inducing agents, etc.
conventionally used in pharmaceutical preparations.
[0278] Each of the above-described formulations may contain
pharmaceutically acceptable carriers or additives. Specific
examples of such carriers or additives include water,
pharmaceutically acceptable organic solvents, collagen, polyvinyl
alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium
alginate, water-soluble dextran, sodium carboxymethyl amylose,
pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerol,
propylene glycol, polyethylene glycol, vaseline, paraffin, stearyl
alcohol, stearic acid, serum albumin, mannitol, sorbitol and
lactose. One or a plurality of these additives are selected or
combined appropriately depending on the form of the
preparation.
[0279] The dosage levels of the therapeutic agent of the invention
will vary depending on the age of the subject, the route of
administration and the frequency and duration of administration and
may be varied over a wide range as suitable for each subject. When
an effective amount of the polypeptide or antibody of the invention
is administered in combination with an appropriate diluent and a
pharmaceutically acceptable carrier, the effective amount of the
polypeptide or antibody is in the range from 0.01 to 1000 mg/kg per
administration, although other amounts are contemplated, as
appropriate. One skilled in the art is capable of determining the
therapeutically effective amount appropriate any given
circumstances. In some embodiments, the therapeutic agent is
administered once a day or in several dosages per day for at least
one day.
[0280] In some embodiments of the present invention, at least one
gene of the present invention is used as an agent for therapy for
immune diseases or disorders. When used as a therapeutic agent, the
gene(s) may be administered systemically or locally. The gene(s)
may be delivered by direct application of the nucleic acid to cells
or tissues.
[0281] In one embodiment, the present invention is used as a gene
therapy agent to treat an inflammatory disease or condition. In one
embodiment, the gene therapy agent of the present invention is
delivered via a viral delivery system. In an alternative
embodiment, the gene therapy agent of the present invention
involves a non-viral delivery system.
[0282] Viral-mediated gene delivery has been shown to be an
effective mechanism for gene delivery for use in gene therapy.
Indeed, methods for viral-mediated gene therapy have recently been
shown to be effective in human and non-human systems (Kordower et
al, Science 290:767-773 [2000]; Lee et al, Nature 408:483-488
[2000]; Cavazzana-Calvo et al, Science 288:669-672 [2000]; Kay et
al., Nature Genetics 24:257-261 [2000]; Amado and Chen, Science
285:674-676 [1999]; Burton et al, Proc. Natl Acad. Sci. USA
96(22):12725-12730 [1999]; Zhang, Cancer Gene Ther., 6(2):113-138
[1999]; Connelly et al, Blood 91(9):3273-3281 [1998]; and Connelly
et al, Blood 88(10):3846-3853 [1996]). A number of viruses have
been demonstrated to be effective or potentially effective tools in
recombinant gene delivery to subjects, including adenovirus
(lentivirus) vectors, adeno-associated virus vectors, herpes virus
vectors, vaccinia virus vectors, and retrovirus vectors. In some
preferred embodiments, the recombinant viral vector comprising the
ARTS-1 gene of the present invention comprises nucleic acid
elements operably linked for the purpose of transcribing and
translating the gene of the invention in cells in a subject. In
preferred embodiments, these nucleic acid elements consist of a
nucleotide sequence encoding the ARTS-1 polypeptide, and operably
linked promoter and enhancer elements for expression of the ARTS-1
gene. In some embodiments, these promoter/enhancer elements are
widely active in all or many cell types, and direct constitutive
expression of the gene (e.g., cytomegalovirus (CMV), SV40 or Rous
sarcoma virus (RSV) promoter/enhancer sequences). In alternative
embodiments, operably linked promoter/enhancer elements are
restricted in activity to a single cell type or tissue (e.g.,
cardiac-specific or liver-specific promoter/enhancers) (Maniatis et
al, Science 236:1237-1245 [1987]; Voss et al., Trends Biochem.
Sci., 11:287 [1986]). In further embodiments, a promoter/enhancer
element that imparts inducible (i.e., conditional) expression of an
operably linked open reading frame (e.g., tetracycline inducible or
repressible promoters) is used. Furthermore, in other embodiments,
operably linked nucleotide sequences include sequences directing
proper translation initiation, post-transcriptional
splicing/editing, and/or polyadenylation. In still other
embodiments, in addition to containing nucleotide sequences
controlling the expression of the ARTS-1 gene, a viral gene therapy
vector further contains the necessary nucleotide sequences for in
vitro replication and propagation of the virus, production of
infective virion particles, and sequences that impart stability of
the DNA in a cellular host (although many viral functions require
the presence of a "helper virus"). Collectively, such sequences are
sometimes referred to as the viral "backbone."
[0283] Additionally, a genetic sequences of the invention may be
enclosed in phospholipid vesicles such as liposomes, and the
resultant liposomes administered to a subject. Liposomes are
biodegradable vesicles containing an internal aqueous region
surrounded by a lipid bilayer. This structure is able to
encapsulate materials (e.g., at least one gene of the present
invention). By mixing at least one gene of the present invention
with phospholipid starting material, a liposome-gene complex will
form. Subsequently, when this complex is cultured with cells, the
gene(s) in the complex is taken into the cells (i.e., via
lipofection). A liposome-gene complex comprising at least one gene
of the present invention can be administered to a subject, either
locally or systemically. In addition to liposomes, a plasmid
encoding the gene(s) of interest may be used.
[0284] Alternatively, direct DNA administration, liposome gene
transfer, or viral vector, all comprising at least one gene of the
present invention, can be used to transfect cells ex vivo (i.e.,
not within the subject), followed by the transplantation of the
recipient cells into the subject. The source of the cells receiving
the gene(s) of the invention can be cells that have been removed
from the subject, or cells from some other source. Following
delivery of the gene(s) of the present invention into these cells,
the cells are then placed into the subject to provide therapeutic
value.
[0285] In some embodiments of the present invention, methods are
contemplated for administering gene(s) of the invention locally to
tissues via surgical or injection protocols, as well as
systemically, such as by intravenous or intra-arterial
administration. Further, an administration method combined with
catheter techniques and surgical operations may also be
employed.
[0286] The dosage levels of the agent for delivering the gene(s) of
the invention vary depending on the age, sex and conditions of the
subject, the route of administration, the number of times of
administration, and the type of the formulation, among other
considerations. One skilled in the art is capable of determining
the therapeutically effective amount appropriate any given
circumstances. Usually, it is appropriate to administer a gene of
the invention in an amount of 0.1-100 mg/adult body/day, although
other concentrations are contemplated, as appropriate.
XII. Diagnostic Agents for Immune Diseases and Disorders
[0287] It is contemplated that the ARTS-1 gene of the present
invention will find use as a diagnostic marker for TNF signaling
activity. Indeed, levels of ARTS-1 mRNA or ARTS-1 polypeptide in
biological samples (e.g., blood, urine, serum or any other body
fluid) are useful indicators of the levels of TNF signaling
activity in vivo. It is contemplated that the presence of elevated
ARTS-1 mRNA and/or polypeptide levels correlate with decreased TNF
signaling activity, while reduced levels of ARTS-1 mRNA or
polypeptide correlate with increased TNF activity. It is
contemplated that excessive or inadequate TNF activity is
indicative of disease or pathological states. Thus, the present
invention provides methods and compositions for rapid quantitation
of ARTS-1 mRNA and polypeptide indicative of TNF signaling
activity, thereby providing useful tools for assessing the immune
condition of an individual suspected of suffering from TNF-mediated
immune disorders or diseases. In contrast, existing methods for the
assay of TNF activity involve lengthy tissue culture assays which
are not readily applicable for use as a rapid diagnostic tool in a
clinical setting (Suffredini et al., J. Immunol, 155:5038-5045
[1995]; Suffredini et al, N. Eng. J. Med., 321:280-287 [1989]; and
Eskandari et al, Immunol Invest., 19:69-79 [1990]).
[0288] The present invention further provides compositions for use
in diagnostic kits which can be used to rapidly assess ARTS-1 mRNA
or polypeptide using either a nucleic acid probe specific for
ARTS-1 mRNA, or PCR primers capable of amplifying ARTS-1 mRNA (all
derived from the nucleic acid of SEQ ID NO: 1) or the antibody
directed against at least a portion of an ARTS-1 polypeptide. Such
kits may be designed to incorporate PCR, nucleic acid probe
hybridization, and/or antibody immunoassay protocols for ARTS-1
marker detection. These kits may further include any reagent(s) or
material(s) which makes possible or facilitates the analysis of a
sample (e.g., apparatus for sample collection, sample tubes,
holders, trays, racks, dishes, plates, instructions to the kit
user, solutions or other chemical reagents, and samples to be used
for standardization, normalization, and or as control samples).
XIII. Identification of Genes Substantially Homologous to
ARTS-1
[0289] In other embodiments, the present invention provides
compositions and methods for the identification of genes
substantially homologous to the ARTS-1 gene (i.e., SEQ ID NO:1). It
is contemplated that genes similar to the gene set forth in SEQ ID
NO:1 have the ability to regulate the cleavage and shedding of
TNFR1 ectodomain. In particular, it is contemplated that the
compositions and methods of the present invention will find use in
stringent hybridization and/or PCR methods to identify genes
substantially homologous to SEQ ID NO:1.
[0290] It is further contemplated that genes similar to the gene
set forth in SEQ ID NO:1 have the ability to regulate the cleavage
and shedding of the ectodomains of other pro-inflammatory cytokine
receptors (e.g., type II IL-1 receptor and IL-6 receptor). As
discussed above, TNF, IL-1 and IL-6 all are multi-functional
pro-inflammatory cytokines which regulate acute phase protein
production during innate immune responses to infection and tissue
injury (Suffredini et al, J. Clin. Immunol, 19:203-214 [1999]).
Consequently, it is contemplated that genes significantly
homologous to ARTS-1 will find utility in the treatment of immune
disorders or diseases mediated by abnormal TNF, IL-1 or IL-6
activity. It is contemplated that the methods and compositions of
the present invention will find use in promoting the cleavage and
shedding of the TNFR1 ectodomain, as well as the ectodomains of
IL-1 and IL-6 cytokine receptors. Although it is not intended that
the present invention be so limited, two methods for isolation of
genes substantially homologous to the ARTS-1 gene are provided
below.
[0291] A) Hybridization to Identify Genes Significantly Homologous
to the ARTS-1 Gene
[0292] In this method, a probe derived from the nucleic acid of SEQ
ID NO:1 is used to screen a phage cDNA library. The probe is
preferably derived from the ARTS-1 gene coding region. The probe
may be an oligonucleotide amplified or excised from a larger
nucleic acid (e.g., from a purified restriction digest product of a
plasmid or other vector) or produced synthetically, recombinantly
or by PCR amplification. There is no limitation on the size of the
probe, although it is preferably longer than 25 nucleotides, and
most preferably encompasses the entire coding region of the ARTS-1
cDNA. This probe may be single or double stranded. In particularly
preferred embodiments the probe is labelled with a "reporter
molecule," so that is detectable in a detection system of choice. A
detection system may include, but is not limited to, enzymatic
detection, fluorescence, radioactivity, and luminescent systems.
