U.S. patent application number 11/087991 was filed with the patent office on 2005-08-25 for novel interleukin-1 receptor intracellular ligand proteins and inhibitors of ligand binding.
This patent application is currently assigned to Genetics Institute, Inc. LLC. Invention is credited to Graham, James, Lin, Lih-Ling.
Application Number | 20050186615 11/087991 |
Document ID | / |
Family ID | 23937751 |
Filed Date | 2005-08-25 |
United States Patent
Application |
20050186615 |
Kind Code |
A1 |
Lin, Lih-Ling ; et
al. |
August 25, 2005 |
Novel interleukin-1 receptor intracellular ligand proteins and
inhibitors of ligand binding
Abstract
Novel IL-1-R intracellular ligand proteins are disclosed.
Polynucleotides encoding the IL-1-R intracellular ligand protein
are also disclosed, along with vectors, host cells, and methods of
making the IL-1-R intracellular ligand protein. Pharmaceutical
compositions containing the IL-1-R intracellular ligand protein,
methods of treating inflammatory conditions, and methods of
inhibiting IL-1-R intracellular domain binding are also disclosed.
Methods of identifying inhibitors of IL-1-R intracellular domain
binding and inhibitors identified by such methods are also
disclosed.
Inventors: |
Lin, Lih-Ling; (Concord,
MA) ; Graham, James; (Somerville, MA) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY
AND POPEO, P.C.
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Assignee: |
Genetics Institute, Inc.
LLC
|
Family ID: |
23937751 |
Appl. No.: |
11/087991 |
Filed: |
March 23, 2005 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11087991 |
Mar 23, 2005 |
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09884319 |
Jun 18, 2001 |
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6899878 |
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09884319 |
Jun 18, 2001 |
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09083516 |
May 22, 1998 |
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6300086 |
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09083516 |
May 22, 1998 |
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08726525 |
Oct 7, 1996 |
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5789181 |
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08726525 |
Oct 7, 1996 |
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08487942 |
Jun 7, 1995 |
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5817476 |
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Current U.S.
Class: |
435/6.16 ;
435/320.1; 435/325; 435/69.1; 514/1.2; 514/12.2; 530/350;
536/23.5 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 29/00 20180101; C07K 14/47 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 514/012; 530/350; 536/023.5 |
International
Class: |
C12Q 001/68; G01N
033/567; C07H 021/04; A61K 038/17; C07K 014/715 |
Claims
What is claimed is:
1. A composition comprising an isolated polynucleotide encoding a
protein having IL-1-R intracellular ligand protein activity.
2. The composition of claim 1 wherein said polynucleotide is
selected from the group consisting of: (a) a polynucleotide
comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide 2
to nucleotide 529; (b) a polynucleotide comprising a fragment of
the nucleotide sequence of SEQ ID NO:1, which encodes a protein
having IL-1-R intracellular ligand protein activity; (c) a
polynucleotide encoding an IL-1-R intracellular ligand protein
comprising the amino acid sequence of SEQ ID NO:2; (d) a
polynucleotide encoding an IL-1-R intracellular ligand protein
comprising a fragment of the amino acid sequence of SEQ ID NO:2 and
having IL-1-R intracellular ligand protein activity; and (e) a
polynucleotide capable of hybridizing under stringent conditions to
any one of the poly nucleotides specified in (a)-(d), which encodes
a protein having IL-1-R intracellular ligand protein activity.
3. The composition of claim 1 wherein said polynucleotide sequence
is selected from the group consisting of: (a) a polynucleotide
comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 2
to nucleotide 961; (b) a polynucleotide comprising a fragment of
the nucleotide sequence of SEQ ID NO:3, which encodes a protein
having IL-1-R intracellular ligand protein activity; (c) a
polynucleotide encoding an IL-1-R intracellular ligand protein
comprising the amino acid sequence of SEQ ID NO:4; (d) a
polynucleotide encoding an IL-1-R intracellular ligand protein
comprising a fragment of the amino acid sequence of SEQ ID NO:4 and
having IL-1-R intracellular ligand protein activity; and (e) a
polynucleotide capable of hybridizing under stringent conditions to
any one of the polynucleotides specified in (a)-(d), which encodes
a protein having IL-1-R intracellular ligand protein activity.
4. The composition of claim 1 wherein said polynucleotide is
selected from the group consisting of: (a) a polynucleotide
comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 2
to nucleotide 754; (b) a polynucleotide comprising a fragment of
the nucleotide sequence of SEQ ID NO:5, which encodes a protein
having IL-1-R intracellular ligand protein activity; (c) a
polynucleotide encoding an IL-1-R intracellular ligand protein
comprising the amino acid sequence of SEQ ID NO:6; (d) a
polynucleotide encoding an IL-1-R intracellular ligand protein
comprising a fragment of the amino acid sequence of SEQ ID NO:6 and
having IL-1-R intracellular ligand protein activity; and (e) a
polynucleotide capable of hybridizing under stringent conditions to
any one of the polynucleotides specified in (a)-(d), which encodes
a protein having IL-1-R intracellular ligand protein activity.
5. A composition comprising a protein having IL-1-R intracellular
ligand protein activity.
6. The composition of claim 5 wherein said protein comprises an
amino acid sequence selected from the group consisting of: (a) the
amino acid sequence of SEQ ID NO:2and (b) fragments of the amino
acid sequence of SEQ ID NO:2; said protein being substantially free
from other mammalian proteins.
7. The composition of claim 5 wherein said protein comprises an
amino acid sequence selected from the group consisting of: (a) the
amino acid sequence of SEQ ID NO:4; and (b) fragments of the amino
acid sequence of SEQ ID NO:4; said protein being substantially free
from other mammalian proteins.
8. The composition of claim 5 wherein said protein comprises an
amino acid sequence selected from the group consisting of: (a) the
amino acid sequence of SEQ ID NO:6; and (b) fragments of the amino
acid sequence of SEQ ID NO:6; said protein being substantially free
from other mammalian proteins.
9. A composition of claim 1 wherein said polynucleotide is operably
linked to an expression control sequence.
10. A host cell transformed with a composition of claim 9.
11. The host cell of claim 10, wherein said cell is a mammalian
cell.
12. A process for producing an IL-1-R intracellular ligand protein,
which comprises: (a) growing a culture of the host cell of claim 10
in a suitable culture medium; and (b) purifying the IL-1-R
intracellular ligand protein from the culture.
13. A method of identifying an inhibitor of IL-1-R intracellular
domain binding which comprises: (a) combining an IL-1-R
intracellular domain protein with a composition of claim 5, said
combination forming a first binding mixture; (b) measuring the
amount of binding between the IL-1-R intracellular domain protein
and the IL-1-R intracellular ligand protein in the first binding
mixture; (c) combining a compound with the IL-1-R intracellular
domain protein and an IL-1-R intracellular ligand protein to form a
second binding mixture; (d) measuring the amount of binding in the
second binding mixture; and (e) comparing the amount of binding in
the first binding mixture with the amount of binding in the second
binding mixture; wherein the compound is capable of inhibiting
IL-1-R intracellular domain binding when a decrease in the amount
of binding of the second binding mixture occurs.
14. The method of claim 13 wherein said IL-1-R intracellular ligand
protein comprises an amino acid sequence selected from the group
consisting of: (a) the amino acid sequence of SEQ ID NO:2; (b)
fragments of the amino acid sequence of SEQ ID NO:2; (c) the amino
acid sequence of SEQ ID NO:4; (d) fragments of the amino acid
sequence of SEQ ID NO:4 (e) the amino acid sequence of SEQ ID NO6;
(f) fragments of the amino acid sequence of SEQ ID NO:6: (g) the
amino acid sequence of SEQ ID NO:7: and (h) fragments of the amino
acid sequence of SEQ ID NO:7.
15. A method of identifying an inhibitor of IL-1-R intracellular
domain binding which comprises: (a) transforming a cell with a
first polynucleotide encoding an IL-1-R intracellular domain
protein, a second polynucleotide encoding an IL-1-R intracellular
ligand protein, and at least one reporter gene herein the
expression of the reporter gene is regulated by the binding of the
IL-1-R intracellular ligand protein encoded by the second
polynucleotide to the IL-1-R intracellular domain protein encoded
by the first polynucleotide: (b) growing the cell in the presence
of and in the absence of a compound; and (c) comparing the degree
of expression of the reporter gene in the presence of and in the
absence of the compound; wherein the compound is capable of
inhibiting IL-1-R intracellular domain binding when a decrease in
the degree of expression of the reporter gene occurs.
16. The method of claim 15 wherein the second polynucleotide is
selected from the group consisting of: (a) a polynucleotide
comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide 2
to nucleotide 529; (b) a polynucleotide comprising a fragment of
the nucleotide sequence of SEQ ID NO:1, which encodes a protein
having IL-1-R intracellular ligand protein activity; (c) a
polynucleotide encoding an IL-1-R intracellular ligand protein
comprising the amino acid sequence of SEQ ID NO:2; (d) a
polynucleotide encoding an IL-1-R intracellular ligand protein
comprising a fragment of the amino acid sequence of SEQ ID NO:2 and
having IL-1-R intracellular ligand protein activity; (e) a
polynucleotide comprising the nucleotide sequence of SEQ ID NO:3
from nucleotide 2 to nucleotide 961. (f) a polynucleotide
comprising, a fragment of the nucleotide sequence of SEQ ID NO:3,
which encodes a protein having IL-1-R intracellular ligand protein
activity; (g) a polynucleotide encoding an IL-1-R intracellular
ligand protein comprising the amino acid sequence of SEQ ID NO:4;
(h) a polynucleotide encoding an IL-1-R intracellular ligand
protein comprising a fragment of the amino acid sequence of SEQ ID
NO:4 and having IL-1-R intracellular ligand protein activity; (i) a
polynucleotide comprising the nucleotide sequence of SEQ ID NO:5
from nucleotide 2 to nucleotide 754; (j) a polynucleotide
comprising a fragment of the nucleotide sequence of SEQ ID NO:5,
which encodes a protein having IL-1-R intracellular ligand protein
activity; (k) a polynucleotide encoding an IL-1-R intracellular
ligand protein comprising the amino acid sequence of SEQ ID NO:6;
(l) a polynucleotide encoding an IL-1-R intracellular ligand
protein comprising a fragment of the amino acid sequence of SEQ ID
NO:6 and having IL-1-R intracellular ligand protein activity; (m) a
polynucleotide encoding an IL-1-R intracellular ligand protein
comprising the amino acid sequence of SEQ ID NO:7; (n) a
polynucleotide encoding an IL-1-R intracellular ligand protein
comprising a fragment of the amino acid sequence of SEQ ID NO:7 and
having IL-1-R intracellular ligand protein activity; and (o) a
polynucleotide capable of hybridizing under stringent conditions to
any one of the polynucleotides specified in (a)-(n), which encodes
a protein having IL-1-R intracellular ligand protein activity.
17. A composition comprising an antibody which specifically reacts
with the IL-1-R intracellular ligand protein of claim 5.
18. The composition of claim 5, further comprising a
pharmaceutically acceptable carrier.
19. A method of preventing or ameliorating an inflammatory
condition which comprises administering a therapeutically effective
amount of a composition of claim 18.
20. A method of inhibiting IL-1-R intracellular domain binding
comprising administering a therapeutically effective amount of a
composition of claim 18.
21. IL-1-R intracellular ligand protein produced according to the
method of claim 12.
22. A composition comprising an inhibitor identified according to
the method of claim 15.
23. The composition of claim 22 further comprising a
pharmaceutically acceptable carrier.
24. A method of preventing or ameliorating an inflammatory
condition comprising administering to a mammalian subject a
therapeutically effective amount of the composition of claim
23.
25. A method of inhibiting IL-1-R intracellular domain binding
comprising administering to a mammalian subject a therapeutically
effective amount of the composition of claim 23.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to the field of
anti-inflammatory substances and other substances which act by
inhibiting binding to the intracellular domain of an interleukin-1
receptor (hereinafter "IL-1-R"), such as, for example, the p80,
type I IL-1 receptor. More particularly, the present invention is
directed to novel ligands which bind to the IL-1-R intracellular
domain and to inhibition or modulation of signal transduction by
this receptor.
[0002] Interleukin-1-.alpha. and interleukin-1-.beta. (herein
collectively "IL-1") are cytokines which produce a wide range of
cellular activities. IL-1 causes an inflammatory response, which
can be beneficial, such as in mounting an immune response to a
pathogen, or when overexpressed can lead to other detrimental
effects of inflammation.
[0003] The cellular effects of IL-1 are initiated by the binding of
IL-1 to its receptors (IL-1-Rs) on the surface of target cells. The
isolation of polynucleotides encoding IL-1-Rs and variant forms of
such receptors has been described in U.S. Pat. Nos. 4,968,607,
5,081,228, 5,180,812, in PCT Publication No. WO91/18982, and by
Sims et al., PNAS, 86, 8946 (1989) (disclosing the p80, type I IL-1
receptor). Processes for purification of IL-1-Rs have also been
disclosed in U.S. Pat. No. 5,296,592.
[0004] Native IL-1-Rs are characterized by distinct extracellular,
transmembrane and intracellular domains. The primary purpose of the
extracellular domain is to present a binding site for IL-1 on the
outside of the cell. When IL-1 is bound to the binding site, a
"signal" is transmitted to the inside of the cell through the
transmembrane and intracellular domains, indicating that binding
has occurred. Transmission or "transduction" of the signal to the
inside of the cell occurs by a change in conformation of the
transmembrane and/or intracellular domains of the receptor. This
signal is "received" by the binding of proteins and other molecules
to the intracellular domain of the receptor resulting in the
effects seen upon IL-1 stimulation.
[0005] While IL-1 binding by IL-1-Rs results in beneficial cellular
effects, it is often desirable to prevent or deter IL-1 binding
from causing other detrimental cellular effects. Although
substantial effort has been expended investigating inhibition of
IL-1 binding to the extracellular domain of IL-1-Rs, examination of
binding of proteins and other molecules to the intracellular domain
of IL-1-Rs has received much less attention.
[0006] However, ligands which bind to the IL-1-R intracellular
domain have yet to be identified. It would be desirable to identify
and isolate such ligands to examine their effects upon IL-1-R
signal transduction and their use as therapeutic agents for
treatment of IL-1-induced conditions. Furthermore, identification
of such ligands would provide a means for screening for inhibitors
of IL-1-R/intracellular ligand binding, which will also be useful
as anti-inflammatory agents.
SUMMARY OF THE INVENTION
[0007] Applicants have for the first time identified novel IL-1-R
intracellular ligand proteins and have isolated polynucleotides
encoding such ligands. Applicants have also identified certain
known proteins which may also bind to the intracellular domain of
IL-1-R.
[0008] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide encoding a
protein having IL-1-R intracellular ligand protein activity. In
preferred embodiments, the polynucleotide is selected from the
group consisting of:
[0009] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:1 from nucleotide 2 to nucleotide 529;
[0010] (b) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:1, which encodes a protein having IL-1-R
intracellular ligand protein activity;
[0011] (c) a polynucleotide encoding an IL-1-R intracellular ligand
protein comprising the amino acid sequence of SEQ ID NO:2;
[0012] (d) a polynucleotide encoding an IL-1-R intracellular ligand
protein comprising a fragment of the amino acid sequence of SEQ ID
NO:2 and having IL-1-R intracellular ligand protein activity;
[0013] (e) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:3 from nucleotide 2 to nucleotide 961;
[0014] (f) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:3, which encodes a protein having IL-1-R
intracellular ligand protein activity;
[0015] (g) a polynucleotide encoding an IL-1-R intracellular ligand
protein comprising the amino acid sequence of SEQ ID NO:4;
[0016] (h) a polynucleotide encoding an IL-1-R intracellular ligand
protein comprising a fragment of the amino acid sequence of SEQ ID
NO:4 and having IL-1-R intracellular ligand protein activity;
[0017] (i) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:5 from nucleotide 2 to nucleotide 754;
[0018] (j) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:5, which encodes a protein having IL-1-R
intracellular ligand protein activity;
[0019] (k) a polynucleotide encoding an IL-1-R intracellular ligand
protein comprising the amino acid sequence of SEQ ID NO:6;
[0020] (l) a polynucleotide encoding an IL-1-R intracellular ligand
protein comprising a fragment of the amino acid sequence of SEQ ID
NO:6 and having IL-1-R intracellular ligand protein activity;
and
[0021] (m) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in (a)-(l),
which encodes a protein having IL-1-R intracellular ligand protein
activity.
[0022] In certain preferred embodiments, the polynucleotide is
operably linked to an expression control sequence. The invention
also provides a host cell, including bacterial, yeast, insect and
mammalian cells, transformed with such polynucleotide
compositions.