Indeed, it is not intended that the present invention be limited to
any particular detection system or label.
[0293] As a source of nucleic acid to be used in the hybridization
screening, it is contemplated that nucleic acid from a wide variety
of eukaryotic sources may be used. However, .lamda.gt10-based cDNA
bacteriophage libraries derived from human sources are preferred
and advantageous in this embodiment of the invention. Nonetheless,
the source of the nucleic acid to be screened is by no means
limited.
[0294] Example 1 provides experimental protocols used in the
development of the present invention, and specifically, a protocol
for the screening of a phage plaque library with a radiolabelled
DNA probe. This same protocol finds use in identification of genes
which are substantially homologous to SEQ ID NO:1. Briefly, a
bacteriophage cDNA library is plated in a lawn of DH-5a host
bacteria. The DNA contained in each of the plaques is replica
plated via a plaque-lift onto a membrane suitable for subsequent
hybridization. The DNA is fixed to the filter, and probed in
solution using the ARTS-1 derived probe under stringent conditions.
Phage plaque DNA with the ability to hybridize to the probe is
isolated, analyzed and sequenced.
[0295] B) PCR to Identify Genes Significantly Homologous to the
ARTS-1 Gene
[0296] PCR may be used to identify genes significantly homologous
to the ARTS-1 gene. In a preferred embodiment, a sense primer and
an anti-sense primer derived from the polypeptide coding region of
the ARTS-1 gene are synthesized and used in a polymerase chain
reaction (PCR). In an alternative embodiment, "degenerate" PCR
primers are used. In these embodiments, the PCR primers have
nucleotide sequences which encode amino acid domains of the ARTS-1
polypeptide, but differ from the ARTS-1 gene nucleotide sequence.
Criteria for designing "degenerate" PCR primers are well known in
the art. It is not intended that the present invention be limited
to any particular primer or primer set.
[0297] It is also contemplated that nucleic acid from a wide
variety of eukaryotic sources may be used as template in the PCR
methods. It is not intended that the present invention be limited
as to the source of the template nucleic acid. However, nucleic
acid template derived from human sources is the most preferred
embodiment of the present invention.
[0298] In preferred PCR methods, a nucleic acid that is
substantially homologous to the ARTS-1 gene is amplified using
materials and methods known in the art. Conditions of the PCR may
be manipulated (e.g., the duration or temperature of the cycle
times may be changed) to amplify a nucleic acid substantially
homologous to the ARTS-1 gene. Indeed, it is contemplated that many
suitable PCR conditions as known in the art will find use in the
present invention. The nucleic acid amplified in the PCR reaction
is visualized, isolated, subcloned, and sequenced using techniques
standard in the art.
[0299] The isolated nucleic acid obtained by either plaque
hybridization or PCR is further examined to determine if the
isolated nucleic acid is "substantially homologous" to the ARTS-1
gene of the present invention based on criteria previously
discussed. In a preferred embodiment, the isolated nucleic acid
which is substantially homologous to the ARTS-1 gene encodes a
polypeptide having the ability to promote the cleavage and shedding
of at least one of the ectodomains of the group of cytokine
receptors consisting of TNFR1, and IL-1 and IL-6 cytokine
receptors. The nucleic acid obtained is then tested using methods
such as those described in Examples 7, 11, 12 and 13. In a most
preferred embodiment, the isolated gene has TNF regulatory
activity, as determined using the protocol supplied in Example
14.
XIV. Methods for Drug Screening
[0300] It is contemplated that compounds which are able to regulate
ARTS-1 activity or receptor ectodomain shedding are candidates for
further development as therapeutically advantageous drugs. Such
drugs find use as immune response modifiers, to either enhance or
attenuate the action of an cytokine signalling (e.g., TNF, IL-1 or
IL-6). The present invention provides compositions and methods for
the screening and identification of test compounds which can
regulate ARTS-1 activity, ARTS-1 transcript or protein expression,
and receptor shedding, and thus, identifies drug candidates for
further development as therapeutics. However, an understanding of
the mechanism(s) of how a particular compound regulates cytokine
signalling and proinflammatory immune responses is not necessary in
order to use the present invention.
[0301] A) Method for Drug Screening to Identify Compounds Having
the Ability to Regulate ARTS-1 Expression
[0302] The present invention provides screening methods to identify
compounds which can regulate the level of ARTS-1 transcript or
protein in a tissue. Test compounds which are able to regulate
ARTS-1 transcript or protein levels in a cell or tissue are
candidates for further development as therapeutic agents. It is
contemplated that compounds which can upregulate or downregulate
ARTS-1 expression also have the ability to downregulate or
upregulate cytokine signalling (i.e., a proinflammatory immune
response), respectively.
[0303] In one embodiment of the present invention, the screening
method uses Northern blotting to assess the levels of the ARTS-1
transcript in a cell culture following exposure of the culture to a
test compound. In this embodiment, cultured cells are exposed to a
test compound, and samples of tissue are collected at intervals
ranging from approximately 0 to 48 hours. RNA is isolated from
these cells and subjected to Northern blot analysis, as described
in Example 3, using a probe specific for the ARTS-1 mRNA.
Comparison of the ARTS-1 transcript levels before and after
exposure to the test compound identifies those compounds that
upregulate or downregulate ARTS-1 transcript levels. It is
contemplated that compounds that can regulate ARTS-1 transcript
levels provide targets for further development as therapeutic
agents.
[0304] In a preferred embodiment, cultured human NCI-H292 pulmonary
mucoepidermoid carcinoma cells are used in the screening. However,
it is not intended that the invention be limited to the use of only
this cell type, as other cell types are equally suitable, and are
known to those in the art. Similarly, it is not intended that the
reagents, methods or apparatus for RNA collection and analysis be
limited to those described herein, as numerous suitable equivalents
are known to those in the art.
[0305] In another embodiment of the screening method of the present
invention, Western immunoblotting is used to assess the levels of
ARTS-1 protein following exposure of a cell culture to a test
compound. In this embodiment, cultured cells were exposed to a test
compound, in this case, 4b-phorbol 12-myristate 13-acetate (PMA),
and samples of tissue were collected at 0, 2, 8 and 24 hours
following the exposure. Membrane proteins were isolated from these
cells and subjected to Western immunob lot analysis, as described
in Examples 4 and 5, using antiserum specific for the ARTS-1
protein. Results of this screening are shown in FIG. 14, Panel A.
Comparison of the ARTS-1 protein levels in the cells before and
after exposure to the test compound demonstrated that treatment of
the cells with PMA resulted in an upregulation of ARTS-1 protein
expression. It is contemplated that compounds that can upregulate
ARTS-1 protein levels are therapeutically advantageous, as such
compounds can suppress a promflammatory immune response by
enhancing receptor ectodomain shedding. However, an understanding
of the mechanism(s) is not required in order to use the present
invention. As indicated herein, PMA is a candidate for further
development as a therapeutic agent.
[0306] In an alternative preferred embodiment, cultured human
NCI-fl292 pulmonary mucoepidermoid carcinoma cells are used in the
screening. However, it is not intended that the invention be
limited to the use of only this cell type, as other cell types are
equally suitable, and are known to those in the art. Similarly, it
is not intended that the reagents, methods or apparatus for protein
collection and Western immunoblot analysis be limited to those
described herein, as numerous suitable equivalents are known to
those in the art.
[0307] B) Method for Drug Screening to Identify Compounds Having
the Ability to Enhance Receptor Ectodomain Shedding
[0308] The present invention provides screening methods to identify
compounds which can regulate receptor ectodomain shedding. Test
compounds which are able to regulate ectodomain shedding are
identified as candidates for further development as therapeutic
agents. It is contemplated that compounds which can upregulate or
downregulate ectodomain shedding also have the ability to
downregulate or upregulate a proinflammatory immune response,
respectively.
[0309] In one embodiment of the present invention, the screening
method uses an ELISA to assess the levels of sTNFR1 in the
supernatant of cell cultures. In the experiments described herein,
an ELISA was used to assess the levels of sTNFR1 in the
supernatants from human NCI-H292 pulmonary mucoepidermoid carcinoma
cells following exposure of the culture to PMA. In this embodiment,
the cultured cells were exposed to the compound, and samples of
supernatant were collected at 0, 2, 8 and 24 hours following the
exposure. The samples were analyzed using an ELISA with an
anti-sTNFR1 antibody as described in Example 11. The results of
this ELISA screening are shown in FIG. 14, Panel B. As can be seen
in the Figure, treatment of the cultured cells resulted in an
increase in sTNFR1 shedding over the course of 24 hours compared to
a control culture (n=5,*P<0.05). Thus, this screening method
identified a compound as a candidate for further drug
development.
[0310] It is contemplated that compounds that can upregulate sTNFR1
ectodomain shedding are therapeutically advantageous, as such
compounds can suppress a proinflammatory immune response. However,
an understanding of the mechanism(s) is not required in order to
use the invention. Based on the criteria set forth and described
herein, PMA is a candidate for further development as a therapeutic
agent.
[0311] It is not intended that the method for drug screening
assessing sTNFR1 ectodomain shedding be limited to analysis of
sTNFR1 shedding. It is contemplated that the analysis of ectodomain
shedding other cytokine receptors, such as type II interleukin-1
cytokine receptor and interleukin-6 cytokine receptor alpha-chain
gp80 also find use with the present invention.
[0312] C) Method for Drug Screening to Identify Compounds Having
the Ability to Regulate the Peptidase Activity of ARTS-1
Protein
[0313] The present invention provides screening methods to identify
compounds which can regulate the peptidase activity of ARTS-1
protein. Test compounds which are able to regulate ARTS-1 peptidase
activity are candidates for further development as therapeutic
agents. It is contemplated that compounds which can upregulate or
downregulate ARTS-1 peptidase activity also have the ability to
downregulate or upregulate a proinflammatory immune response,
respectively.
[0314] In one embodiment of the present invention, the screening
method uses amino acid p-nitroanilide substrates to assess the
aminopeptidase activity (described in Example 7) of purified
recombinant GST-ARTS-1 fusion protein (described in Example 6). In
this screening method, the amino acid p-nitroanilide hydrolysis
reaction is conducted in the absence and presence of a test
compound, according to the reaction conditions provided in Example
7, and the rate of amide bond hydrolysis by ARTS-1 is determined.
It is then observed whether a test compound has the ability to
regulate the rate of amide bond hydrolysis.
[0315] It is contemplated that compounds that can regulate ARTS-1
amide bond hydrolysis are therapeutically advantageous, as such
compounds can regulate a proinflammatory immune response, and are
targets for further development as therapeutic agents. However, an
understanding of the mechanism(s) is not required in order to use
the present invention.