[0023] Processes are also provided for producing an IL-1-R
intracellular ligand protein, which comprises:
[0024] (a) growing a culture of the host cell transformed with such
polynucleotide compositions in a suitable culture medium; and
[0025] (b) purifying the IL-1-R intracellular ligand protein from
the culture.
[0026] The ligand protein produced according to such methods is
also provided by the present invention.
[0027] Compositions comprising a protein having IL-1-R
intracellular ligand protein activity are also disclosed. In
preferred embodiments the protein comprises an amino acid sequence
selected from the group consisting of:
[0028] (a) the amino acid sequence of SEQ ID NO:2;
[0029] (b) fragments of the amino acid sequence of SEQ ID NO:2;
[0030] (c) the amino acid sequence of SEQ ID NO:4;
[0031] (d) fragments of the amino acid sequence of SEQ ID NO:4;
[0032] (e) the amino acid sequence of SEQ ID NO:6; and
[0033] (f) fragments of the amino acid sequence of SEQ ID NO:6; the
protein being substantially free from other mammalian proteins.
Such compositions may further comprise a pharmaceutically
acceptable carrier.
[0034] Compositions comprising an antibody which specifically
reacts with such IL-1-R intracellular ligand protein are also
provided by the present invention.
[0035] Methods are also provided for identifying an inhibitor of
IL-1-R intracellular domain binding which comprise:
[0036] (a) combining an IL-1-R intracellular domain protein with an
IL-1-R intracellular ligand protein, said combination forming a
first binding mixture;
[0037] (b) measuring the amount of binding between the IL-1-R
intracellular domain protein and the IL-1-R intracellular ligand
protein in the first binding mixture;
[0038] (c) combining a compound with the IL-1-R intracellular
domain protein and an IL-1-R intracellular ligand protein to form a
second binding mixture;
[0039] (d) measuring the amount of binding in the second binding
mixture; and
[0040] (e) comparing the amount of binding in the first binding
mixture with the amount of binding in the second binding mixture;
wherein the compound is capable of inhibiting IL-1-R intracellular
domain binding when a decrease in the amount of binding of the
second binding mixture occurs. In certain preferred embodiments the
IL-1-R intracellular ligand protein used in such method comprises
an amino acid sequence selected from the group consisting of:
[0041] (a) the amino acid sequence of SEQ ID NO:2;
[0042] (b) fragments of the amino acid sequence of SEQ ID NO:2
[0043] (c) the amino acid sequence of SEQ ID NO:4;
[0044] (d) fragments of the amino acid sequence of SEQ ID NO:4;
[0045] (e) the amino acid sequence of SEQ ID NO:6;
[0046] (f) fragments of the amino acid sequence of SEQ ID NO:6;
[0047] (g) the amino acid sequence of SEQ ID NO:7; and
[0048] (h) fragments of the amino acid sequence of SEQ ID NO:7.
[0049] Compositions comprising inhibitors identified according to
such method are also provided. Such compositions may include
pharmaceutically acceptable carriers.
[0050] Methods are also provided for preventing or ameliorating an
inflammatory condition which comprises administering a
therapeutically effective amount of a composition comprising a
protein having IL-1-R intracellular ligand protein activity and a
pharmaceutically acceptable carrier.
[0051] Other embodiments provide methods of inhibiting IL-1-R
intracellular domain binding comprising administering a
therapeutically effective amount of a composition comprising a
protein having IL-1-R intracellular ligand protein activity and a
pharmaceutically acceptable carrier.
[0052] Methods of preventing or ameliorating an inflammatory
condition or of inhibiting IL-1-R intracellular domain binding are
provided which comprise administering to a mammalian subject a
therapeutically effective amount of inhibitors of IL-1-R
intracellular domain binding, are also provided.
[0053] Methods of identifying an inhibitor of IL-1-R intracellular
domain binding are also provided by the present invention which
comprise:
[0054] (a) transforming a cell with a first polynucleotide encoding
an IL-1-R intracellular domain protein, a second polynucleotide
encoding an IL-1-R intracellular ligand protein, and at least one
reporter gene wherein the expression of the reporter gene is
regulated by the binding of the IL-1-R intracellular ligand protein
encoded by the second polynucleotide to the IL-1-R intracellular
domain protein encoded by the first polynucleotide;
[0055] 1(b) growing the cell in the presence of and in the absence
of a compound; and
[0056] (c) comparing the degree of expression of the reporter gene
in the presence of and in the absence of the compound;
[0057] wherein the compound is capable of inhibiting IL-1-R
intracellular domain binding when a decrease in the degree of
expression of the reporter gene occurs. In preferred embodiments,
the cell is a yeast cell and the second polynucleotide is selected
from the group consisting of:
[0058] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:1 from nucleotide 2 to nucleotide 529;
[0059] (b) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:1, which encodes a protein having IL-1-R
intracellular ligand protein activity;
[0060] (c) a polynucleotide encoding an IL-1-R intracellular ligand
protein comprising the amino acid sequence of SEQ ID NO:2;
[0061] (d) a polynucleotide encoding an IL-1-R intracellular ligand
protein comprising a fragment of the amino acid sequence of SEQ ID
NO:2 and having IL-1-R intracellular ligand protein activity;
[0062] (e) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:3 from nucleotide 2 to nucleotide 961;
[0063] (f) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:3, which encodes a protein having IL-1-R
intracellular ligand protein activity;
[0064] (g) a polynucleotide encoding an IL-1-R intracellular ligand
protein comprising the amino acid sequence of SEQ ID NO:4;
[0065] (h) a polynucleotide encoding an IL-1-R intracellular ligand
protein comprising a fragment of the amino acid sequence of SEQ ID
NO:4 and having IL-1-R intracellular ligand protein activity;
[0066] (i) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:5 from nucleotide 2 to nucleotide 754;
[0067] (j) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:5, which encodes a protein having IL-1-R
intracellular ligand protein activity;
[0068] (k) a polynucleotide encoding an IL-1-R intracellular ligand
protein comprising the amino acid sequence of SEQ ID NO:6;
[0069] (l) a polynucleotide encoding an IL-1-R intracellular ligand
protein comprising a fragment of the amino acid sequence of SEQ ID
NO:6 and having IL-1-R intracellular ligand protein activity;
[0070] (m) a polynucleotide encoding an IL-1-R intracellular ligand
protein comprising the amino acid sequence of SEQ ID NO:7;
[0071] (n) a polynucleotide encoding an IL-1-R intracellular ligand
protein comprising a fragment of the amino acid sequence of SEQ ID
NO:7 and having IL-1-R intracellular ligand protein activity;
and
[0072] (o) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in (a)-(n),
which encodes a protein having IL-1-R intracellular ligand protein
activity.
BRIEF DESCRIPTION OF THE FIGURES
[0073] FIG. 1 depicts an autoradiograph demonstrating the
expression of IL-1-R intracellular ligand proteins of the present
invention in mammalian cells. The expression of flag-14w, -31w and
-19w was detected by an anti-flag antibody, M2, as described
below.
[0074] FIG. 2 demonstrates the effects of the clone 19w product on
JNK1 activation. Top panel: HA-tagged JNK1 was coexpressed in COS
cells with either pED flag vector of pED flag-19w. After 48 hr, the
cells were treated with different concentrations of IL-1.alpha. for
15 min. JNK1 was isolated by immunoprecipitation with 12CA5
antibody and JNK activity was measured using an immune complex
kinase assay with the substrate GST-c-jun (1-79). Middle panel: The
expression and recovery if HA-JNK1 from immunoprecipitation was
examined by Western blot analysis wit 12CA5 antibody. Bottom panel:
The expression of clone 19w was detected by Western blot analysis
of cell lysate using M2 antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0075] The present inventors have for the first time identified and
isolated novel polynucleotides which encode proteins which bind to
the IL-1-R intracellular domain. As used herein "IL-1-R" includes
all receptors for interleukin-I. The type 1, p80 IL-1-R is the
preferred receptor for practicing the present invention.
[0076] The sequence of a polynucleotide encoding one such protein
is set forth in SEQ ID NO:1 from nucleotide 2 to 529. This
polynucleotide has been identified as "clone 19w." The amino acid
sequence of the IL-1-R intracellular ligand protein encoded by
clone 19w is set forth in SEQ ID NO:2. It is believed that clone
19w is a partial cDNA clone of a longer full length coding
sequence. However, as demonstrated herein the protein encoded by
clone 19w does bind the intracellular domain of IL-1-R (i.e., has
"IL-1-R intracellular ligand protein activity" as defined herein).
Clone 19w was deposited with the American Type Culture Collection
on Mar. 31, 1995 and given the accession number ATCC 69774. The
protein encoded by clone 19w is 176 amino acids in length. No
identical or closely related sequences were found using database
searches. Therefore, clone 19w encodes a novel protein. However,
using an extensive FASTA search, a significant homology to amino
acids 330 to 390 of thrombospondin (41% identity in 59 amino acids)
is found in the C-terminal portion of the 19w protein. Moreover, a
significant homology to the Ca.sup.- binding domain, EF hand of
calmodulin (25% in 65 amino acids) is observed in the region
between amino acids 40 and 110 of the protein encoded by clone
19w.
[0077] The sequence of a polynucleotide encoding another such
protein is set forth in SEQ ID NO:3 from nucleotide 2 to 961. This
polynucleotide has been identified as "clone 31w." The amino acid
sequence of the IL-1-R intracellular ligand protein encoded by
clone 31w is set forth in SEQ ID NO:4. It is believed that clone
31w is a partial cDNA clone of a longer full length coding
sequence. However, as demonstrated herein the protein encoded by
clone 31w does bind the intracellular domain of IL-1-R (i.e., has
"IL-1-R intracellular ligand protein activity" as defined herein).
Clone 31w was deposited with the American Type Culture Collection
on Mar. 31, 1995 and given the accession number ATCC 69775. The
protein encoded by clone 31w is 320 amino acids in length. No
identical or closely related sequences were found using
BLASTN/BLASTX or FASTA searches. Therefore, clone 31w encodes a
novel protein.
[0078] The sequence of a polynucleotide encoding another such
protein is set forth in SEQ ID NO:5 from nucleotides 2 to 754. This
polynucleotide has been identified as "clone 14w." The amino acid
sequence of the IL-1-R intracellular ligand protein encoded by
clone 14w is set forth in SEQ ID NO:6. It is believed that clone
14w is a partial cDNA clone of a longer full length coding
sequence. However, as demonstrated herein the protein encoded by
clone 14w does bind the intracellular domain of IL-1-R (i.e., has
"IL-1-R intracellular ligand protein activity" as defined herein).
CLone 14w was deposited with the American Type Culture Collection
on Mar. 31, 1995 and given the accession number ATCC 69773.
[0079] The protein encoded by clone 14w is identical to the
sequence of amino acids 449 to 700 of calcium activated neutral
protease (CANP), with the exception of an amino acid change (Val to
Phe) at position 553 of CANP. The sequence of CANP is disclosed in
Imajoh et al., Biochemistry 1988, 27, 8122-8128, which is
incorporated herein by reference (accession no. A31218). The amino
acid sequence of CANP is set forth in SEQ ID NO:7. Based upon this
sequence homology, CANP and certain fragments thereof will exhibit
IL-1-R intracellular ligand binding activity (as defined
herein).
[0080] For the purposes of the present application, "IL-1-R
intracellular ligand protein" includes proteins which exhibit
IL-1-R intracellular ligand protein activity.
[0081] For the purposes of the present application, a protein is
defined as having "IL-1-R intracellular ligand protein activity"
when it binds to a protein derived from the IL-1-R intracellular
domain. Activity can be measured by using any assay which will
detect binding to an IL-1-R intracellular domain protein. Examples
of such assays include without limitation the interaction trap
assays and assays in which IL-1-R intracellular domain protein
which is affixed to a surface in a manner conducive to observing
binding, including without limitation those described in Examples 1
and 3. As used herein an "IL-1-R intracellular domain protein"
includes the entire intracellular domain or fragments thereof.
[0082] Fragments of the IL-1-R intracellular ligand protein which
are capable of interacting with the IL-1-R intracellular domain or
which are capable of inhibiting IL-1-R intracellular domain binding
(i.e., exhibit IL-1-R intracellular ligand protein activity) are
also encompassed by the present invention. Fragments of the IL-1-R
intracellular ligand protein may be in linear form or they may be
cyclized using known methods, for example, as described in H. U.
Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S.
McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both
of which are incorporated herein by reference. Such fragments may
be fused to carrier molecules such as immunoglobulins for many
purposes, including increasing the valency of IL-1-R intracellular
ligand protein binding sites. For example, fragments of the IL-1-R
intracellular ligand protein may be fused through "linker"
sequences to the Fc portion of an immnunoglobulin. For a bivalent
form of the IL-1-R intracellular ligand protein, such a fusion
could be to the Fc portion of an IgG molecule. Other immunoglobulin
isotypes may also be used to generate such fusions. For example, an
IL-1-P intracellular ligand protein--IgM fusion would generate a
decavalent form of the IL-1-R intracellular ligand protein of the
invention.
[0083] The isolated polynucleotide of the invention may be operably
linked to an expression control sequence such as the pMT2 or pED
expression vectors disclosed in Kaufman et al., Nucleic Acids Res.
19, 4485-4490 (1991), in order to produce the IL-1-R intracellular
ligand protein recombinantly. Many suitable expression control
sequences are known in the art. General methods of expressing
recombinant proteins are also known and are exemplified in R.
Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined
herein "operably linked" means that the isolated polynucleotide of
the invention and the expression control sequence are situated
within a vector or cell in such a way that the IL-1-R intracellular
ligand protein is expressed by a host cell which has been
transformed (transfected) with the ligated
polynucleotide/expression control sequence.
[0084] A number of types of cells may act as suitable host cells
for expression of the IL-1-R intracellular ligand protein. Host
cells include, for example. monkey COS cells, Chinese Hamster Ovary
(CHO) cells, human kidney 293 cells, human epidermal A431 cells,
human Colo205 cells,3T3 cells, CV-1 cells, other transformed
primate cell lines, normal diploid cells, cell strains derived from
in vitro culture of primary tissue, primary explants, HeLa cells,
mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
[0085] The IL-1-R intracellular ligand protein may also be produced
by operably linking the isolated polynucleotide of the invention to
suitable control sequences in one or more insect expression
vectors, and employing an insect expression system. Materials and
methods for baculovirus/insect cell expression systems are
commercially available in kit form from, e.g., Invitrogen, San
Diego, Calif., U.S.A. (the MaxBac.RTM. kit), and such methods are
well known in the art, as described in Summers and Smith, Texas
Agricultural Experiment Station Bulletin No. 1555 (1987)
incorporated herein by reference.
[0086] Alternatively, it may be possible to produce the IL-1-R
intracellular ligand protein in lower eukaryotes such as yeast or
in prokaryotes such as bacteria. Potentially suitable yeast strains
include Saccharomyces cerevisiae, Schizosaccharomyces pombe,
Kluyveromyces strains, Candida, or any yeast strain capable of
expressing heterologous proteins. Potentially suitable bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella
typhimurium, or any bacterial strain capable of expressing
heterologous proteins. If the IL-1-R intracellular ligand protein
is made in yeast or bacteria, it may be necessary to modify the
protein produced therein, for example by phosphorylation or
glycosylation of the appropriate sites, in order to obtain the
functional IL-1-R intracellular ligand protein. Such covalent
attachments may be accomplished using known chemical or enzymatic
methods.
[0087] The IL-1-R intracellular ligand protein of the invention may
also be expressed as a product of transgenic animals, e.g., as a
component of the milk of transgenic cows, goats, pigs, or sheep
which are characterized by somatic or germ cells containing a
nucleotide sequence encoding the IL-1-R intracellular ligand
protein.
[0088] The IL-1-R intracellular ligand protein of the invention may
be prepared by culturing transformed host cells under culture
conditions suitable to express the recombinant protein. The
resulting expressed protein may then be purified from such culture
(i.e., from culture medium or cell extracts) using known
purification processes, such as gel filtration and ion exchange
chromatography. The purification of the IL-1-R intracellular ligand
protein may also include an affinity column containing the IL-1-R
intracellular domain or other IL-1-R intracellular domain protein;
one or more column steps over such affinity resins as concanavalin
A-agarose, heparin-toyopearl.RTM. or Cibacrom blue 3GA
Sepharose.RTM.; one or more steps involving hydrophobic interaction
chromatography using such resins as phenyl ether, butyl ether, or
propyl ether; or immunoaffinity chromatography.
[0089] Alternatively, the IL-1-R intracellular ligand protein of
the invention may also be expressed in a form which will facilitate
purification. For example, it may be expressed as a fusion protein,
such as those of maltose binding protein (MBP) or
glutathione-S-transferase (GST). Kits for expression and
purification of such fusion proteins are commercially available
from New England BioLab (Beverly, Mass.) and Pharmacia (Piscataway,
N.J.), respectively. The IL-1-R ligand protein can also be tagged
with an epitope and subsequently purified by using a specific
antibody directed to such epitope. One such epitope ("Flag") is
commercially available from Kodak (New Haven, Conn.).