[0316] It is not intended that this method for drug screening be
limited to those reagents itemized in Example 7. For example, it is
contemplated that purified ARTS-1 proteins in addition to
ARTS-1-GST also find use with the present invention. Furthermore,
it is contemplated that more than one amino acid p-nitroanilide
substrate finds use with the screening method, as isoleucine
p-nitroanilide, phenylalanine p-nitroanilide and glycine
p-nitroanilide substrates can all be used in the hydrolysis
reaction.
Experimental
[0317] In the experimental disclosure which follows, the following
abbreviations apply: eq (equivalents); M (Molar); jaM (micromolar);
N (Normal); mol (moles); mmol (millimoles); umol (micromoles); nmol
(nanomoles); g (grams); mg (milligrams); u.g (micrograms); ng
(nanograms); l or L (liters); ml (milliliters); jil (microliters);
cm (centimeters); mm (millimeters); jam (micrometers); nm
(nanometers); .degree. C. (degrees Centigrade); Accurate Chemical
and Scientific Corporation (Accurate Chemical and Scientific
Corporation, Westbury, N.Y.); Advanced Biotechnologies (Advanced
Biotechnologies Incorporated, Columbia, Md.); Amersham/Pharmacia
(Amersham/Pharmacia Biotech, Piscataway, N.J.); ATCC (American Type
Culture Collection, Rockville, Md.); Bachem (Bachem, King of
Prussia, Pa.); Biofluids (Biofluids, Inc, Rockville, Md.);
Boehringer Mannheim (Roche/Boehringer Mannheim Corporation,
Indianapolis, Ind.); Calbiochem (Calbiochem-Novabiochem Corp, San
Diego, Calif.); Clontech (Clontech Laboratories, Inc., Palo Alto,
Calif.); Genzyme (Genzyme Corporation, Cambridge, Mass.); Life
Technologies (Life Technologies/Gibco/BRL, Gaithersburg, Md.);
Novex (Novex/Invitrogen, Carlsbad, Calif.); Pierce (Pierce,
Rockford, Ill.); Promega (Promega Corporation, Madison, Wis.); R
& D Systems (R & D Systems, Minneapolis, Minn.); Research
Genetics (Research Genetics, Huntsville, Ala.); Roche
(Roche/Boehringer Mannheim Corporation, Indianapolis, Ind.); Sigma
(Sigma Chemical Co., St. Louis, Mo.); Stratagene (Stratagene, La
Jolla, Calif.).
[0318] The following Examples are provided in order to demonstrate
and further illustrate certain preferred embodiments and aspects of
the present invention and are not to be construed as limiting the
scope thereof.
EXAMPLE 1
Identification of ARTS-1, a Novel Human Aminopeptidase Regulator of
Type-1 Tumor Necrosis Factor Receptor (TNFR1) Shedding
A) Yeast Two-Hybrid Library Screening
[0319] A yeast two-hybrid library screening was conducted to
identify proteins that interact with or are capable of interacting
with the extracellular domain of the type I, 55 kDa TNF receptor
(TNFR1). A human lung cDNA library (Clontech), cloned into pGADIO
(GAL4 DNA-activation domain vector), was screened to detect
interactions with a GAL4BD-TNFR1 fusion protein using a Matchmaker
System 2 (Clontech) using methods known in the art as well as the
manufacturer's recommended protocols. Amino acids 26 to 216 of
TNFR1, corresponding to the extracellular domain (i.e., the
ectodomain) located between the putative leader domain and the
transmembrane domain (Nophar et al, EMBO J., 10:3269-3278 [1990])
were cloned into pAS2-1 (GAL4 DNA-binding domain vector) to
generate the bait GAL4 DNA-binding domain fusion protein,
GAL4BD-TNFR1. The Y190 yeast strain was transformed with the pAS2-1
GAL4BD-TNFR1 bait plasmid by the lithium acetate method utilizing
the Yeastmaker Transformation System (Clontech). Transformed yeast
were selected on synthetic drop-plates deficient in tryptophan,
leucine and histidine in the presence of 25 mM 3
amino-1,2,4-triazole (SIGMA). His.sup.+ colonies were then
subjected to p-galactosidase colony-lift filter assays and
p-galactosidase producing colonies were selected and restreaked on
synthetic drop-out plates. Approximately 7.11.times.10.sup.6
transformants were screened, p-galactosidase positive colonies
which produced a blue signal within 2 hours were selected for
further study. Selected colonies were analyzed for expression of
the GAL4 binding domain (GAL4BD) fusion protein by immunoblotting
using an anti-GAL4BD monoclonal antibody (Clontech). Thirty three
positive clones were identified which activated the P-galactosidase
reporter construct. All 33 positive clones were sequenced using an
AB1 Perkin Elmer 377 automated fluorescent sequencer. One positive
clone, L26C-53A, encoding a consensus zinc metalloprotease
catalytic motif was selected for further study. This clone
contained a 2355-bp insert containing an open reading frame of 631
amino acids, but did not contain a poly(A) tail or a putative
translation initiation site. The gene encoded by this insert was
entitled aminopeptidase regulator of type-1, 55 kDa tumor necrosis
factor receptor shedding (or ARTS-1). The L26C-53A sequence
corresponds to ARTS-1 bases 1044 to 3082.
B) ARTS-1 cDNA Cloning/Phage Plaque Hybridization
[0320] Using .sup.32P-labelled DNA derived from clone L26C-53A as a
probe, a PMA-stimulated NCI-H292 cell line cDNA library
(Stratagene) was screened to obtain ARTS-1 cDNA clones. The
NCI-H292 cell line was derived from human pulmonary mucoepidermoid
carcinoma cells and has been demonstrated to shed cell surface
sTNFR1 in response to PMA stimulation (Levine et al., Am. J.
Respir. Cell Mol. BioL, 14:254-261 [1996]). The uni-ZAP XR phage
cDNA library (Stratagene) was constructed with NCI-H292 cell poly
(A.sup.+) mRNA isolated following 24 hours of stimulation with 1 uM
PMA (phorbol 12-myristate, 13-acetate, Sigma).
[0321] Bacteriophage from the uni-ZAP library were plated in a lawn
of XL 1-Blue (MRF-) E. coli (Stratagene) at a density of 50,000 pfu
per 150 mm plate and incubated overnight at 37.degree. C. Plaques
were transferred to Hybond N+ filters (Amersham/Pharmacia) and
denatured for 5 minutes in 1.5M sodium chloride, 0.5 M sodium
hydroxide. Filters were then neutralized in two washes of 5 minutes
each in 1.5M sodium chloride, 1M Tris-base, rinsed in 3.times.SSC
and UV cross-linked. Filters were pre-hybridized in 10.times.
Denhardt's solution, 6.times.SSPE, 1% SDS at 42.degree. C. for 4
hours in hybridization buffer. Filters were hybridized overnight at
42.degree. C. with approximately 10.sup.7 CPM of .sup.32P-labelled
L26C-53A insert which was generated by random priming. Filters were
then washed in 2.times.SSC, 0.5% SDS for 20 minutes at room
temperature, followed by two 1 hour washes in 1.times.SSC, 0.1% SDS
at 65.degree. C. Filters were then exposed to x-ray film overnight
and positive plaques were selected. Positive plaques were subjected
to two additional rounds of plaque hybridization prior to
sequencing. Positive plaques were recovered via in vivo excision
utilizing the ExAssist helper phage (Stratagene).
[0322] Following 3 rounds of screening, four hybridizing phage
clones were identified, then sequenced utilizing the
ThermoSequenase cycle sequencing kit (Amersham/Pharmacia). These
four hybridizing clones overlapped the L26C-53A sequence, but none
encoded a full-length cDNA. Sequencing revealed one phage clone (bp
1-1777) which contained the putative 5' UTR and three phage clones
which contained the putative 3' UTR and the poly(A) tail (bp
2181-4845). cDNA sequence encoding the portion of the gene lying
between these 5' and 3' terminal clones was obtained via PCR from
Marathon-Ready.TM. human lung cDNA (Clontech) utilizing Pfu Turbo
DNA Polymerase (Stratagene) and the following primers:
TABLE-US-00005 (SEQ ID NO:3) 5'-ATAACCATCACAGTGAGGGGGAGG-3'
(1684-2279) and (SEQ ID NO:4) 5'-TAGTTGACTCCGCAGCATTCGCTC-3'
(2257-2280)
The cDNA segment amplified with these primers was cloned into
pGemTEasy (Promega) and both strands were sequenced by automated
fluorescent sequencing using an ABI Perkin Elmer 377 automated
fluorescent sequencer. Sequences of all overlapping regions from
the original two-hybrid clone L26C-53A (bp 1044 to 3082), the four
NCI-H292 cDNA library clones and the PCR product (bp 1684 to 2280)
showed no discrepancies in the nucleotide sequence in their
overlapping regions.
EXAMPLE 2
Molecular Analysis of the ARTS-1 cDNA and Predicted Polypeptide
[0323] The complete cDNA corresponding to the human ARTS-1
transcription unit contains 4845 nucleotides and encodes an open
reading frame of 2823 bp, as shown in FIG. 1 and set forth in SEQ
ID NO:1. The first in-frame ATG codon, located at nucleotide 88,
follows an in-frame stop codon, located at nucleotide 76, and
matches the -3 and +4 nucleotides of the consensus Kozak sequence
consistent with a strong initiator codon (aagatgg) (Kozak, J. Cell
Biol, 108:229-241 [1989]). Sequence analysis revealed five
potential N-glycosylation sites and a TAA stop codon, located at
nucleotide 2911. The putative 3' untranslated region contained a
consensus polyadenylation site (AATAAA), located at nucleotides
4876 to 4800, which is 18 nucleotides upstream of a 27 nucleotide
poly(A) tail. The 3' untranslated region also included two ATTTA
sites, located at nucleotides 3929 and 4457; this sites have been
identified as an mRNA destabilization motif in mRNA's for
cytokines, oncogenes and transcriptional activating factors
(Stephens et al, J. Biol. Chem., 267:8336-8341 [1992]).
[0324] The open reading frame predicted from the human ARTS-1 cDNA
encodes a protein of 941 amino acid residues (See, FIG. 1 and SEQ
ID NO:2) with a calculated molecular weight of 107,227 Da and an
estimated pi of 6.0. Sequence analysis, including Kyte-Doolittle
hydropathy prediction (Kyte and Doolittle, J. Mol. Biol.,
157:105-132 [1982]) was performed using MacVector 7.0 software
(Oxford Molecular). The location of the putative hydrophobic
transmembrane a-helical domain was predicted utilizing several
web-based analysis programs (MEMSAT2 (McGuffin et al., Bioinform.,
16:404-405 [2000]; Sosui (Hirokawa et al, Bioinform., 14:378-379
[1998]; TMAP (Persson and Argos, J. Mol Biol, 237:182-192 [1994]);
TMpred (Hofmann and Stoffel, Biol. Chem. Hoppe-Seyler 374:166
[1993]; and TopPred2 (von Heijne, J. Mol. Biol, 225:487-494
[1992]). In sum, ARTS-1 is predicted to be a type II integral
membrane protein with a single hydrophobic transmembrane a-helical
domain, located between amino acids 5 and 28 (See, FIGS. 1 and 3),
and a very short hydrophobic intracellular amino-terminal domain
(See, McGuffin et al, Bioinform., 16:404-405 [2000]; Hirokawa et
al, Bioinform., 14:378-379 [1998]; Persson and Argos, /. Mol Biol,
237:182-192 [1994]); Hofmann and Stoffel, Biol. Chem. Hoppe-Seyler
374:166 [1993]; and von Heijne, J. Mol Biol, 225:487-494
[1992]).
[0325] The protein sequence analysis using MacVector software
revealed a zinc metalloprotease consensus catalytic motif
(HEXXH(Y).sub.18E) (SEQ ID NO: 10), which is predictive of an
active metalloprotease (Hooper, Zinc Metalloproteases in Health and
Disease, Taylor & Francis, London, England [1996], p.1-21).
Utilizing various web-based analysis programs, a putative amino
acid transmembrane domain (as predicted by von Heijne, J. Mol.
Biol, 20:487-494 [1992]), extending from amino acids 5 to 28, was
identified. These features suggest that human ARTS-1 is a type II
integral membrane protein with a single hydrophobic transmembrane
a-helical domain, located between amino acids 5 and 28 (See, FIGS.
1 and 2), and a very short, hydrophobic intracellular ammo-terminal
domain (See, McGuffin et al, Bioinform., 16:404-405 [2000];
Hirokawa et al, Bioinform., 14:378-379 [1998]; Persson and Argos,
J. Mol. Biol, 237:182-192 [1994]); Hofmann and Stoffel, Biol. Chem.
Hoppe-Seyler 374:166 [1993]; and von Heijne, J. Mol. Biol.,
225:487-494 [1992]).
[0326] A BLAST protein homology comparison revealed that the human
ARTS-1 contained significant homology with several members of the
aminopeptidase family of gluzincin zinc metalloproteases (See,
Tables 1 and 2, supra) (Wang and Cooper, Zinc Metalloproteases in
Health and Disease, Taylor & Francis, London, England [1996]).
As has previously been reported for other aminopeptidase family
members, the human ARTS-1 was found to contain a 375 amino acid
domain that is highly conserved with human placental leucine
aminopeptidase (FLAP) (Rogi et al, J. Biol Chem., 271:56-61
[1996]), rat insulin-regulated aminopeptidase (IRAP) (Keller et al,
J. Biol. Chem., 270:23612-23618 [1995]), human aminopeptidase A
(AMP A) (Nanus et al, Proc. Natl. Acad. Sci. USA 90:7069-7073
[1993]; Li et al, Genomics 17:657-664 [1993]), human aminopeptidase
N (AMP N) (Olsen et al, FEBS Lett., 238:307-314 [1988]), human
puromycin sensitive aminopeptidase (PSA) (Tobler et al, J.
Neurochem., 68:889-897 [1997]), rat thyrotropin-releasing hormone
degrading enzyme (TRH DE) (Schauder et al, Proc. Natl Acad. Sci.
USA 91:9534-9538 [1994]), Saccharomyces cerevisiae aminopeptidase
YSCII (Garcia-Alvarez et al, Eur. J Biochem., 202:993-1002 [1991]),
C. elegans cosmid F49E8.3 gene product (Wilson et al, Nature
368:32-38 [1994]), and Lactococcus lactis aminopeptidase N (Tan et
al, FEBS Lett., 306:9-16 [1992]). This highly conserved domain
contains the putative consensus zinc binding domain and catalytic
site (T.sup.350VAHELAHQWFG (SEQ ID NO:8) and L.sup.372WLNEGFA (SEQ
ID NO:9) which is characteristic of aminopeptidase family members
(Wang and Cooper, Zinc Metalloproteases in Health and Disease,
Taylor & Francis, London, England [1996]; Keller et al, J. Biol
Chem., 270:23612-23618 [1995]). Furthermore, PLAP, AMP A, AMP N,
IRAP and TRH DE share structural similarity with human ARTS-1,
based upon the presence of a transmembrane domain and a short
intracytoplasmic tail, consistent with a type II integral membrane
protein.
EXAMPLE 3
ARTS-1 mRNA Expression Analysis
[0327] Following analysis of the ARTS-1 cDNA, the expression
pattern of endogenous of ARTS-1 mRNA was investigated using a
Northern immunoblotting protocol. RT-PCR was performed on human
lung poly(A.sup.+) mRNA (Clontech) to generate the human ARTS-1
cDNA coding sequence utilizing the following primers:
TABLE-US-00006 (SEQ ID NO:5) 5'-GCAAGAAGATGGTGTTTCTGCCCCTC-3'
(80-105) and (SEQ ID NO:6) 5'-TTACATACGTTCAAGCTTTTCACT-3'
(2890-2913).
The full length ARTS-1 coding sequence amplified by these primers
was cloned into the pTarget mammalian expression vector (Promega).
The DNA sequence of this cloned open reading frame was obtained
from both strands by automated fluorescent sequencing, as known in
the art. There were no discrepancies between the sequence obtained
from these two strands, nor from the sequence obtained from the
PMA-stimulated NCI-H292 cell line cDNA library. A .sup.32P-labelled
cDNA probe, corresponding to the full length ARTS-1 coding
sequence, was utilized as a probe for Northern blotting of human
multiple tissue Northern blots (Clontech) according to the
manufacturer's protocol.
[0328] As shown in FIG. 3, Northern blot analysis utilizing
poly(A+) mRNA from multiple human tissues revealed that the human
ARTS-1 transcript was expressed in multiple tissues, including
spleen, thymus, small and large intestine, peripheral blood
leukocyte, heart, placenta, lung, skeletal muscle, kidney and
pancreas. In these tissues, an approximately 5.7 kB predominant
mRNA species was detected (See, FIG. 3, top panel). Also shown is
the same blot following stripping and rehybridization to a probe
specific for the human GAPDH transcript as a reference for RNA
loading normalization (See, FIG. 3, bottom panel).
EXAMPLE 4
The Generation of Polyclonal Anti-ARTS-1 Antiserum
[0329] In order to conduct studies of the ARTS-1 polypeptide,
polyclonal antiserum was generated against the ARTS-1 polypeptide.
Specifically, a 17 amino acid ARTS-1 synthetic peptide was used to
immunogenize New Zealand white rabbits and subsequently collect
immune serum using a commercial service (Research Genetics). This
peptide corresponded to amino acids 538 to 554 of the ARTS-1
protein and had the sequence: TABLE-US-00007 (SEQ ID NO:7)
R.sup.538GRNVHMKQEHYMKGSD.sup.554
This particular peptide was chosen based upon its antigenic
potential and its lack of homology with other protein sequences via
BLAST homology search.
[0330] Rabbits were immunized with this peptide using standard
techniques. The ARTS-1 peptide was conjugated to KLH and mixed with
an equal volume of Freund's complete adjuvant. The amount of
antigen utilized per immunization was 0.1 mg, which was injected
into three subcutaneous dorsal sites. The animals received boosts
at weeks 2, 6, and 8. Bleeds were obtained at weeks 4, 8 and 10 and
tested for the presence of anti-ARTS-1 antibody. In subsequent
experiments, the antiserum obtained from the 10 week bleed was
used.
EXAMPLE 5
Analysis of ARTS-1 Polypeptide Expression in Cultured Cell Lines
and Primary Cells
[0331] Following the production of ARTS-1 polyclonal antiserum, the
expression of endogenous ARTS-1 polypeptide in cultured cell lines
and primary cells was investigated using standard Western
immunoblotting as known in the art. Protein concentrations were
determined via the BCA protein assay (Pierce). Following protein
quantitation, 20 microgram samples of protein were boiled for 5
minutes in Laemmli buffer. Samples were then resolved via SDS-PAGE
(6% polyacrylamide) and electroblotted onto nitrocellulose (Novex).
Blots were incubated overnight in blocking buffer (5% wt/vol nonfat
dry milk in PBS/0.1% Tween-20), then incubated for 2 hours with
ARTS-1 antiserum at a 1:20,000 dilution in blocking buffer.
Membranes were washed three times for 5 minutes each wash in
PBS/0.1% Tween; incubated for 2 hours with 0.8 mg/ml horseradish
peroxidase conjugated goat anti-rabbit IgG (Life Technologies)
diluted to 1:5,000 in blocking buffer, then washed three times for
5 minutes each wash in PBS/0.1% Tween and finally washed three
times for 5 minutes each wash in PBS/0.3% Tween. Membranes were
then incubated in chemiluminescent detection substrate for 1 minute
and signal detected on X-ray film.
[0332] Crude homogenates made from NCI-H292 cells for immunoblot
analysis were produced by cell lysis in homogenization buffer
consisting of 200 jal of 50 mM Tris-HCl, pH 7.2, containing 0.1%
Triton X-100 and Complete.TM. protease inhibitor cocktail tablet
(Boehringer Mannheim) and sonicated for four times at 15 seconds
each using a microprobe. The homogenate was centrifuged at
1,000.times.g for 5 minutes to remove nuclei, unbroken cells and
debris. The low speed supernatant was either utilized as a whole
cell lysate or was further fractionated by ultracentrifugation at
100,000.times.g for 1 hour to generate a crude cytosolic and
membrane fractions. The crude membrane fraction was resolubilized
in homogenization buffer prior to immunoblotting.
[0333] Specificity of the resulting polyclonal antiserum was first
tested using crude whole cell homogenates, and membrane and
cytosolic fractions prepared from cultured NCI-H292 cells. These
results are shown in FIG. 4. Comparing Panels A and B of FIG. 4,
the antiserum was shown to detect a predominant 100 kDa membrane
form and a predominant 68 kDa cytosolic form from the NCI-H292
cells. The whole cell extracts revealed a mixture of these two
forms. The preimmune serum showed no reactivity towards the same
samples when used at the same concentration.
[0334] Specificity of the immune serum was further demonstrated in
competition experiments as shown in Panels C and D of FIG. 4. In
these competition experiments, 1 ul of the ARTS-1 antiserum was
pre-incubated with 1 mg of either bovine serum albumin or
RGRNVHMKQEHYMKGSD peptide (SEQ ID NO:7) for two hours prior to
utilization for immunoblotting. Preincubation of the immune serum
with the peptide against which the polyclonal antiserum was raised
resulted in almost complete attenuation of the immune signal (Panel
D). In contrast, preincubation of the immune serum with bovine
serum albumin resulted in minimal attenuation of immune signal
(Panel C).