[0090] Finally, one or more reverse-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify the IL-1-R intracellular ligand
protein. Some or all of the foregoing purification steps, in
various combinations, can also be employed to provide a
substantially homogeneous isolated recombinant protein. The IL-1-R
intracellular ligand protein thus purified is substantially free of
other mammalian proteins and is defined in accordance with the
present invention as an "isolated IL-1-R intracellular ligand
protein."
[0091] IL-1-R intracellular ligand proteins may also be produced by
known conventional chemical synthesis. Methods for constructing the
proteins of the present invention by synthetic means are known to
those skilled, in the art. The synthetically-constructed protein
sequences, by virtue of sharing primary, secondary or tertiary
structural and/or conformational characteristics with IL-1-R
intracellular ligand proteins may possess biological properties in
common therewith, including IL-1-R intracellular ligand protein
activity. Thus, they may be employed as biologically active or
immunological substitutes for natural, purified IL-1-R
intracellular ligand proteins in screening of therapeutic compounds
and in immunological processes for the development of
antibodies.
[0092] The IL-1-R intracellular ligand proteins provided herein
also include proteins characterized by amino acid sequences similar
to those of purified IL-1-R intracellular ligand proteins but into
which modification are naturally provided or deliberately
engineered. For example, modifications in the peptide or DNA
sequences can be made by those skilled in the art using known
techniques. Modifications of interest in the IL-1-R intracellular
ligand protein sequences may include the replacement, insertion or
deletion of a selected amino acid residue in the coding sequence.
For example, one or more of the cysteine residues may be deleted or
replaced with another amino acid to alter the conformation of the
molecule. Mutagenic techniques for such replacement, insertion or
deletion are well known to those skilled in the art (see, e.g.,
U.S. Pat. No. 4,518,584).
[0093] Other fragments and derivatives of the sequences of IL-1-R
intracellular ligand proteins which would be expected to retain
IL-1-R intracellular ligand protein activity in whole or in part
and may thus be useful for screening or other immunological
methodologies may also be easily made by those skilled in the art
given the disclosures herein. Such modifications are believed to be
encompassed by the present invention.
[0094] IL-1-R intracellular ligand protein of the invention may
also be used to screen for agents which are capable of inhibiting
or blocking binding of an IL-1-R intracellular ligand protein to
the intracellular domain of IL-1-R, and thus may act as inhibitors
of IL-1-R intracellular domain binding and/or IL-1 activity.
Binding assays using a desired binding protein, immobilized or not,
are well known in the art and may be used for this purpose using
the IL-1-R intracellular ligand protein of the invention. Examples
1 and 3 describe examples of such assays. Appropriate screening
assays may be cell-based or cell-free. Alternatively, purified
protein based screening assays may be used to identify such agents.
For example, IL-1-R intracellular ligand protein may be immobilized
in purified form on a carrier and binding to purified IL-1-R
intracellular domain may be measured in the presence and in the
absence of potential inhibiting agents. A suitable binding assay
may alternatively employ purified IL-1-R intracellular domain
immobilized on a carrier with a soluble form of a IL-1-R
intracellular ligand protein of the invention. Any IL-1-R
intracellular ligand protein may be used in the screening assays
described above.
[0095] In such a screening assay, a first binding mixture is formed
by combining IL-1-R intracellular domain protein and IL-1-R
intracellular ligand protein, and the amount of binding in the
first binding mixture (B.sub.o) is measured. A second binding
mixture is also formed by combining IL-1-R intracellular domain
protein, IL-1-R intracellular ligand protein, and the compound or
agent to be screened, and the amount of binding in the second
binding mixture (B) is measured. The amounts of binding in the
first and second binding mixtures are compared, for example, by
performing a B/B.sub.o calculation. A compound or agent is
considered to be capable of inhibiting IL-1-R intracellular domain
binding if a decrease in binding in the second binding mixture as
compared to the first binding mixture is observed. The formulation
and optimization of binding mixtures is within the level of skill
in the art. Such binding mixtures may also contain buffers and
salts necessary to enhance or to optimize binding, and additional
control assays may be included in the screening assay of the
invention.
[0096] Alternatively, appropriate screening assays may be cell
based. For example, the binding or interaction between an IL-1-R
ligand protein and the IL-1-R intracellular domain can be measured
in yeast as described below in Examples 1 and 3.
[0097] Compounds found to reduce, preferably by at least about 10%,
more preferably greater than about 50% or more, the binding
activitv of IL-1-R intracellular ligand protein to IL-1-R
intracellular domain may thus be identified and then secondarily
screened in other binding assays, including in vivo assays. By
these means compounds having inhibitory activity for IL-1-R
intracellular domain binding which may be suitable as
anti-inflammatory agents may be identified.
[0098] Isolated IL-1-R intracellular ligand protein may be useful
in treating, preventing or ameliorating inflammatory conditions and
other conditions such as osteoporosis, colitis, myelogenous
leukemia, diabetes, wasting and atherosclerosis. Isolated IL-1-R
intracellular ligand protein may be used itself as an inhibitor of
IL-1-R intracellular domain binding or to design inhibitors of
IL-1-R intracellular domain binding. Inhibitors of binding of
IL-1-R intracellular ligand protein to the IL-1-R intracellular
domain ("IL-1-R intracellular binding inhibitors") are also useful
for treating such conditions.
[0099] The present invention encompasses both pharmaceutical
compositions and therapeutic methods of treatment or use which
employ isolated IL-1-R intracellular ligand protein and/or binding
inhibitors of IL-1-R intracellular binding.
[0100] Isolated IL-1-R intracellular ligand protein or binding
inhibitors (from whatever source derived, including without
limitation from recombinant and non-recombinant cell lines) may be
used in a pharmaceutical composition when combined with a
pharmaceutically acceptable carrier. Such a composition may also
contain (in addition to IL-1-R intracellular ligand protein or
binding inhibitor and a carrier) diluents, fillers, salts, buffers,
stabilizers, solubilizers,and other materials well known in the
art. The term "pharmaceutically acceptable" means a non-toxic
material that does not interfere with the effectiveness of the
biological activity of the active ingredient(s). The
characteristics of the carrier will depend on the route of
administration. The pharmaceutical composition of the invention may
also contain cytokines, lymphokines, or other hematopoletic factors
such as M-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-11, IL-12, G-CSF, Meg-CSF, stem cell factor,
and erythropoietin. The pharmaceutical composition may further
contain other anti-inflammatory agents. Such additional factors
and/or agents may be included in the pharmaceutical composition to
produce a synergistic effect with isolated IL-1-R intracellular
ligand protein or binding inhibitor, or to minimize side effects
caused by the isolated IL-1-R intracellular ligand protein or
binding inhibitor. Conversely, isolated IL-1-R intracellular ligand
protein or binding inhibitor may be included in formulations of the
particular cytokine, lymphokine, other hematopoietic factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent
to minimize side effects of the cytokine lymphokine, other
hematopoietic factor, thrombolytic or anti-thrombotic factor or
anti-inflammatory agent.
[0101] The pharmaceutical composition of the invention may be in
the form of a liposome in which isolated IL-1-R intracellular
ligand protein or binding inhibitor is combined, in addition to
other pharmaceutically acceptable carriers with amphipathic agents
such as lipids which exist in aggregated form as micelles insoluble
monolayers, liquid crystals or lamellar layers in aqueous solution.
Suitable lipids for liposomal formulation include, without
limitation, monoglycerides, diglycerides, sulfatides, lysolecithin,
phospholipids, saponin, bile acids, and the like. Preparation of
such liposomal formulations is within the level of skill in the
art, as disclosed, for example, in U.S. Pat. No. 4,235,871; U.S.
Pat. No. 4,501,728; U.S. Pat. No. 5 4,837,028; and U.S. Pat. No.
4,737,323, all of which are incorporated herein by reference.
[0102] As used herein, the term "therapeutically effective amount"
means the total amount of each active component of the
pharmaceutical composition or method that is sufficient to show a
meaningful patient benefit, i.e., treatment, healing, prevention or
amelioration of an inflammatory response or condition, or an
increase in rate of treatment, healing, prevention or amelioration
of such conditions. When applied to an individual active
ingredient, administered alone, the term refers to that ingredient
alone. When applied to a combination, the term refers to combined
amounts of the active ingredients that result in the therapeutic
effect, whether administered in combination, serially or
simultaneously.