[0335] The expression of endogenous ARTS-1 polypeptide in primary
cells and other cell lines was further investigated using the
anti-ARTS-1 antiserum and Western immunoblot technique, as shown in
FIG. 5. These experiments analyzed membrane and cytosolic fractions
made from human bronchial brushing-specimens, the airway epithelial
cell lines BEAS-2B and BET-1A, human lung carcinoma cell line A549,
cultured NCI-H292 cells, primary cultures of normal human bronchial
epithelial cells (NHBE), human umbilical vein endothelial cells
(HUVEC) and human fibroblasts. These experiments also revealed
multiple sized forms of ARTS-1 polypeptide on the Western
immunoblot, including 132, 100 and 68 kDa forms which may localize
to various subcellular fractions. These multiple sized forms may be
due to regulated processing of the ARTS-1 polypeptide, and
furthermore may indicate that this processing is
compartmentalized.
EXAMPLE 6
Expression and Purification of Recombinant GST-ARTS-1
Polypeptide
[0336] The cDNA sequence of the ARTS-1 extracellular domain (i.e.,
the ectodomain; ammo acids 30-941) was PCR amplified from the
pTarget plasmid containing the full length ARTS-1 coding sequence.
The ARTS-1 extracellular domain was then cloned into the pGEX-6P-1
plasmid (Amersham/Pharmacia) and used to transform the BL21 E. coli
host strain. Colonies expressing GST-ARTS-1 fusion protein were
selected by Western blotting utilizing an anti-GST antibody
(Amersham/Pharmacia). Positive clones were grown at room
temperature, stimulated for 4 hours with 0.6 mM isopropyl
(3-D-thiogalactoside (IPTG) and subsequently lysed with B-PER
protein extraction reagent (Pierce). Cells were centrifuged at
27,000.times.g for 15 minutes to separate the soluble from the
insoluble fractions. Following treatment with 0.2 mg/ml lysozyme
for 5 minutes, the GST-ARTS-1 fusion protein was isolated from the
insoluble fraction by denaturation with 6M urea in PBS. The
GST-ARTS-1 fusion protein was refolded by serial dialysis against
PBS baths containing decreasing urea concentrations (5 M to 0 M).
The GST-ARTS-1 fusion protein was purified utilizing a glutathione
sepharose 4B affinity column and eluted with reduced glutathione
buffer using techniques known in the art.
[0337] To assess the purity of the eluted recombinant GST-ARTS-1
fusion protein, samples were subjected to SDS-PAGE on a 4% -12 %
gradient gel (Novex) and stained with Coomassie brilliant blue.
FIG. 6A shows the result of this experiment. In this Figure,
soluble and insoluble protein fractions from BL21 E. coli
transformed with empty pGEX-6P-1 vector (lanes 1 and 2) or the
pGEX-6P-1-ARTS-1 vector (lanes 3 and 4) are shown. The GST-ARTS-1
fusion protein following elution is shown in lane 5 as a
predominant 132 kDa band, corresponding to the predicted molecular
weight of a GST-ARTS-1 fusion protein (104 kDa ARTS-1 extracellular
domain plus 26 kDa GST tag). The control purified GST tag is
revealed as a predicted 26 kDa band in lane 6.
[0338] Purified recombinant GST-ARTS-1 fusion protein samples were
further subjected to FPLC analysis (LKB LCC-500 plus,
Amersham/Pharmacia) utilizing a Superose 6 FIR 10/30 gel filtration
column (Amersham/Pharmacia). Recombinant purified GST-ARTS-1 fusion
protein samples were eluted with PBS at a flow rate of 0.5 ml/min
and fractions were collected every 1 minute. Absorbance was
recorded at 280 mn with a chart speed of 0.25 cm/min. FIG. 6B shows
the result of this analysis. In this figure, FPLC analysis of the
purified GST-ARTS-1 revealed a single major protein elution peak
which eluted at approximately 40 minutes.
[0339] Using the aminopeptidase activity assay described below,
FPLC fractions were assessed for aminopeptidase activity utilizing
a phenylalanine p-nitroaniline substrate. As shown in FIG. 6C, the
single major protein peak revealed by FPLC analysis coeluted with a
peak of aminopeptidase activity against a phenylalanine
p-nitroaniline substrate in pooled fractions from 38-44
minutes.
EXAMPLE 7
ARTS-1 Aminopeptidase Activity Assay
[0340] Aminopeptidase activity of the recombinant GST-ARTS-1 fusion
protein was assessed by determination of the rate of amide bond
hydrolysis of amino acid p-nitroanilide substrates (Bachem). Amino
acid p-nitroanilides (final concentrations 0.25 to 8 mM) were
incubated at room temperature with 24 pmoles of GST-ARTS-1 fusion
protein in 200 1 of 50 mM Tris, pH 7.5 for 1 hour. Reactions were
terminated by addition of 280 ul of 3M sodium acetate (pH 5.2). The
rate of amide bond hydrolysis was determined by measuring the
absorbance of p-nitroaniline at 380 nm (See, Table 3, supra).
Spontaneous hydrolysis of the substrate was corrected for by
subtracting the absorbance of control incubations which were
terminated immediately. Kinetic constants were determined by
Lineweaver-Burk analysis. Each experimental point was assayed in
triplicate and each determination utilized six concentrations of
each amino acid p-nitroanilide substrate. Correlation coefficients
for each line generated were greater than 0.997.
[0341] As shown in Table 3, recombinant GST-ARTS-1 protein
possessed selective aminopeptidase activity against non-polar amino
acid substrates with a four-fold range of enzyme activity.
Isoleucine-pNA was found to be the most favorable amino acid
substrate based upon k.sub.cJK.sub.m determination, followed by
Phe>Gly>Cys>Leu>Met>Ala>Pro>Val. Recombinant
GST-ARTS-1 had no activity against either acidic (Asp or Glu) or
basic (Arg, His, or Lys) amino acid substrates.
EXAMPLE 8
ARTS-1 Endopeptidase Activity Assay
[0342] The endopeptidase activity of recombinant GST-ARTS-1 fusion
protein was also assessed. In this assay, 5 O,g of recombinant
GST-ARTS-1 fusion protein was incubated with 10 ng of either bovine
serum albumin, human albumin, rabbit myosin heavy chain or
transferrin overnight. Sample were then subjected to SDS-PAGE
utilizing 4 to 12% gradient gels (Novex) and stained with Coomassie
brilliant blue. In this assay, recombinant GST-ARTS-1 was shown to
have no demonstrable endopeptidase activity against bovine serum
albumin, human albumin, rabbit myosin heavy chain or human
transferrin.
EXAMPLE 9
Construction of Stably Transfected Cell Lines Expressing Sense and
Antisense ARTS-1 cDNA's
[0343] In light of the ability of the ARTS-1 polypeptide to bind to
the TNFR1 ectodomain in the yeast two-hybrid interaction assay, the
following experiment was conducted in order to determine whether
ARTS-1 has the ability promote the cleavage and shedding of the
TNFR1 ectodomain from the surface of human cells in culture. This
experiment was done in two phases. The first phase involved
construction of stably transfected cell lines which expressed
either reduced or elevated levels of ARTS-1 polypeptide. The
NCI-H292 cell line was stably transfected with one of three
constructs, all based on the pTarget vector (Promega). Transfection
was by the Fugene system (Roche). The pTarget vector expresses a
gene product (encoded by the neo gene) which imparts resistance to
the antibiotic G-418, which kills both prokaryotic and eukaryotic
cells. The construct also contains a constitutively active CMV
promoter which will express cloned DNA inserts in mammalian cells.
The pTarget vectors used to transfect the NCI-H292 cells were:
[0344] 1) an empty pTarget vector, [0345] 2) pTarget vector
containing the full length ARTS-1 cDNA coding region in the sense
orientation, [0346] 3) pTarget vector containing ARTS-1 cDNA bases
61 to 213 in the anti-sense orientation. This region overlaps the
putative transcription start site and intracellular and
transmembrane domains. Following the transfection of these
constructs into the host cells, the transfectants were cultured
under selective pressure in RPM-1640 media supplemented with 10%
heat-inactivated fetal calf serum and Ix antibiotic-antimycotic
(Biofluids) and 500 | g/ml of the G-418 (Promega). Two independent
clones of stable transfectants containing either sense or
anti-sense ARTS-1 plasmid were then generated via limiting
dilutions. Both sense and anti-sense clones were screened by
immunoblotting (described above) utilizing anti-ARTS-1 polyclonal
serum to select duplicate clones for subsequent analysis. Cell
lines were then selected based upon enhanced or suppressed ARTS-1
protein expression as determined by the immunoblotting of cell
membrane fractions, as shown in FIG. 7. Integration of the empty
pTarget vector (indicated "Mock") had little effect on endogenous
ARTS-1 expression compared to cells that do not contain any stably
integrated plasmid (WT). The two cell lines expressing the full
length ARTS-1 cDNA in the sense orientation (ARTS-1) showed a
significant increase in ARTS-1 protein expression, while the two
cell lines expressing ARTS-1 antisense sequence (AS)-showed
significant reduction in ARTS-1 protein expression. These stably
integrated cell lines were examined for sTNFR1 shedding activity
(See, Example 11, below).
EXAMPLE 10
Construction of Stably Transfected Cell Lines Expressing Mutant
ARTS-1 cDNA's
[0347] The predicted ARTS-1 polypeptide contains a
peptidase/protease consensus motif found in the aminopeptidase
family of gluzincin zinc metalloproteases. It was determined if
this peptidase/protease catalytic motif was necessary for the
ability of ARTS-1 to promote shedding of the TNFR1 ectodomain. To
conduct this experiment, a series of ARTS-1 mutants were
constructed which contain point mutations predicted to abolish the
ARTS-1 peptidase/protease activity (Devault et al., FEES Lett.,
23154-23158 [1988]; Devault et al., J. Biol. Chem., 263:4033-4040
[1988]; Vallee and Auld, FEES Lett., 257:138-140 [1989]; Vallee and
Auld, Biochemistry 29:5647-5659 [1990]; Wang and Cooper, Proc.
Natl. Acad. Sci. USA 90:1222-1226 [1993]). These mutations lie
within the zinc metalloprotease family zinc-binding/catalytic
domain consensus HEXXH(Y).sub.18E (SEQ ID NO: 10; consisting of a
zinc binding and catalytic site domains). In the ARTS-1
polypeptide, this motif is located at
H.sup.353ELAH(Y).sub.18E.sup.376 (SEQ ID NO:11). The mutations made
were H353P, E354V, H353P and E354V in combination, and H357V.
Mutagenesis of the ARTS-1 gene open reading frame was performed
using a QuikChange Site-Directed Mutagenesis Kit (Stratagene)
according to the manufacturer's instructions.
[0348] The NCI-H292 cell line was stably transfected with one of
six constructs, all based on the pTarget expression vector. The
method used to produce stably transfected cell lines containing
these constructs is the same as that provided in Example 9. These
constructs (and resulting cell lines) were: [0349] 1) an empty
pTarget vector, [0350] 2) the ARTS-1 cDNA (WT) coding region,
[0351] 3) the ARTS-1 cDNA encoding a H353P mutation, [0352] 4) the
ARTS-1 cDNA encoding a E354V mutation, [0353] 5) the ARTS-1 cDNA
encoding a H353P and E354V double mutation, and [0354] 6) the
ARTS-1 cDNA encoding a H357V mutation.
EXAMPLE 11
TNFR1 Ectodomain Shedding Assay
[0355] The amount of TNFR1 ectodomain shedding occurring in each of
the stably transfected cell lines described in Example 9 was
assessed. The levels of sTNFR1 ectodomain in cell culture
supernatants from these cell lines were assayed by a commercially
available sandwich-enzyme-linked immunosorbent assay (ELISA)
technique (R & D Systems) with a lower limit of detection of
7.8 pg/ml. The protocol used was according to the manufacturer's
instructions. The results of this assay are depicted in FIG. 8 as
the mean of 5 independent experiments, with accompanying SEM
(standard error of the mean) indicated above the bar. As can be
seen in FIG. 8, the cell lines with increased ARTS-1 protein
expression also showed a significant increase in the amount of
sTNFR1 present in cell culture supernatants as compared to cells
transfected with the empty pTarget vector (Mock) or nontransfected
(WT) controls. This change in sTNFR1 concentration represents an
approximately 200% increase in sTNFR1 shedding resulting from
overexpression of the ARTS-1 protein.
[0356] Conversely, cell lines showing decreased ARTS-1 protein
expression showed significantly decreased levels of sTNFR1 in cell
culture supernatants as compared to cells transfected with the
empty pTarget vector, as shown in FIG. 8. This change in
concentration represents an approximately 80% decrease in sTNFR1
shedding resulting from expression of an ARTS-1 anti-sense
transcript encompassing the putative translation start site.
[0357] A similar experiment analyzing the ability of ARTS-1
overexpression to potentiate the cleavage and shedding of TNFR
ectodomain from the surface of NCI-H292 cells in response to PMA
stimulation using these same cell lines is shown in FIG. 9. Cell
lines overexpressing full length ARTS-1 mRNA were stimulated with
0.1 |iM phorbol 12-myristate 13-acetate (PMA), which has previously
been shown to upregulate sTNFR1 shedding in NCI-H292 cells (Levine
et al., Am. J. Respir. Cell Mol Biol, 14:254-261 [1996]). As shown
in FIG. 9, the cell line containing only the empty pTarget vector
showed only a modest increase in sTNFR1 shedding following 24 hours
of PMA treatment, increasing from approximately 300 pg/ml to
415.3.+-.4.5 pg/ml. However, the cell line overexpressing the
ARTS-1 cDNA showed a more dramatic increase in sTNFR1 shedding
following 24 hours of PMA treatment, increasing from 485.+-.16.9
pg/ml to 914.2.+-.9.5 pg/ml
[0358] This assay was further used to measure the sTNFR1 ectodomain
shedding activity of ARTS-1 peptidase/protease catalytic mutants.
The construction of these mutations (and resulting mutant cell
lines) are described in Example 10. The ability of theses mutants
to regulate sTNFR1 shedding was ascertained by measuring the
concentration of sTNFR1 in the cell culture supernatant as
described above in this Example. These results are depicted in FIG.
10 as the mean of five independent experiments, with accompanying
SEM (standard error of the mean).
[0359] As shown in FIG. 10, the cell line overexpressing the ARTS-1
cDNA (ARTS-1) showed a significantly elevated level of sTNFR1 in
the culture supernatant compared to control cell lines containing
no integrated DNA (WT) or containing the empty pTarget vector
(MOCK). Unexpectedly, each of the cell lines containing mutant
forms of the ARTS-1 polypeptide showed elevated levels of sTNFR1
compared to the control lines (i.e., WT and MOCK). This experiment
demonstrates the unexpected property where the peptidase/protease
activity of the ARTS-1 polypeptide is not required for the sTNFR1
shedding regulatory activity of ARTS-1 polypeptide.
[0360] Although not described here, IL-1 and IL-6 receptor
ectodomain shedding can also be measured by ELISA-based assays
using commercially available kits that measure soluble forms of the
IL-1 and IL-6 receptors (R & D Systems, Catalog numbers DRIBOO
and DR600, respectively). These ELISA-based assays measure the
concentration of sIL-1RII and sIL-6R, both with a lower limit of
detection of 31 pg/ml.
EXAMPLE 12
Effect of ARTS-1 Expression on Membrane-Associated TNFR1
[0361] The degree of TNFR1 ectodomain shedding as a function of
ARTS-1 protein expression was also indirectly assessed by
determining the relative amounts of membrane-bound TNFR1 fragment
in each of the stably transfected cell lines described in Example 9
using Western immunoblotting.
[0362] Crude membrane fractions from the stably transfected
NCI-H292 cells described in Example 9 were prepared and protein
concentrations were quantitated, as described above. Samples from
these membrane-derived protein preparations were resolved by
SDS-PAGE and analyzed by Western immunoblotting as described above
in Example 5 using a murine anti-human TNFR1 monoclonal primary
antibody which detected the membrane fragment of the receptor (R
& D System). This Western blot is shown in FIG. 11. Two
independent strains of each cell line were analyzed in
parallel.
[0363] As shown in FIG. 11, cell lines over-expressing ARTS-1
(ARTS-1) demonstrated a decrease in membrane-associated TNFR1
relative to non-transfected (WT) or control transfected (Mock) cell
lines, consistent with an increase in constitutive TNFR1 ectodomain
shedding. Conversely, cell lines expressing anti-sense ARTS-1 IMRNA
(AS) demonstrated an increase in membrane-associated TNFR1 relative
to non-transfected (WT) or control transfected (Mock) cell lines,
consistent with a reduction in constitutive TNFR1 ectodomain
shedding.
EXAMPLE 13
ARTS-1/TNFR1 in vivo Co-Immunoprecipitation Assays
[0364] The ability of ARTS-1 to directly interact with TNFR1
ectodomain was assessed in vivo using a co-immunoprecipitation
assay. This assay utilized anti-ARTS-1 antiserum and monoclonal
anti-TNFR1 antibodies. The immunoprecipitated proteins were
visualized by Western immunoblotting.
[0365] Crude membrane fractions from cultured NCI-H292 cells were
prepared and protein concentrations were quantitated as described
above. From these membrane-derived protein preparations, 200 .mu.g
samples were incubated with 20 jag of murine anti-human TNFR1
monoclonal antibody (R & D System) or 1 ml of anti-ARTS-1
antiserum overnight at 4.degree. C. in immunoprecipitation buffer
(50 mM Tris-HCl, 120 mM NaCl, 0.1% Triton X-100 and COMPLETE.TM.
protease inhibitor (Roche), pH 7.2). Following the incubation, the
resulting antibody complexes were immunoprecipitated by binding to
200 ul of immobilized protein A/G beads (Pierce) for 2 hours at
room temperature. Proteins contained in the samples were then
resolved by SDS-PAGE and analyzed by Western immunoblotting as
described above.
[0366] Two different combinations of precipitation and
immunoblotting antibody were used. The results of these
immunoprecipitation experiments are shown in FIG. 12. In one
experiment (FIG. 12, top panel), the anti-TNFR1 antibody was used
in the immunoprecipitation (indicated as "IP" in the Figure), and
the anti-ARTS-1 antiserum was used as the primary antibody in the
immunoblotting (indicated as "IB" in the Figure). In a second
experiment (FIG. 12, bottom panel), the antibodies were reversed,
where the anti-ARTS-1 antiserum was used in the
immunoprecipitation, while the anti-TNFR1 antibody was used as the
primary antibody in the immunoblotting. Also in these experiments,
anti-ARTS-1 pre-immune serum (written "PI") and a purified murine
IgG1 isotype (written "IgG1") were used as negative controls.
[0367] As shown in the top panel of FIG. 12, immunoprecipitation of
the NCI-H292 cell membrane proteins with an anti-TNFR1 monoclonal
antibody resulted in the coprecipitation of the 100 kDa ARTS-1
species and, conversely, as seen in the bottom panel,
immunoprecipitation with anti-ARTS-1 antiserum coprecipitated the
55 kDa TNFR1. These results indicate an in vivo protein-protein
interaction between ARTS-1 and TNFR1 proteins.
[0368] Similar immunoprecipitation experiments were also performed
using the stably-transfected NCI-H292 cell lines described in
Example 9. In these experiments, the anti-TNFR1 antibody was used
to immunoprecipitate protein from the various membrane protein
fractions, and the resulting immunoprecipitate was examined by
Western immunoblotting using anti-ARTS-1 antiserum as the primary
antibody. As shown in FIG. 13, immunoprecipitation using an
anti-TNFR1 monoclonal antibody of cell membrane protein derived
from the anti-sense ARTS-1 cell line (AS) showed decreased amounts
of ARTS-1 protein as compared to control-transfected (Mock) or
non-transfected (WT) cells, consistent with decreased ARTS-1
protein expression in anti-sense ARTS-1 cells. No increase in
ARTS-1 protein levels relative to control cell lines was detected
following immunoprecipitation of ARTS-1 overexpressing cell lines
with an anti-TNFR1 monoclonal antibody, which likely reflects
increased TNFR1 shedding related to ARTS-1 over-expression.
EXAMPLE 14
Methods for Drug Screening
[0369] The present invention provides two examples of methods for
drug screening. These methods identify test compounds which are
able to regulate ARTS-1 protein expression in a tissue, or regulate
sTNFR1 shedding in a cell culture system.
[0370] In the first method, cultured human NCI-H292 pulmonary
mucoepidermoid carcinoma cells were exposed to a test compound, in
this case, 4b-phorbol 12-myristate 13-acetate (PMA) at a
concentration of 0.1 uM. Cells were harvested and the membrane
protein fraction isolated at intervals from prior to exposure to
the test compound (Time=0) to 24 hours following exposure to the
compound. The protein samples were analyzed in a Western immunoblot
using the anti-ARTS-1 antiserum at the primary detection antibody,
as described in Examples 4 and 5. The protein fractions were
analyzed in duplicate, and the relative densitometry units for each
lane are shown beneath the columns. Each immunoblot is
representative of 3 independent experiments. The results of this
screening are shown in FIG. 14, Panel A. As can be seen in the
Figure, treatment of the cultured cells resulted in an increase in
ARTS-1 protein expression over the course of 24 hours. Thus, this
screening method identified a compound that is a candidate for
further drug development.
[0371] Results from a second screening method are illustrated in
FIG. 14, Panel B. In this screening method, cultured human NCI-H292
pulmonary mucoepidermoid carcinoma cells were again exposed to a
test compound, PMA, at a concentration of 0.1 .mu.M. Culture medium
supernatant was collected from the cultures at intervals between 0
and 24 hours post treatment. The samples were analyzed by the TNFR1
ectodomain shedding assay using an ELISA as described in Example
11. In this assay, anti-sTNFR1 antibody was used to quantitate the
concentrations of sTNFR1 in the cell culture supernatants. The
results of this ELISA screening are shown in FIG. 14, Panel B. As
can be seen in this Figure, treatment of the cultured cells
resulted in an increase in sTNFR1 shedding over the course of 24
hours compared to a control culture (n=5,*P<0.05). Thus, this
screening method also identified a compound that is a candidate for
further drug development.