[0103] In practicing the method of treatment or use of the present
invention, a therapeutically effective amount of isolated IL-1-R
intracellular ligand protein or binding inhibitor is administered
to a mammal having a condition to be treated.
[0104] Isolated IL-1-R intracellular ligand protein or binding
inhibitor may be administered in accordance with the method of the
invention either alone or in combination with other therapies such
as treatments employing cytokines, lymphokines or other
hematopoietic factors. When co-administered with one or more
cytokines. lymphokines or other hematopoietic factors, isolated
IL-1-R intracellular ligand protein or binding inhibitor may be
administered either simultaneously with the cytokine(s),
lymphokine(s), other hematopoietic factor(s), thrombolytic or
anti-thrombotic factors, or sequentially. If administered
sequentially, the attending physician will decide on the
appropriate sequence of administering isolated IL-1-R intracellular
ligand protein or binding inhibitor in combination with
cytokine(s), lymphokine(s), other hematopoietic factor(s),
thrombolytic or anti-thrombotic factors.
[0105] Administration of isolated IL-1-R intracellular ligand
protein or binding inhibitor used in the pharmaceutical composition
or to practice the method of the present invention can be carried
out in a variety of conventional ways such as oral ingestion,
inhalation, or cutaneous, subcutaneous, or intravenous injection.
Intravenous administration to the patient is preferred.
[0106] When a therapeutically effective amount of isolated IL-1-R
intracellular ligand protein or binding inhibitor is administered
orally, isolated IL-1-R intracellular ligand protein or binding
inhibitor will be in the form of a tablet, capsule, powder,
solution or elixir. When administered in tablet form, the
pharmaceutical composition of the invention may additionally
contain a solid carrier such as a gelatin or an adjuvant. The
tablet, capsule, and powder contain from about 5 to 95% isolated
IL-1-R intracellular ligand protein or binding inhibitor, and
preferably from about 25 to 90% isolated IL-1-R intracellular
ligand protein or binding inhibitor. When administered in liquid
form, a liquid carrier such as water, petroleum, oils of animal or
plant origin such as peanut oil, mineral oil, soybean oil, or
sesame oil, or synthetic oils may be added. The liquid form of the
pharmaceutical composition may further contain physiological saline
solution, dextrose or other saccharide solution, or glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When
administered in liquid form, the pharmaceutical composition
contains from about 0.5 to 90% by weight of isolated IL-1-R
intracellular ligand protein or binding inhibitor, and preferably
from about 1 to 50% isolated IL-1-R intracellular ligand protein or
binding inhibitor.
[0107] When a therapeutically effective amount of isolated IL-1-R
intracellular ligand protein or binding inhibitor is administered
by intravenous, cutaneous or subcutaneous injection, isolated
IL-1-R intracellular ligand protein or binding inhibitor will be in
the form of a pyrogen-free parenterally acceptable aqueous
solution. The preparation of such parenterally acceptable protein
solutions, having due regard to pH, isotonicity, stability, and the
like, is within the skill in the art. A preferred pharmaceutical
composition for intravenous, cutaneous, or subcutaneous injection
should contain, in addition to isolated IL-1-R intracellular ligand
protein or binding inhibitor, an isotonic vehicle such as Sodium
Chloride Injection, Ringer's Injection, Dextrose Injection,
Dextrose and Sodium Chloride Injection, Lactated Ringer's
Injection, or other vehicle as known in the art. The pharmaceutical
composition of the present invention may also contain stabilizers,
preservatives buffers, antioxidants, or other additives known to
those of skill in the art.
[0108] The amount of isolated IL-1-R intracellular ligand protein
or binding inhibitor in the pharmaceutical composition of the
present invention will depend upon the nature and severity of the
condition being treated, and on the nature of prior treatments
which the patient has undergone. Ultimately, the attending
physician will decide the amount of isolated IL-1-R intracellular
ligand protein or binding inhibitor with which to treat each
individual patient. Initially, the attending physician will
administer low doses of isolated IL-1-R intracellular ligand
protein or binding inhibitor and observe the patient's response.
Larger doses of isolated IL-1-R intracellular ligand protein or
binding inhibitor may be administered until the optimal therapeutic
effect is obtained for the patient, and at that point the dosage is
not increased further. It is contemplated that the various
pharmaceutical compositions used to practice the method of the
present invention should contain about 0.1 .mu.g to about 100 mg of
isolated IL-1-R intracellular ligand protein or binding inhibitor
per kg body weight.
[0109] The duration of intravenous therapy using the pharmaceutical
composition of the present invention will vary, depending on the
severity of the disease being treated and the condition and
potential idiosyncratic response of each individual patient. It is
contemplated that the duration of each application of the isolated
IL-1-R intracellular ligand protein or binding inhibitor will be in
the range of 12 to 24 hours of continuous intravenous
administration. Ultimately the attending physician will decide on
the appropriate duration of intravenous therapy using the
pharmaceutical composition of the present invention.
[0110] Isolated IL-1-R intracellular ligand protein of the
invention may also be used to immunize animals to obtain polyclonal
and monoclonal antibodies which specifically react with the IL-1-R
intracellular ligand protein and which may inhibit IL-1-R
intracellular domain binding. Such antibodies may be obtained using
either the entire IL-1-R intracellular ligand protein or fragments
of IL-1-R intracellular ligand protein as an immunogen. The peptide
immunogens additionally may contain a cysteine residue at the
carboxyl terminus, and are conjugated to a hapten such as keyhole
limpet hemocyanin (KLH). Methods for synthesizing such peptides are
known in the art, for example, as in R. P. Merrifield, J. Amer.
Chem. Soc. 85, 2149-2154 (1963); J. L. Krstenansky, et al., FEBS
Lett. 211, 10 (1987).
[0111] Monoclonal antibodies binding to IL-1-R intracellular ligand
protein or to complex carbohydrate moieties characteristic of the
IL-1-R intracellular ligand glycoprotein may be useful diagnostic
agents for the immunodetection of IL-1-R ligand protein.
[0112] Neutralizing monoclonal antibodies binding to IL-1-R
intracellular ligand protein or to complex carbohydrates
characteristic of IL-1-R intracellular ligand glycoprotein may also
be useful therapeutics for both inflammatory conditions and also in
the treatment of some forms of cancer where abnormal expression of
IL-1-R intracellular ligand protein is involved. These neutralizing
monoclonal antibodies are capable of blocking the signaling
function of the IL-1-R intracellular ligand protein. By blocking
the binding of IL-1-R intracellular ligand protein, certain
biological responses to IL-1 are either abolished or markedly
reduced. In the case of cancerous cells or leukemic cells,
neutralizing monoclonal antibodies against IL-1-R intracellular
ligand protein may be useful in detecting and preventing the
metastatic spread of the cancerous cells, which may be mediated by
the IL-1-R intracellular ligand protein.
[0113] Due to the similarity of its sequence to SEQ ID NO:6, CANP
and fragments thereof which bind to the IL-1-R intracellular domain
are proteins having IL-1-R intracellular ligand protein activity as
defined herein. As a result, they are also useful in pharmaceutical
compositions, for treating inflammatory conditions and for
inhibiting IL-1-R intracellular domain binding as described above
for IL-1-R intracellular ligand proteins generally.
EXAMPLE 1
Cloning of IL-1-R Intracellular Ligand Protein Encoding
Polynucleotide
[0114] A yeast genetic selection method, the "interaction trap"
[Gyuris et al, Cell 75:791-803, 1993, which is incorporated herein
by reference], was used to screen W138 and HeLa cell cDNA libraries
(preparation see below) for proteins that interact with IL-1-R-1c,
the cytoplasmic portion (intracellular domain) of the interleukin-1
receptor p80, or type I. The IL-1-R-1c DNA, encoding amino acids
340 to 552 of the type I IL-1 receptor, was obtained via the
polymerase chain reaction (PCR) of a human W138 cell cDNA library.
This IL-1-R-1c DNA was then cloned into pEG202 by an EcoRI site,
generating the bait plasmid, pEG202-IL-1-R-1c. This plasmid
contains the HIS3 selectable marker, and expression of the bait,
the LexA-IL-1-R-1c fusion protein, is from the strong constitutive
ADH1 promoter. To create the reporter strain carrying the bait
protein, yeast strain EGY48, containing the reporter sequence
LexAop-Leu2 in place of the chromosomal LEU2, was transformed with
pEG202-IL-1-R-1c and pSH18-34 (Ura+), which carries another
reporter sequence, LexAop-lacZ. For screening cDNAs encoding
proteins that interact with IL-1-R-1c, the expression vector pJG4-5
(TRP1), containing either a W138 or HeLa cell cDNA library (see
below for the cDNA library construction), was transformed into the
above strain (EGY48/pEG202-IL-1-R-1c/pSH18-34) according to the
method described by Gietz et al., Nucleic Acids Res., 20, 1425,
1992.
[0115] The bait used in obtaining clones 14w, 19w and 31w was
constructed by cloning the DNA sequences encoding amino acids 477
to 527 of IL-1 receptor p80 into the EcoRI and NotI sites of EG202.
The resulting plasmid was named EG202-IL1R (477-527). This region
of the IL-1 receptor is believed to be essential for signaling.
[0116] cDNA Library Construction:
[0117] W138 cell cDNA library: Double stranded cDNA was prepared
from 3 .mu.g of W138 mRNA using reagents provided by the
Superscript Choice System (Gibco/BRL, Gaithersberg, Md.) with the
following substitutions: the first strand synthesis was primed
using an oligo dT/Xhol primer/linker, and the dNTP mix was
substituted with a mix containing methyl dCTP (Stratagene, LaJolla,
Calif.). The cDNA was modified at both ends by addition of an
EcoRI/NotI/SalI adapter linker and subsequently digested with Xhol.
This produced cDNA molecules possessing an EcoRI/NotI/SalI overhang
at the 5' end of the gene and an Xhol overhang at the 3 end. These
fragments were then ligated into the yeast expression/fusion vector
pJG4-5 (Gyuris et al., Cell, 75, 791-803, 1993), which contains at
its amino terminus, the influenza virus HA1 epitope tag, the B42
acidic transcription activation domain, and the SV40 nuclear
localization signal, all under the control of the
galactose-dependent GAL1 promoter. The resulting plasmids were then
electroporated into DH10B cells (Gibco/BRL). A total of
7.1.times.10.sup.6 colonies were plated on LB plates containing 100
ug/ml of ampicillin. These E. coli were scraped, pooled, and a
large scale plasmid prep was performed using the Wizard Maxi Prep
kit (Promega, Madison, Wis.), yielding 3.2 mg of supercoiled
plasmid DNA.
[0118] HeLa cell cDNA: HeLa cell cDNA preparation methods are
described in Gyuris et al., Cell, 75, 791-803, 1993, which is
incorporated herein by reference.
[0119] HeLa Cell cDNA Screening Results:
[0120] 2.times.10.sup.5 transformants were obtained on glucose
Ura.sup.-His.sup.-Trp.sup.- plates. These transformants were pooled
and resuspended in a solution of 65% glycerol, 10 nM Tris-HCl (pH
7.5), 10 mM MgCl.sub.2 and stored at -80.degree. C. in 1 mL
aliquots. For screening purposes, aliquots of these were diluted
10-fold into Ura.sup.-His.sup.-Trp.sup.- CM dropout gal/raff medium
(containing 2% galactose, 1% raffinose), which induces the
expresssion of the library encoded proteins, and incubated at
30.degree. C. for 4 hours. 2.times.10.sup.6 colony forming units
(CFUs) were then plated on standard 10 cm galactose X-Gal
Ura.sup.-His.sup.-Trp.sup.-Leu.sup.- plates at a density of
2.times.10.sup.5 CFU/plate. After 4 days at 30.degree. C., colonies
that were strong LacZ.sup.+ were chosen for further processing. In
order to test if the Leu.sup.+/LacZ.sup.+ phenotype was due to the
library-encoded protein, the galactose dependency of the phenotype
was tested. Expression of the library-encoded proteins was turned
off by growth on glucose Ura.sup.-His.sup.-Trp.sup.- master plates
and then retested for galactose-dependency on glucose
Ura.sup.-His.sup.-Trp.sup.-L- eu.sup.-, galactose
Ura.sup.-His.sup.-Trp.sup.-Leu.sup.-, glucose X-Gal
Ura.sup.-His.sup.-Trp.sup.-, and galactose X-Gal
Ura.sup.-His.sup.-Trp.su- p.- plates. Of these, many colonies
showed galactose-dependent growth on Leu.sup.- plates and
galactose-dependent blue color on X-Gal-containing medium
(LacZ.sup.- phenotype). Total yeast DNA was prepared from these
colonies according to the method described previously (Hoffman and
Winston, 1987). In order to analyze the cDNA sequences. PCR
reactions were performed using the above yeast DNA as a template
and oligo primers specific for the vector pJG4-5, flanking the cDNA
insertion point. PCR products were purified (Qiagen PCR
purification kit), subjected to restriction digest with the enzyme
HaeIII, run on 1.8% agarose gels, and the restriction patterns
compared. Similar and identical restriction patterns were grouped
and representatives of each group were sequenced and compared to
Genbank and other databases to identify any sequence
homologies.
[0121] W138 Cell cDNA Screening Results:
[0122] This screen was performed as above with the following
exceptions: 1) 1.times.10.sup.6 transformants were obtained on
glucose Ura.sup.-His.sup.-Trp.sup.- plates and pooled. 2)
11.times.10.sup.6 CFU were screened. Of these, 0.5% were Leu.sup.-
and of those, 1% were LacZ.sup.+. This gave a frequency of 50
double positives per 10.sup.6 transformants screened. Colonies,
exhibiting a strong LacZ.sup.+ phenotype (as judged by the strength
of blue color on X-Gal containing medium), were chosen for further
processing. Clones with the strongest LacZ+ phenotype were chosen
for further specificity tests as described above.
[0123] A W138 cDNA library was transformed into the reporter strain
(EGY48/pSH18-34) containing the bait plasmid EG202-IL1R(477-527).
1.3 million primary transformants were harvested and 7 million
colonies were screened. 192 galactose-dependent colonies were
isolated. Among these, 51 clones were bait specific (i.e.,
interacted specifically with the original bait, but not with an
unrelated bait, bicoid). These clones were then subjected to DNA
sequence analysis. Clones 19w was isolated 6, times, clone 31w
twice and clone 14w once.
EXAMPLE 2
Expression of the IL-1-R Intracellular Ligand Protein
[0124] cDNAs encoding IL-1-R intracellular ligand proteins were
released from the pJG4-5 vector with the appropriate restriction
enzymes. For example. EcoRI and XhoI were used to release cDNA from
the relevant clone. Where the restriction sites were also present
in the internal sequence of the cDNA. PCR was performed to obtain
the cDNA. These cDNAs were then cloned into various expression
vectors. These included pGEX (Pharmacia) or pMAL (New England
Biolabs) for expression as a GST (Glutathione-S-transferase) or MBP
(maltose binding protein) fusion protein in E. coli, a pED-based
vector for mammalian expression, and pVL or pBlueBacHis
(Invitrogen) for baculovirus/insect expression. For the
immunodetection of IL-1-R intracellular ligand expression in
mammalian cells. an epitope sequence, "Flag," was inserted into the
translational start site of the pED vector, generating the pED-Flag
vector. cDNAs were then inserted into the pED-Flag vector. Thus,
the expression of cDNA from pED-Flag yields a protein with an amino
terminal Met, followed by the "Flag" sequence,
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys. Standard DEAE-Dextran or
lipofectamine methods were used to transfect COS or CHO dukx cells.
Immunodetection of Flag-tagged proteins was achieved using the M2
antibody (Kodak). Moreover, an immunoaffinity column using the M2
antibody, followed by elution with the "Flag" peptide, can be used
for the rapid purification of the flag-tagged protein. Similarly,
affinity purification of GST-, MBP- or His-tagged fusion proteins
can be performed using glutathione, amylose, or nickel columns.
Detailed purification protocols are provided by the manufacturers.
For many fusion proteins, the IL-1-R intracellular ligand can be
released by the action of thrombin, factor Xa, or enterokinase
cleavage. In the case where highly purified material is required,
standard purification procedures such as ion-exchange, hydrophobic,
and gel filtration chromatography will be applied in addition to
the affinity purification step.
[0125] FIG. 1 depicts an autoradiograph demonstrating the
expression of IL-1-R intracellular ligand proteins in mammalian
cells. FIG. 1 shows the results of expression of Flag-14w, -19w and
-31w in COS cells. COS cells were transfected with either pED-Flag
(vector control), Flag-14w, -19w or -31w plasmid by the
lipofectamine method. Thirty .mu.g of each cell lysate were
prepared and subjected to 4-20% SDS gel electrophoresis, followed
by Western blot analysis using M2 antibody (Kodak). A
Flag-containing protein, Flag-BAP (Kodak) was also loaded as a
standard. The bands in the Flag-14w, -19w and -31w indicate
significant expression of the respective IL-1-R intracellular
ligand proteins.