EXAMPLE 15
Tumor Necrosis Factor Bioactivity (Cytotoxicity) Assay
[0372] The bioactivity of tumor necrosis factor (TNF) is measured
by a cell cytotoxicity assay utilizing the WEHI 164 clone-13 mouse
fibrosarcoma cell line (ATCC, CRL 1751). This cell line has been
shown to be highly sensitive to the cytotoxic effects of human
tumor necrosis factor at concentrations as low as 0.1 pg/ml TNF
following pretreatment with actinomycin D (Eskandari et al.,
Immunol. Invest., 19:69-79 [1990]). In this assay, WEHI 164 cells
are seeded into 96-well microtiter plates at a density of 40,000
cells per well in RPMI-1640 supplemented with penicillin (100
units/ml) (Advanced Biotechnologies), streptomycin (100 ng/ml)
(Advanced Biotechnologies), L-glutamine (2 mM) (Advanced
Biotechnologies), 10% heat-inactivated fetal-calf serum (Inovar),
and actinomycin-D (0.5 fig/ml) (Calbiochem). Test samples (serum,
plasma or any other body fluid) to be tested for TNF activity are
diluted 6-fold in RPMI-1640 culture medium (Advanced
Biotechnologies), heat-inactivated at 56.degree. C. for 30 minutes,
and sterile filtered. A volume of 50 f l of the diluted,
heat-inactivated sample is added to the microtiter plate wells
containing the WEHI cells. Duplicate test samples are incubated in
the presence of polyclonal rabbit anti-human TNF antiserum
(Genzyme) or control rabbit serum (Life Technologies). Cells are
incubated for 20 hours, after which time 20 ul of a 5 mg/ml stock
solution of the tetrazolium salt
3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (also
called MTT or Thiazolyl blue; Sigma, Catalog Number M2128) in
phosphate buffered saline (PBS) is added to each well. Following an
additional 4 hours incubation, the plates are spun at 950.times.g
for 10 minutes, the media from each well is aspirated, and 100 ul
of 0.04N HCI/2-propanol is added to each well, and the plates are
incubated overnight in the dark at room temperature. In the
morning, an additional 100 ul of 0.04N HCU/2-propanol is added to
each well and incubated for two hours in the dark at room
temperature. Cell survival is then measured colorimetrically using
a microplate reader with a 570 nm wavelength test filter and a 630
nm reference filter. Cell survival in each test well is determined
as a percentage of the optical density of control wells.
TNF-specific killing is defined as the difference in cell killing
with or without the anti-TNF antisenim and is compared with
standard curves produced with recombinant human TNF-a (R & D
Systems).
EXAMPLE 15
Statistical Analysis
[0373] Data are presented as mean+standard error of the mean.
Comparisons were made utilizing a paired two-tailed student's T
test with a Bonneferoni correction for multiple comparisons. A P
value<0.005 was considered significant.
[0374] These experiments demonstrate that expression of ARTS-1
protein directly correlates with and is necessary for soluble TNFR1
ectodomain shedding. Furthermore, ARTS-1 represents the first gene
and protein which have been identified which have the ability
regulate TNFR1 ectodomain shedding. In addition, the present
invention provides the nucleic acid and amino acid sequences of
this gene and protein.
[0375] Furthermore, the compositions and methods of the present
invention provide therapeutic applications for the treatment of a
wide variety of disorders of the immune system which arise as a
result of improper TNF activity.
[0376] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described methods and
compositions of the present 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 described modes for carrying out the invention which are
obvious to those skilled in molecular biology and immunology and/or
related fields are intended to be within the scope of the present
invention.
Sequence CWU 1
1
11 1 4845 DNA Homo sapiens 1 gcacgagagc taggccggcg gcagtggtgg
tggcggcggc gcaagggtga gggcggcccc 60 agaaccccag gtaggtagag
caagaagatg gtgtttctgc ccctcaaatg gtcccttgca 120 accatgtcat
ttctactttc ctcactgttg gctctcttaa ctgtgtccac tccttcatgg 180
tgtcagagca ctgaagcatc tccaaaacgt agtgatggga caccatttcc ttggaataaa
240 atacgacttc ctgagtacgt catcccagtt cattatgatc tcttgatcca
tgcaaacctt 300 accacgctga ccttctgggg aaccacgaaa gtagaaatca
cagccagtca gcccaccagc 360 accatcatcc tgcatagtca ccacctgcag
atatctaggg ccaccctcag gaagggagct 420 ggagagaggc tatcggaaga
acccctgcag gtcctggaac acccccgtca ggagcaaatt 480 gcactgctgg
ctcccgagcc cctccttgtc gggctcccgt acacagttgt cattcactat 540
gctggcaatc tttcggagac tttccacgga ttttacaaaa gcacctacag aaccaaggaa
600 ggggaactga ggatactagc atcaacacaa tttgaaccca ctgcagctag
aatggccttt 660 ccctgctttg atgaacctgc cttcaaagca agtttctcaa
tcaaaattag aagagagcca 720 aggcacctag ccatctccaa tatgccattg
gtgaaatctg tgactgttgc tgaaggactc 780 atagaagacc attttgatgt
cactgtgaag atgagcacct atctggtggc cttcatcatt 840 tcagattttg
agtctgtcag caagataacc aagagtggag tcaaggtttc tgtttatgct 900
gtgccagaca agataaatca agcagattat gcactggatg ctgcggtgac tcttctagaa
960 ttttatgagg attatttcag cataccgtat cccctaccca aacaagatct
tgctgctatt 1020 cccgactttc agtctggtgc tatggaaaac tggggactga
caacatatag agaatctgct 1080 ctgttgtttg atgcagaaaa gtcttctgca
tcaagtaagc ttggcatcac aatgactgtg 1140 gcccatgaac tggctcacca
gtggtttggg aacctggtca ctatggaatg gtggaatgat 1200 ctttggctaa
atgaaggatt tgccaaattt atggagtttg tgtctgtcag tgtgacccat 1260
cctgaactga aagttggaga ttatttcttt ggcaaatgtt ttgacgcaat ggaggtagat
1320 gctttaaatt cctcacaccc tgtgtctaca cctgtggaaa atcctgctca
gatccgggag 1380 atgtttgatg atgtttctta tgataaggga gcttgtattc
tgaatatgct aagggagtat 1440 cttagtgctg acgcatttaa aagtggtatt
gtacagtatc tccagaagca tagctataaa 1500 aatacaaaaa acgaggacct
gtgggatagt atggcaagta tttgccctac agatggtgta 1560 aaagggatgg
atggcttttg ctctagaagt caacattcat cttcatcctc acattggcat 1620
caggaagggg tggatgtgaa aaccatgatg aacacttgga cactgcagaa gggttttccc
1680 ctaataacca tcacagtgag ggggaggaat gtacacatga agcaagagca
ctacatgaag 1740 ggctctgacg gcgccccgga cactgggtac ctgtggcatg
ttccattgac attcatcacc 1800 agcaaatccg acatggtcca tcgatttttg
ctaaaaacaa aaacagatgt gctcatcctc 1860 ccagaagagg tggaatggat
caaatttaat gtgggcatga atggctatta cattgtgcat 1920 tacgaggatg
atggatggga ctctttgact ggccttttaa aaggaacaca cacagcagtc 1980
agcagtaatg atcgggcgag tctcattaac aatgcatttc agctcgtcag cattgggaag
2040 ctgtccattg aaaaggcctt ggatttatcc ctgtacttga aacatgaaac
tgaaattatg 2100 cccgtgtttc aaggtttgaa tgagctgatt cctatgtata
agttaatgga gaaaagagat 2160 atgaatgaag tggaaactca attcaaggcc
ttcctcatca ggctgctaag ggacctcatt 2220 gataagcaga catggacaga
cgagggctca gtctcagagc gaatgctgcg gagtcaacta 2280 ctactcctcg
cctgtgtgca caactatcag ccgtgcgtac agagggcaga aggctatttc 2340
agaaagtgga aggaatccaa tggaaacttg agcctgcctg tcgacgtgac cttggcagtg
2400 tttgctgtgg gggcccagag cacagaaggc tgggattttc tttatagtaa
atatcagttt 2460 tctttgtcca gtactgagaa aagccaaatt gaatttgccc
tctgcagaac ccaaaataag 2520 gaaaagcttc aatggctact agatgaaagc
tttaagggag ataaaataaa aactcaggag 2580 tttccacaaa ttcttacact
cattggcagg aacccagtag gatacccact ggcctggcaa 2640 tttctgagga
aaaactggaa caaacttgta caaaagtttg aacttggctc atcttccata 2700
gcccacatgg taatgggtac aacaaatcaa ttctccacaa gaacacggct tgaagaggta
2760 aaaggattct tcagctcttt gaaagaaaat ggttctcagc tccgttgtgt
ccaacagaca 2820 attgaaacca ttgaagaaaa catcggttgg atggataaga
attttgataa aatcagagtg 2880 tggctgcaaa gtgaaaagct tgaacgtatg
taaaaattcc tcccttgcca ggttcctgtt 2940 atctctaatc accaacattt
tgttgagtgt attttcaaac tagagatggc tgttttggct 3000 ccaactggag
atactttttt cccttcaact cattttttga ctatccctgt gaaaagaata 3060
gctgttagtt tttcatgaat gggctatcgc taccatgtgt tttgttcatc acaggtgttg