EXAMPLE 3
Assays of IL-1-R Intracellular Domain Binding
[0126] Two different methods were used to assay for IL-1-R
intracellular ligand protein activity. The first assay measures
binding in the yeast strain in "interaction trap," the system used
here to screen for IL-1-R-1c interacting proteins. In this system,
the expression of reporter genes from both LexAop-Leu2 and
LexAop-LacZ relies on the interaction between the bait protein, in
this case IL-1-R-1c, and the prey, the IL-1-R intracellular ligand.
Thus, one can measure the strength of the interaction by the level
of Leu2 or LacZ expression. The most simple method is to measure
the activity of the LacZ encoded protein, .beta.-galactosidase.
This activity can be judged by the degree of blueness on the X-Gal
containing medium or filter. For the quantitative measurement of
.beta.-galactosidase activity, standard assays can be found in
"Methods in Yeast Genetics" Cold Spring Harbor, New York, 1990 (by
Rose, M. D., Winston, F., and Hieter, P.).
[0127] The second assay for measuring binding is a cell-free
system. An example of a typical assay is described below. Purified
MBP-IL-1-R-1c fusion protein (2 .mu.g) was mixed with
glutathione-Sepharose 4 B beads bound with a GST-IL-1-R-1c
intracellular ligand for 2 hour at 4.degree. C. The mixture was
then centrifuged to separate bound (remained with the beads) and
unbound (remained in the supernatant) MBP-IL-1-R-1c. After
extensive washing, the bound MBP-IL-1-R-1c was eluted with
glutathione and detected by Western blot analysis using an MBP
antibody. The IL-1-R-1c or the intracellular ligand can also be
immobilized on other solid supports, such as on plates or
fluorobeads. The binding can then be measured using ELISA or SPA
(scintillation proximity assay).
EXAMPLE 4
Characterization of IL-1-R Intracellular Ligand Protein
[0128] Mapping the Interaction Site in IL-1-R-1c
[0129] Many of the key amino acids for IL-1-R signaling have been
determined by site-directed mutagenesis (Heguy et al., 1992, JBC,
267, 2605-2609). These amino acids are conserved between IL-1-R and
the Drosophila Toll protein, which is required for transducing
dorsoventral positional information to cells in the developing
embryo. In order to test if the IL-1-R intracellular proteins
interact with these residues, these residues were mutagenized and
the ability of the mutant protein to interact with the
intracellular ligand in the "interaction trap" system was tested.
Mutations that abolish IL-1R signaling were introduced into the
original bait plasmid, EG202-IL-1R (477-527) (with following amino
acid substitutions: F513A, W514A, K515R, R518K, and Y519S) and the
ability of the IL-1R intracellular ligands to interact with these
mutant proteins was tested in the interaction trap. EGY48 carrying
pSH18-34 (lexAop-LacZ) were cotransformed with two plasmids: one
carrying 14w, 19w or 31w; the other with bait, EG202-IL1R
(477-527), either wild-type or one of the mutants transformants
were then streaked onto CM ura.sup.-his.sup.-trp.sup.- plates
containing galactose/raffinose and .beta.-gal. The strength of
interaction (as indicated by the number of "+" signs) was judged by
the blueness in the plates (indicator of LacZ expression). The
results are summarized in Table I.
1 TABLE I bait clone WT F513A W514A K515R R518K Y519S 14 w +++ ++ +
+++ -++ - 19 w + -- + ++ -- -- 31 w ++ -- -- +++ -- --
[0130] Clone 14w interacted with mutant baits W514A and Y519S much
more weakly than with wild-type bait. Clone 19w interacted
differentially with wild-type and manu of the mutant baits. It
appeared to interact with the mutant bait K515R more strongly than
with wild-type, while reduced interaction was observed with mutant
baits F513R, R518K and Y519S. The interaction of clone 31w was
significantly reduced by mutations F513A, W514A, R518K and Y519S.
The change in the interaction strength by these mutations suggests
that these residues are the site(s) of interaction therefore, these
data suggest that clones 14w, 19w and 31w interact with many of the
signaling residues and may play a role in IL-1R signaling.
[0131] Effect on the IL1-Mediated Response
[0132] The effect of the IL-1-R intracellular ligands on the
IL-1-mediated response can be evaluated in cells overexpressing the
ligands. A number of IL-1 mediated responses, including transient
or prolonged responses, can be measured. For example, IL-1-induced
kinase activity toward either MBP (myelin basic protein) or the
N-terminus (amino acids 1-79) of c-jun can be measured in COS cells
or CHO cells either transiently or stably overexpressing IL-1R
intracellular ligand proteins. Alternatively, other functional
assays, such as the induction of gene expression or PGE.sub.2
production after prolonged incubation with IL-1, can also be used
to measure the IL-1 mediated response. Conversely, the significance
of the IL-1-R intracellular ligand proteins in IL-1 signaling can
be established by lowering or eliminating the expression of the
ligands. These experiments can be performed using antisense
expression or transgenic mice.
[0133] IL-1 mediated JNK (c-jun NH.sub.2-terminal kinase, Derjard
et al., Cell 1994, 76, 1025-1037) activation was used to study the
effect of the IL-1R intracellular ligands on IL-1 signaling. COS
cells were transfected with both pEDflag plasmid containing one of
the clones (e.g., 19w) and HA-JNK1 plasmid by the DEAE-dextran
method. 48 hrs after transfection, cells were starved in 0.1% BSA
for 1 hr and treated with various amounts of IL-1.alpha. for 15
min. Cells were then lysed, centrifuged and immunoprecipitation
with anti-HA monoclonal antibody, 12CA5 (Boehringer Mannheim). JNK
activity was performed at 30.degree. C. for 20 min using 5 .mu.g
GST-c-jun (1-79 amino acids), 20 .mu.M ATP, and 5 .mu.Ci
[.gamma.-.sup.32P]ATP in 40 .mu.l of kinase buffer (25 mM HEPES, pH
7.5, 20 mM MgCl.sub.2, 20 mM .beta.-glycerophosphate, 0.1 mM sodium
orthovanadate, 2 mM DTT). The reactions were terminated using
laemmli sample buffer and the products were resolved by SDS-PAGE
(4-20%).
[0134] As shown in FIG. 2, expression of clone 19w stimulated JNK
activity in all IL-1 concentrations tested as compared to the pED
flag vector transfected cells. It also enhanced JNK activity even
in the absence of IL-1. These data strongly suggest that clone 19w,
through its interaction with the signaling domain of IL-1 receptor
(i.e., amino acids 477-527 of IL-1R), may indeed participate in the
signaling event.
[0135] Enzymatic or Functional Assays
[0136] The signal transduction events initiated by IL-1 binding to
its receptor are still largely unknown. However, one major result
of IL-1 binding is the stimulation of cellular serine/threonine
kinase activity. In addition IL-1 has been shown to stimulate the
activity of PC-PLC, PLA.sub.2, and sphingomyelinase. Therefore,
some of the IL-1-R intracellular ligand proteins may possess
intrinsic enzymatic activity that is responsible for these
activities. Therefore, enzymatic assays can be performed to test
this possibility, particularly with those clones that encode
proteins with sequence homology to known enzymes. In addition to
enzymatic activity, based on the sequence homology to proteins with
known function, other functional assays, for instance, ATP
binding/transporter activity for the full length protein of clone
140, can also be measured.
EXAMPLE 5
Isolation of Full Length Clones
[0137] In many cases, cDNAs obtained from the interaction trap
method each encode only a portion of the full length protein.
Therefore, it is desirable to isolate full length clones. The cDNAs
obtained from the screening are used as probes, and the cDNA
libraries described herein, or alternatively phage cDNA libraries,
are screened to obtain full length clones in accordance with known
methods (see for example, "Molecular Cloning, A Laboratory Manual",
by Sarnbrook et al. 1989 Cold Spring Harbor).
EXAMPLE 6
Antibodies Specific for IL-1-R Intracellular Ligand Protein
[0138] Antibodies specific for IL-1-R intracellular ligand proteins
can be produced using purified recombinant protein, as described in
Example 2. as antigen. Both polyclonal and monoclonal antibodies
will be produced using standard techniques, such as those described
in "Antibodies, a Laboratory Manual" by Ed Harlow and David Lane
(1988), Cold Spring Harbor Laboratory.
* * * * *