3120 ccctgcaacg taaacccaag tgttgggttc cctgccacag aagaataaag
taccttattc 3180 ttctcatttt atagtttatg cttaagcacc cgtgtccaaa
accctgtacc ccatgtttat 3240 cattcataaa ctgtttcatc agtctcctcg
aaagactctg aatagtcgac tactgaacaa 3300 tgaacacctg gatctgagac
taagccggac gatgactggg ttaaagctct cccggctcac 3360 ccctccagac
ccgctgccca tccctcttcc ttgctccatg cccaggggct gacttgtaaa 3420
ggccaagtca tcaagctttc ttgccctttg gatgttggtc agtggggagc cggagagctg
3480 gagctggggt cggaggaggt agtaggtgga ggtgttcttc cctgattccc
ttgcgggatg 3540 cctcgggctg gcctcccctg agggttttag ctccgagagg
ggaccctctt ttccacacag 3600 ccttctccac ctctggattt tggtaactgc
tccctcctca tcccttcagg attagtggcc 3660 tcagtgggag tctggctttt
actagtcctg gcggacttgt ggtttctaca taatgtgctc 3720 gcacttttgc
aaaaaatctt ctttttatag aaccctcctc agataattct gagtgtcatc 3780
tatttccctg actggtacag tatctcttct gaaaaagcag agtgcattca agtctgtagg
3840 aaaacccttt tcttagggag gtgatttttt ttctctctct gcttcttatt
tggcctactt 3900 tacaatttct aactaactag ttattggcat ttactgacag
taaattattg cagtcaccaa 3960 taaatgatag tacattgtga aacaaaatat
ttgctcatat tagcaaatag gacattcttt 4020 ggctttgaag tctttctttt
gtgaagactt cacacacggt tgcttcagca cacagttgct 4080 gctcaggttt
tatgtataga tgataataat agaaagcaca gtttactaac atggtaaacc 4140
aacggagttc aagtcaagtc agttaatacc ctaagaatta gattttattt cttattctga
4200 aaacttgcta cacagggact tatctaaccc atagtgtgct ctgttgctga
cttgattcaa 4260 gttgcagcgt gttttgcgct gactctaagg tgcggaaatc
ctcacacctg gcaaaggaga 4320 attcaaactg aactttttga atataaggca
aaaacttcaa gataagggaa tatgattgat 4380 gattggtacg aaaaatgtca
aaatgtgttc ccctaataca cgacaaaata gagtgacttc 4440 tggacataaa
tctgccattt attaaaccat tcactacaac aaataaatag gtataaaagt 4500
ggaattggaa tttttatact tatttgttgt agtgaatggt ttaataaaaa tagaaatcac
4560 tggtaatttc caccccaaac taaactattt cccttctttt aaaaaaatac
acaaccaaga 4620 ttttaatgta aaatattttg ctttaattgt attttatgcc
ttgattaatg aaacatggaa 4680 atattgattt tcagttttgg tcacctgagg
aacctatctt tgtttgcttt tggaaaagcc 4740 cattttctaa acagatacaa
tattgccaca acaatgtgca gaaacctttt tgataataaa 4800 aaattgttct
ttgcctctaa aaaaaaaaaa aaaaaaaaaa aaaaa 4845 2 941 PRT Homo sapiens
2 Met Val Phe Leu Pro Leu Lys Trp Ser Leu Ala Thr Met Ser Phe Leu 1
5 10 15 Leu Ser Ser Leu Leu Ala Leu Leu Thr Val Ser Thr Pro Ser Trp
Cys 20 25 30 Gln Ser Thr Glu Ala Ser Pro Lys Arg Ser Asp Gly Thr
Pro Phe Pro 35 40 45 Trp Asn Lys Ile Arg Leu Pro Glu Tyr Val Ile
Pro Val His Tyr Asp 50 55 60 Leu Leu Ile His Ala Asn Leu Thr Thr
Leu Thr Phe Trp Gly Thr Thr 65 70 75 80 Lys Val Glu Ile Thr Ala Ser
Gln Pro Thr Ser Thr Ile Ile Leu His 85 90 95 Ser His His Leu Gln
Ile Ser Arg Ala Thr Leu Arg Lys Gly Ala Gly 100 105 110 Glu Arg Leu
Ser Glu Glu Pro Leu Gln Val Leu Glu His Pro Arg Gln 115 120 125 Glu
Gln Ile Ala Leu Leu Ala Pro Glu Pro Leu Leu Val Gly Leu Pro 130 135
140 Tyr Thr Val Val Ile His Tyr Ala Gly Asn Leu Ser Glu Thr Phe His
145 150 155 160 Gly Phe Tyr Lys Ser Thr Tyr Arg Thr Lys Glu Gly Glu
Leu Arg Ile 165 170 175 Leu Ala Ser Thr Gln Phe Glu Pro Thr Ala Ala
Arg Met Ala Phe Pro 180 185 190 Cys Phe Asp Glu Pro Ala Phe Lys Ala
Ser Phe Ser Ile Lys Ile Arg 195 200 205 Arg Glu Pro Arg His Leu Ala
Ile Ser Asn Met Pro Leu Val Lys Ser 210 215 220 Val Thr Val Ala Glu
Gly Leu Ile Glu Asp His Phe Asp Val Thr Val 225 230 235 240 Lys Met
Ser Thr Tyr Leu Val Ala Phe Ile Ile Ser Asp Phe Glu Ser 245 250 255
Val Ser Lys Ile Thr Lys Ser Gly Val Lys Val Ser Val Tyr Ala Val 260
265 270 Pro Asp Lys Ile Asn Gln Ala Asp Tyr Ala Leu Asp Ala Ala Val
Thr 275 280 285 Leu Leu Glu Phe Tyr Glu Asp Tyr Phe Ser Ile Pro Tyr
Pro Leu Pro 290 295 300 Lys Gln Asp Leu Ala Ala Ile Pro Asp Phe Gln
Ser Gly Ala Met Glu 305 310 315 320 Asn Trp Gly Leu Thr Thr Tyr Arg
Glu Ser Ala Leu Leu Phe Asp Ala 325 330 335 Glu Lys Ser Ser Ala Ser
Ser Lys Leu Gly Ile Thr Met Thr Val Ala 340 345 350 His Glu Leu Ala
His Gln Trp Phe Gly Asn Leu Val Thr Met Glu Trp 355 360 365 Trp Asn
Asp Leu Trp Leu Asn Glu Gly Phe Ala Lys Phe Met Glu Phe 370 375 380
Val Ser Val Ser Val Thr His Pro Glu Leu Lys Val Gly Asp Tyr Phe 385
390 395 400 Phe Gly Lys Cys Phe Asp Ala Met Glu Val Asp Ala Leu Asn
Ser Ser 405 410 415 His Pro Val Ser Thr Pro Val Glu Asn Pro Ala Gln
Ile Arg Glu Met 420 425 430 Phe Asp Asp Val Ser Tyr Asp Lys Gly Ala
Cys Ile Leu Asn Met Leu 435 440 445 Arg Glu Tyr Leu Ser Ala Asp Ala
Phe Lys Ser Gly Ile Val Gln Tyr 450 455 460 Leu Gln Lys His Ser Tyr
Lys Asn Thr Lys Asn Glu Asp Leu Trp Asp 465 470 475 480 Ser Met Ala
Ser Ile Cys Pro Thr Asp Gly Val Lys Gly Met Asp Gly 485 490 495 Phe
Cys Ser Arg Ser Gln His Ser Ser Ser Ser Ser His Trp His Gln 500 505
510 Glu Gly Val Asp Val Lys Thr Met Met Asn Thr Trp Thr Leu Gln Lys
515 520 525 Gly Phe Pro Leu Ile Thr Ile Thr Val Arg Gly Arg Asn Val
His Met 530 535 540 Lys Gln Glu His Tyr Met Lys Gly Ser Asp Gly Ala
Pro Asp Thr Gly 545 550 555 560 Tyr Leu Trp His Val Pro Leu Thr Phe
Ile Thr Ser Lys Ser Asp Met 565 570 575 Val His Arg Phe Leu Leu Lys
Thr Lys Thr Asp Val Leu Ile Leu Pro 580 585 590 Glu Glu Val Glu Trp
Ile Lys Phe Asn Val Gly Met Asn Gly Tyr Tyr 595 600 605 Ile Val His
Tyr Glu Asp Asp Gly Trp Asp Ser Leu Thr Gly Leu Leu 610 615 620 Lys
Gly Thr His Thr Ala Val Ser Ser Asn Asp Arg Ala Ser Leu Ile 625 630
635 640 Asn Asn Ala Phe Gln Leu Val Ser Ile Gly Lys Leu Ser Ile Glu
Lys 645 650 655 Ala Leu Asp Leu Ser Leu Tyr Leu Lys His Glu Thr Glu
Ile Met Pro 660 665 670 Val Phe Gln Gly Leu Asn Glu Leu Ile Pro Met
Tyr Lys Leu Met Glu 675 680 685 Lys Arg Asp Met Asn Glu Val Glu Thr
Gln Phe Lys Ala Phe Leu Ile 690 695 700 Arg Leu Leu Arg Asp Leu Ile
Asp Lys Gln Thr Trp Thr Asp Glu Gly 705 710 715 720 Ser Val Ser Glu
Arg Met Leu Arg Ser Gln Leu Leu Leu Leu Ala Cys 725 730 735 Val His
Asn Tyr Gln Pro Cys Val Gln Arg Ala Glu Gly Tyr Phe Arg 740 745 750
Lys Trp Lys Glu Ser Asn Gly Asn Leu Ser Leu Pro Val Asp Val Thr 755
760 765 Leu Ala Val Phe Ala Val Gly Ala Gln Ser Thr Glu Gly Trp Asp
Phe 770 775 780 Leu Tyr Ser Lys Tyr Gln Phe Ser Leu Ser Ser Thr Glu
Lys Ser Gln 785 790 795 800 Ile Glu Phe Ala Leu Cys Arg Thr Gln Asn
Lys Glu Lys Leu Gln Trp 805 810 815 Leu Leu Asp Glu Ser Phe Lys Gly
Asp Lys Ile Lys Thr Gln Glu Phe 820 825 830 Pro Gln Ile Leu Thr Leu
Ile Gly Arg Asn Pro Val Gly Tyr Pro Leu 835 840 845 Ala Trp Gln Phe
Leu Arg Lys Asn Trp Asn Lys Leu Val Gln Lys Phe 850 855 860 Glu Leu
Gly Ser Ser Ser Ile Ala His Met Val Met Gly Thr Thr Asn 865 870 875
880 Gln Phe Ser Thr Arg Thr Arg Leu Glu Glu Val Lys Gly Phe Phe Ser
885 890 895 Ser Leu Lys Glu Asn Gly Ser Gln Leu Arg Cys Val Gln Gln
Thr Ile 900 905 910 Glu Thr Ile Glu Glu Asn Ile Gly Trp Met Asp Lys
Asn Phe Asp Lys 915 920 925 Ile Arg Val Trp Leu Gln Ser Glu Lys Leu
Glu Arg Met 930 935 940 3 24 DNA Artificial Synthetic 3 ataaccatca
cagtgagggg gagg 24 4 24 DNA Artificial Synthetic 4 tagttgactc
cgcagcattc gctc 24 5 26 DNA Artificial Synthetic 5 gcaagaagat
ggtgtttctg cccctc 26 6 24 DNA Artificial Synthetic 6 ttacatacgt
tcaagctttt cact 24 7 17 PRT Artificial Synthetic 7 Arg Gly Arg Asn
Val His Met Lys Gln Glu His Tyr Met Lys Gly Ser 1 5 10 15 Asp 8 12
PRT Artificial Synthetic 8 Thr Val Ala His Glu Leu Ala His Gln Trp
Phe Gly 1 5 10 9 8 PRT Artificial Synthetic 9 Leu Trp Leu Asn Glu
Gly Phe Ala 1 5 10 24 PRT Artificial Synthetic MISC_FEATURE
(3)..(4) XAA at these positions can be any amino acid. 10 His Glu
Xaa Xaa His Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr 1 5 10 15
Tyr Tyr Tyr Tyr Tyr Tyr Tyr Glu 20 11 24 PRT Artificial
Artificial/Unknown 11 His Glu Leu Ala His Tyr Tyr Tyr Tyr Tyr Tyr
Tyr Tyr Tyr Tyr Tyr 1 5 10 15 Tyr Tyr Tyr Tyr Tyr Tyr Tyr Glu
20
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